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WO2025036241A1 - Composés et procédés de modulation de l'épissage de l'arn - Google Patents

Composés et procédés de modulation de l'épissage de l'arn Download PDF

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Publication number
WO2025036241A1
WO2025036241A1 PCT/CN2024/110654 CN2024110654W WO2025036241A1 WO 2025036241 A1 WO2025036241 A1 WO 2025036241A1 CN 2024110654 W CN2024110654 W CN 2024110654W WO 2025036241 A1 WO2025036241 A1 WO 2025036241A1
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Prior art keywords
compound
rna
splicing
disease
pharmaceutically acceptable
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Chao FANG
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Ribopeutic Inc
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Ribopeutic Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present disclosure relates to compounds and related compositions that modulates nucleic acid splicing, as well as methods of use of the compounds for modulating splicing and treating diseases and conditions.
  • RNA splicing is the process for editing the nascent pre-messenger RNA (pre-mRNA) transcript, and introns are removed and exons are ligated after the splicing.
  • RNA splicing is an essential process required for gene expression in most animals. For nuclear-encoded genes, splicing takes place within the nucleus either co-transcriptionally or immediately after transcription. For many eukaryotic introns, splicing is carried out in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs) .
  • snRNPs small nuclear ribonucleoproteins
  • RNA splicing can enable cells to achieve a wide variety of adaptations and differentiation, while dysregulated or incorrect RNA splicing can lead to disease occurrence and progression. Therefore, regulation of RNA splicing using small molecules offers great potential for treatment of relevant diseases.
  • the present disclosure discloses a compound of Formula (I) :
  • A is S, O or NH
  • M and G is O or NR 1 , and the other is CR 2 R 2a or CR 3 R 3a CR 4 R 4a ;
  • each of Q, E and J is independently C or N;
  • Y is optionally substituted heteroaryl or heterocyclic group
  • each of R a , R b and R c is independently absent, H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, aryl, cyano, oxo, OR’ , -NR’ 2 , -NR’C (O) R’ , -NO 2 , -C (O) NR’ 2 , -C (O) R’ , -C (O) OR’ , S (O) R’ , or -S (O) OR’ , each of which is optionally substituted;
  • each of R 1 , R 2 , R 2a , R 3 , R 3a , R 4 and R 4a is independently selected from H, D, C1-C6 alkyl, C1-C6 heteroalkyl, cycloalkyl, or heterocycloalkyl, each of which is optionally substituted; or R 2 and R 2a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted; or R 3 and R 3a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted; or R 4 and R 4a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted;
  • R 5 is H, optionally substituted alkyl, or optionally substituted cycloalkyl
  • each of R 6 , R 7 , R 8 and R 9 is independently optionally substituted cycloalkyl, or optionally substituted heterocycloalkyl;
  • R 5 , R 6 and the N atom to which they are attached are taken together to form a 3-10-membered ring, which may be a single ring, spiro ring, or fused ring, and the ring is optionally substituted;
  • each R’ is independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted; or two R’and the atoms to which they are attached are taken together to form an optionally substituted 3-7-membered ring.
  • the present disclosure discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and a pharmaceutically acceptable excipient.
  • the present disclosure provides methods for modulating splicing, e.g., splicing of a nucleic acid (e.g., a DNA or RNA, e.g., a pre-mRNA) with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
  • a nucleic acid e.g., a DNA or RNA, e.g., a pre-mRNA
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., splicing of a nucleic acid (e.g., a DNA or RNA, e.g., a pre-mRNA) with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
  • compositions for use in modulating splicing e.g., splicing of a nucleic acid (e.g., a DNA or RNA, e.g., a pre-mRNA) with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
  • Modulation of splicing may comprise impacting any step involved in splicing and may include an event upstream or downstream of a splicing event.
  • the compound of Formula (I) binds to a target, e.g., a target nucleic acid (e.g., DNA or RNA, e.g., a precursor RNA, e.g., a pre-mRNA) , a target protein, or combination thereof (e.g., an snRNP and a pre-mRNA) .
  • a target may include a splice site in a pre-mRNA or a component of the splicing machinery, such as the U1 snRNP.
  • the compound of Formula (I) alters a target nucleic acid (e.g., DNA or RNA, e.g., a precursor RNA, e.g., a pre-mRNA) , target protein, or combination thereof.
  • a target nucleic acid e.g., DNA or RNA, e.g., a precursor RNA, e.g., a pre-mRNA
  • target protein e.g., a pre-mRNA
  • the compound of Formula (I) increases or decreases splicing at a splice site on a target nucleic acid (e.g., an RNA, e.g., a precursor RNA, e.g., a pre-mRNA) by about 0.5%or more (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more) , relative to a reference (e.g., the absence of a compound of Formula (I) , e.g., in a healthy or diseased cell or tissue) .
  • a target nucleic acid e.g., an RNA, e.g., a precursor RNA, e.g., a pre-mRNA
  • a reference e.g., the absence of a compound of Formula (I) , e.g., in a healthy or diseased cell or tissue
  • the presence of a compound of Formula (I) results an increase or decrease of transcription of a target nucleic acid (e.g., an RNA) by about 0.5%or more (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more) , relative to a reference (e.g., the absence of a compound of Formula (I) , e.g., in a healthy or diseased cell or tissue) .
  • a target nucleic acid e.g., an RNA
  • a reference e.g., the absence of a compound of Formula (I) , e.g., in a healthy or diseased cell or tissue
  • the present disclosure provides methods of down-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject.
  • the present disclosure provides methods of up-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject.
  • the present disclosure provides methods of altering the isoform of a target protein with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject.
  • Another aspect of the disclosure relates to methods of inhibiting the activity of a target protein in a biological sample or subject.
  • administration of a compound of Formula (I) to a biological sample, a cell, or a subject comprises inhibition of cell growth or induction of cell death.
  • the present disclosure discloses a method of forming a complex comprising a component of a spliceosome (e.g., a major spliceosome component or a minor spliceosome component) , a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) , and a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof described herein, or the pharmaceutical composition described herein, comprising contacting the nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) with the compound or the pharmaceutical composition.
  • a spliceosome e.g., a major spliceosome component or a minor spliceosome component
  • a nucleic acid e.g., a DNA, RNA, e.g., a pre-mRNA
  • the present disclosure discloses a method of altering the conformation of a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) comprising contacting the nucleic acid with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof described herein, or the pharmaceutical composition described herein.
  • the altering comprises forming a bulge in the nucleic acid.
  • the altering comprises stabilizing or destabilizing a bulge in the nucleic acid.
  • the altering comprises reducing a bulge in the nucleic acid.
  • the nucleic acid comprises a splice site.
  • the present disclosure discloses a method of treating disease or disorder in a subject in need thereof, comprising administering to the subject the compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or the pharmaceutical composition described herein.
  • the present disclosure discloses a use of the compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or the pharmaceutical composition described herein in preparation of medication for treating disease or disorder.
  • the disease or disorder entails unwanted or aberrant splicing.
  • the disease or disorder is a proliferative disease, disorder, or condition.
  • Exemplary proliferative diseases include cancer, a benign neoplasm, or angiogenesis.
  • the present disclosure provides methods for treating and/or preventing a non-proliferative disease, disorder, or condition.
  • the present disclosure provides methods for treating and/or preventing a neurological disease or disorder, autoimmune disease or disorder, immunodeficiency disease or disorder, lysosomal storage disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder, respiratory disease or disorder, renal disease or disorder, or infectious disease.
  • C1-C6 alkyl is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.
  • C1-C4 alkyl is intended to encompass, C1, C2, C3, C4, C1-C4, C1-C3, C1-C2, C2-C4, C2-C3, and C3-C4 alkyl.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms ( “C1-C24 alkyl” ) .
  • an alkyl group has 1 to 12 carbon atoms ( “C1-C12 alkyl” ) .
  • an alkyl group has 1 to 8 carbon atoms ( “C1-C8 alkyl” ) .
  • an alkyl group has 1 to 6 carbon atoms ( “C1-C6 alkyl” ) .
  • an alkyl group has 2 to 6 carbon atoms ( “C2-C6 alkyl” ) .
  • an alkyl group has 1 carbon atom ( “C1 alkyl” ) .
  • C1-6 alkyl groups include methyl (C1) , ethyl (C2) , n-propyl (C3) , isopropyl (C3) , n-butyl (C4) , tert-butyl (C4) , sec-butyl (C4) , iso-butyl (C4) , n-pentyl (C5) , 3-pentanyl (C5) , amyl (C5) , neopentyl (C5) , 3-methyl-2-butanyl (C5) , tertiary amyl (C5) , and n-hexyl (C6) .
  • alkyl groups include n-heptyl (C7) , n-octyl (C8) and the like.
  • Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl” ) or substituted (a “substituted alkyl” ) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkyl group is unsubstituted C1-C10 alkyl (e.g., -CH 3 ) .
  • the alkyl group is substituted C1-C6 alkyl.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ( “C2-C24 alkenyl” ) .
  • an alkenyl group has 2 to 10 carbon atoms ( “C2-C10 alkenyl” ) .
  • an alkenyl group has 2 to 8 carbon atoms ( “C2-C8 alkenyl” ) .
  • an alkenyl group has 2 to 6 carbon atoms ( “C2-C6 alkenyl” ) .
  • an alkenyl group has 2 carbon atoms ( “C2 alkenyl” ) .
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl) .
  • Examples of C2-C4 alkenyl groups include ethenyl (C2) , 1-propenyl (C3) , 2-propenyl (C3) , 1-butenyl (C4) , 2-butenyl (C4) , butadienyl (C4) , and the like.
  • C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5) , pentadienyl (C5) , hexenyl (C6) , and the like. Additional examples of alkenyl include heptenyl (C7) , octenyl (C8) , octatrienyl (C8) , and the like.
  • Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl” ) or substituted (a “substituted alkenyl” ) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkenyl group is unsubstituted C2-C10 alkenyl.
  • the alkenyl group is substituted C2-C6 alkenyl.
  • alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ( “C2-C24 alkenyl” ) .
  • an alkynyl group has 2 to 10 carbon atoms ( “C2-C10 alkynyl” ) .
  • an alkynyl group has 2 to 8 carbon atoms ( “C2-C8 alkynyl” ) .
  • an alkynyl group has 2 to 6 carbon atoms ( “C2-C6 alkynyl” ) .
  • an alkynyl group has 2 carbon atoms ( “C2 alkynyl” ) .
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl) .
  • Examples of C2-C4 alkynyl groups include ethynyl (C2) , 1-propynyl (C3) , 2-propynyl (C3) , 1-butynyl (C4) , 2-butynyl (C4) , and the like.
  • Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl” ) or substituted (a “substituted alkynyl” ) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkynyl group is unsubstituted C2-10 alkynyl.
  • the alkynyl group is substituted C2-C6 alkynyl.
  • heteroalkyl refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized.
  • the heteroatom (s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group.
  • heteroalkyl Up to two or three heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and -CH 2 -O-Si (CH 3 ) 3 .
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -CH 2 O, -NR C R D , or the like, it will be understood that the terms heteroalkyl and -CH 2 O or -NR C R D are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity.
  • heteroalkyl should not be interpreted herein as excluding specific heteroalkyl groups, such as -CH 2 O, -NR C R D , or the like.
  • Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl” ) or substituted (a “substituted heteroalkyl” ) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ( “C6-C14 aryl” ) .
  • an aryl group has six ring carbon atoms ( “C6 aryl” ; e.g., phenyl) .
  • an aryl group has ten ring carbon atoms (“C10 aryl” ; e.g., naphthyl such as 1-naphthyl and 2-naphthyl) .
  • an aryl group has fourteen ring carbon atoms ( “C14 aryl” ; e.g., anthracyl) .
  • An aryl group may be described as, e.g., a 6-10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
  • Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
  • Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl” ) or substituted (a “substituted aryl” ) with one or more substituents.
  • the aryl group is unsubstituted C6-C14 aryl.
  • the aryl group is substituted C6-C14 aryl.
  • heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 ⁇ electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ( “5-10 membered heteroaryl” ) .
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.
  • Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl) .
  • a heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
  • Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl” ) or substituted (a “substituted heteroaryl” ) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5, 6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6, 6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • cycloalkyl refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms ( “C3-C10 cycloalkyl” ) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ( “C3-C8 cycloalkyl” ) . In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ( “C3-C6 cycloalkyl” ) .
  • a cycloalkyl group has 3 to 6 ring carbon atoms ( “C3-C6 cycloalkyl” ) . In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ( “C5-C10 cycloalkyl” ) .
  • a cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
  • Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3) , cyclopropenyl (C3) , cyclobutyl (C4) , cyclobutenyl (C4) , cyclopentyl (C5) , cyclopentenyl (C5) , cyclohexyl (C6) , cyclohexenyl (C6) , cyclohexadienyl (C6) , and the like.
  • Exemplary C3-C8 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7) , cycloheptenyl (C7) , cycloheptadienyl (C7) , cycloheptatrienyl (C7) , cyclooctyl (C8) , cyclooctenyl (C8) , cubanyl (C8) , bicyclo [l. l.
  • Exemplary C3-C10 cycloalkyl groups include, without limitation, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9) , cyclononenyl (C9) , cyclodecyl (C10) , cyclodecenyl (C10) , octahydro-1H -indenyl (C9) , decahydronaphthalenyl (C10) , spiro [4.5] decanyl (C10) , and the like.
  • the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl” ) or contain a fused, bridged or spiro ring system such as a bicyclic system ( “bicyclic cycloalkyl” ) and can be saturated or can be partially unsaturated.
  • “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system.
  • Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl” ) or substituted (a “substituted cycloalkyl” ) with one or more substituents.
  • the cycloalkyl group is unsubstituted C3-C10 cycloalkyl.
  • the cycloalkyl group is a substituted C3-C10 cycloalkyl.
  • heterocyclyl or “heterocycloalkyl” can be used interchangeably herein and refers to a radical of a 3-to 16-membered non-aromatic ring system having ring carbon atoms and 1 to 8 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ( “3-16 membered heterocyclyl” ) .
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic ( “monocyclic heterocyclyl” ) or a fused, bridged or spiro ring system such as a bicyclic system ( “bicyclic heterocyclyl” ) , and can be saturated or can be partially unsaturated.
  • Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • a heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety.
  • Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl” ) or substituted (a “substituted heterocyclyl” ) with one or more substituents.
  • the heterocyclyl group is unsubstituted 3-16 membered heterocyclyl.
  • the heterocyclyl group is substituted 3-16 membered heterocyclyl.
  • Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione.
  • Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.
  • Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl (e.g., 2, 2, 6, 6-tetramethylpiperidinyl) , tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., l-methylpyridin2-onyl) , and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl) , pyrimidinonyl (e.g., l-methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl) , dithianyl, dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
  • Exemplary 5-membered heterocyclyl groups fused to a heterocyclyl ring include, without limitation, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo [3, 4-c] pyrrolyl) , and the like.
  • Exemplary 6-membered heterocyclyl groups fused to a heterocyclyl ring include, without limitation, diazaspirononanyl (e.g., 2, 7-diazaspiro [3.5] nonanyl) .
  • Exemplary 6-membered heterocyclyl groups fused to an aryl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
  • Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring include, without limitation, azabicyclooctanyl (e.g., (l, 5) -8-azabicyclo [3.2.1] octanyl) .
  • Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring include, without limitation, azabicyclononanyl (e.g., 9-azabicyclo [3.3.1] nonanyl) .
  • alkylene alkenylene
  • alkynylene haloalkylene
  • heteroalkylene cycloalkylene
  • heterocyclylene alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, haloalkylene, heteroalkylene, cycloalkyl, or heterocyclyl respectively.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, or heterocyclylene group may be described as, e.g., a C1-C6-membered alkylene, C2-C6-membered alkenylene, C2-C6-membered alkynylene, C1-C6-membered haloalkylene, C1-C6-membered heteroalkylene, C3-C8-membered cycloalkylene, or C3-C8-membered heterocyclylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like) . Still further, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C (O) 2 R’ -may represent both -C (O) 2 R’ -and -R’ C (O) 2 -.
  • cyano or “-CN” as used herein refer to a substituent having a carbon atom joined to a nitrogen atom by a triple bond, e.g., C ⁇ N.
  • halogen or “halo” as used herein refer to fluorine, chlorine, bromine or iodine.
  • hydroxy refers to -OH.
  • nitro refers to a substituent having two oxygen atoms bound to a nitrogen atom, e.g., -NO 2 .
  • oxo refers to a carbonyl, i.e., -C (O) -.
  • Alkyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted.
  • substituted whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound.
  • the present disclosure contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocyclyl groups.
  • Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
  • the ring-forming substituents are attached to adjacent members of the base structure.
  • two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring-forming substituents are attached to non-adjacent members of the base structure.
  • the compounds provided herein may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to:cis-and trans-forms; E-and Z-forms; endo-and exo-forms; R-, S-, and meso-forms; D-and L-forms; d-and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal-and anticlinal-forms; ⁇ -and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or “isomeric forms” ) .
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • the stereochemistry depicted in a compound is relative rather than absolute.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high-pressure liquid chromatography
  • a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess) .
  • an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form.
  • enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75%by weight, more than 80%by weight, more than 85%by weight, more than 90%by weight, more than 91%by weight, more than 92%by weight, more than 93%by weight, more than 94%by weight, more than 95%by weight, more than 96%by weight, more than 97%by weight, more than 98%by weight, more than 99%by weight, more than 99.5%by weight, or more than 99.9%by weight, of the enantiomer.
  • the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
  • an enantiomerically pure compound can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising an enantiomerically pure R-compound can comprise, for example, about 90%excipient and about 10%enantiomerically pure R-compound.
  • the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95%by weight R-compound and at most about 5%by weight S-compound, by total weight of the compound.
  • a pharmaceutical composition comprising an enantiomerically pure S-compound can comprise, for example, about 90%excipient and about 10%enantiomerically pure S-compound.
  • the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95%by weight S-compound and at most about 5%by weight R-compound, by total weight of the compound.
  • a diastereomerically pure compound can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising a diastereometerically pure exo compound can comprise, for example, about 90%excipient and about 10%diastereometerically pure exo compound.
  • the diastereometerically pure exo compound in such compositions can, for example, comprise, at least about 95%by weight exo compound and at most about 5%by weight endo compound, by total weight of the compound.
  • a pharmaceutical composition comprising a diastereometerically pure endo compound can comprise, for example, about 90%excipient and about 10%diastereometerically pure endo compound.
  • the diastereometerically pure endo compound in such compositions can, for example, comprise, at least about 95%by weight endo compound and at most about 5%by weight exo compound, by total weight of the compound.
  • an isomerically pure compound can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising an isomerically pure exo compound can comprise, for example, about 90%excipient and about 10%isomerically pure exo compound.
  • the isomerically pure exo compound in such compositions can, for example, comprise, at least about 95%by weight exo compound and at most about 5%by weight endo compound, by total weight of the compound.
  • a pharmaceutical composition comprising an isomerically pure endo compound can comprise, for example, about 90%excipient and about 10%isomerically pure endo compound.
  • the isomerically pure endo compound in such compositions can, for example, comprise, at least about 95%by weight endo compound and at most about 5%by weight exo compound, by total weight of the compound.
  • the active ingredient can be formulated with little or no excipient or carrier.
  • H may be in any isotopic form, including 1 H, 2 H (D or deuterium) , and 3 H (T or tritium) ;
  • C may be in any isotopic form, including 12 C, 13 C, and 14 C;
  • O may be in any isotopic form, including 16 O and 18 O;
  • N may be in any isotopic form, including 14 N and 15 N;
  • F may be in any isotopic form, including 18 F, 19 F, and the like.
  • pharmaceutically acceptable salt is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like.
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those skilled in the art.
  • Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention.
  • prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • solvate refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding.
  • Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • the compounds of Formula (I) may be prepared, e.g., in crystalline form, and may be solvated.
  • Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid.
  • “Solvate” encompasses both solution-phase and isolable solvates.
  • Representative solvates include hydrates, ethanolates, and methanolates.
  • hydrate refers to a compound which is associated with water.
  • the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R x H 2 O, wherein R is the compound and wherein x is a number greater than 0.
  • a given compound may form more than one type of hydrates, including, e.g., monohydrates (x is 1) , lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R ⁇ 0.5 H 2 O) ) , and polyhydrates (x is a number greater than 1, e.g., dihydrates (R ⁇ 2 H 2 O) and hexahydrates (R ⁇ 6 H 2 O) ) .
  • monohydrates x is 1
  • lower hydrates x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R ⁇ 0.5 H 2 O)
  • polyhydrates x is a number greater than 1, e.g., dihydrates (R ⁇ 2 H 2 O) and hexahydrates (R ⁇ 6 H 2 O) ) .
  • tautomer refers to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of ⁇ electrons and an atom (usually H) .
  • enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base.
  • Another example of tautomerism is the aci-and nitro-forms of phenylnitromethane that are likewise formed by treatment with acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
  • acquire or “acquiring” as used herein refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample) , by “directly acquiring” or “indirectly acquiring” the value or physical entity.
  • “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity.
  • “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value) .
  • Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject.
  • Directly acquiring a value includes performing a process that uses a machine or device, e.g., mass spectrometer to acquire mass spectrometry data
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof.
  • co-administration are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.
  • an “effective amount” of a compound of Formula (I) refers to an amount sufficient to elicit the desired biological response, i.e., treating the condition.
  • the effective amount of a compound of Formula (I) may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject.
  • An effective amount encompasses therapeutic and prophylactic treatment.
  • an effective amount of an inventive compound may reduce the tumor burden or stop the growth or spread of a tumor.
  • a “therapeutically effective amount” of a compound of Formula (I) is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • prevention refers to a treatment that comprises administering a therapy, e.g., administering a compound described herein (e.g., a compound of Formula (I) ) prior to the onset of a disease, disorder, or condition in order to preclude the physical manifestation of said disease, disorder, or condition.
  • a therapy e.g., administering a compound described herein (e.g., a compound of Formula (I) ) prior to the onset of a disease, disorder, or condition in order to preclude the physical manifestation of said disease, disorder, or condition.
  • prevention e.g., “prevent” , and “preventing” require that signs or symptoms of the disease, disorder, or condition have not yet developed or have not yet been observed.
  • treatment comprises prevention and in other embodiments it does not.
  • a “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult) ) and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys) ; commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys) .
  • the animal is a mammal.
  • the animal may be a male or female and at any stage of development.
  • a non-human animal may be a transgenic animal.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause of a disease, disorder, or condition (e.g., as described herein) , e.g., by administering a therapy, e.g., administering a compound described herein (e.g., a compound of Formula (I) ) .
  • treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a symptom of a disease, disorder, or condition.
  • treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a manifestation of a disease, disorder, or condition.
  • treating comprises reducing, reversing, alleviating, reducing, or delaying the onset of, an underlying cause of a disease, disorder, or condition.
  • “treatment” , “treat” , and “treating” require that signs or symptoms of the disease, disorder, or condition have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors) . Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment comprises prevention and in other embodiments it does not.
  • nucleobase is a nitrogen-containing biological compounds found linked to a sugar within a nucleoside -the basic building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) .
  • the primary, or naturally occurring, nucleobases are cytosine (DNA and RNA) , guanine (DNA and RNA) , adenine (DNA and RNA) , thymine (DNA) and uracil (RNA) , abbreviated as C, G, A, T, and U, respectively. Because A, G, C, and T appear in the DNA, these molecules are called DNA-bases; A, G, C, and U are called RNA-bases.
  • Adenine and guanine belong to the double-ringed class of molecules called purines (abbreviated as R) .
  • Cytosine, thymine, and uracil are all pyrimidines.
  • Other nucleobases that do not function as normal parts of the genetic code, are termed non-naturally occurring.
  • a nucleobase may be chemically modified, for example, with an alkyl (e.g., methyl) , halo, -O-alkyl, or other modification.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form.
  • nucleic acid includes a gene, cDNA, pre-mRNA, or an mRNA.
  • the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementarity sequences as well as the sequence explicitly indicated.
  • polypeptide refers to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains.
  • polypeptide refers to a polymer of at least two amino acid monomers joined together through amide bonds.
  • An amino acid may be the L-optical isomer or the D-optical isomer.
  • polypeptide refers to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein.
  • Proteins are essential for the structure, function, and regulation ofthe body's cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof.
  • a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein.
  • a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein.
  • a protein or a variant thereof can be naturally occurring or recombinant.
  • polynucleotide as used herein generally refers to a molecule comprising two or more linked nucleic acid subunits, e.g., nucleotides, and can be used interchangeably with “oligonucleotide” .
  • a polynucleotide may include one or more nucleotides selected from adenosine (A) , cytosine (C) , guanine (G) , thymine (T) and uracil (U) , or variants thereof.
  • a nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO 3 ) groups.
  • Methods for detection and/or measurement of polypeptides in biological material include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques.
  • An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen.
  • composition and “pharmaceutical formulation” (or “formulation” ) are used interchangeably and denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject, e.g., a human in need thereof.
  • pharmaceutical combination means a product that results from mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • fixed combination means that the active ingredients, e.g., a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredients, e.g. a compound described herein and a co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient.
  • cocktail therapy e.g., administration of three or more active ingredients.
  • pharmaceutically acceptable denotes an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use.
  • “Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • pharmaceutically acceptable excipient can be used interchangeably and denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives or lubricants used in formulating pharmaceutical products.
  • pharmaceutically acceptable salts denotes salts which are not biologically or otherwise undesirable.
  • Pharmaceutically acceptable salts include both acid and base addition salts.
  • a “pharmaceutically acceptable salt” can refer to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and/or does not abrogate the biological activity and properties of the compound.
  • intron refers to both the DNA sequence within a gene and the corresponding sequence in the unprocessed RNA transcript. As part of the RNA processing pathway, introns can be removed by RNA splicing either shortly after or concurrent with transcription. Introns are found in the genes of most organisms and many viruses. They can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA) , and transfer RNA (tRNA) .
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • an “exon” can be any part of a gene that encodes a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing.
  • the term “exon” refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts.
  • a “spliceosome” can be assembled from snRNAs and protein complexes.
  • the spliceosome can remove introns from a transcribed pre-mRNA.
  • the present disclosure provides compounds of Formula (I) :
  • A is S, O or NH
  • M and G is O or NR 1 , and the other is CR 2 R 2a or CR 3 R 3a CR 4 R 4a ;
  • each of Q, E and J is independently C or N;
  • Y is optionally substituted heteroaryl or heterocyclic group
  • each of R a , R b and R c is independently absent, H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, aryl, cyano, oxo, OR’ , -NR’ 2 , -NR’C (O) R’ , -NO 2 , -C (O) NR’ 2 , -C (O) R’ , -C (O) OR’ , S (O) R’ , or -S (O) OR’ , each of which is optionally substituted;
  • each of R 1 , R 2 , R 2a , R 3 , R 3a , R 4 and R 4a is independently selected from H, D, C1-C6 alkyl, C1-C6 heteroalkyl, cycloalkyl, or heterocycloalkyl, each of which is optionally substituted; or R 2 and R 2a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted; or R 3 and R 3a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted; or R 4 and R 4a together with the atom to which they are attached, form a 3-10 membered ring system, and the ring is optionally substituted;
  • R 5 is H, optionally substituted alkyl, or optionally substituted cycloalkyl
  • each of R 6 , R 7 , R 8 and R 9 is independently optionally substituted cycloalkyl, or optionally substituted heterocycloalkyl;
  • R 5 , R 6 and the N atom to which they are attached are taken together to form a 3-10-membered ring, which may be a single ring, spiro ring, or fused ring, and the ring is optionally substituted;
  • each R’ is independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted; or two R’and the atoms to which they are attached are taken together to form an optionally substituted 3-7-membered ring.
  • A is S, O or NH; one of M and G is O or NR 1 , and the other is CR 2 R 2a or CR 3 R 3a CR 4 R 4a ; Q, E and J are each independently C or N.
  • A is S or O; one of M and G is O or NR 1 , and the other is CR 2 R 2a ; Q, E and J are each independently C or N. In some embodiments, A is S or O. In some embodiments, A is S.
  • the compound of Formula (I) is a compound of formula wherein M, G, Q, E, J, X, Y, R a , R b , R c are as described herein.
  • one of M and G is O, and the other is CR 2 R 2a or CR 3 R 3a CR 4 R 4a ; wherein preferably, each of R 2 , R 2a , R 3 , R 3a , R 4 , and R 4a is independently H or C1-C4 alkyl, preferably methyl. In some embodiments, each of R 2a , R 3a and R 4a is H. In some embodiments, R 2 and R 2a together with the carbon atom to which they are attached form an optionally substituted 3-10 membered ring system, preferably a 3-membered ring. In some embodiments, one of M and G is O, and the other is CHR 2 ; wherein preferably, R 2 is C1-C4 alkyl, preferably methyl.
  • none, one, two, or three of Q, E and J are each independently N, preferably none or one of Q, E and J is N. In some embodiments, Q, E and J are C; or only one of Q, E and J is N and the other two are C.
  • each of R 1 , R 2 , R 2a , R 3 , R 3a , R 4 and R 4a is independently H, optionally substituted C1-C4 alkyl, or optionally substituted C1-C4 heteroalkyl.
  • R a , R b and R c are independently absent, H, D, or halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, aryl, cyano, oxo, OR’ , -NR’ 2 , -NR’ C (O) R’ , -NO 2 , -C (O) NR’ 2 , -C (O) R’ , -C (O) OR’ , S (O) R’ , or -S (O) OR’ , each of which is optionally substituted.
  • R’ is independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted; or two R’and the atoms to which they are attached are taken together to form an optionally substituted 3-7-membered ring.
  • each of R a , R b and R c is independently absent, H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, 3-6-menbered cycloalkyl, 3-6-menbered heterocycloalkyl, 6-12-menbered heteroaryl, C6-12 aryl, cyano, oxo, OR’ , -NR’ 2 , -NR’ C (O) R’ , -NO 2 , -C (O) NR’ 2 , -C (O) R’ , -C (O) OR’ , S (O) R’ , or -S (O) OR’ .
  • each of R a , R b and R c is independently absent, H, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 heteroalkyl, C1-C4 haloalkyl, cyano, oxo, OR’ , -NR’ 2 , -NR’ C (O) R’ , -NO 2 , -C (O) NR’ 2 , -C (O) R’ , -C (O) OR’ , S (O) R’ , or -S (O) OR’ .
  • each of R a , R b and R c is independently absent, H, halogen, or C1-C6 alkyl. In some embodiments, each of R a , R b and R c is independently absent, H, or halogen.
  • each of R a , R b and R c is independently absent or H.
  • R a is halogen, each of R b and R c is independently H or absent. In some embodiments, R b is halogen, each of R a and R c is independently H or absent. In some embodiments, R c is halogen, each of R a and R b is independently H or absent.
  • each of R a , R b and R c is independently absent, H or F.
  • R a is F
  • R b and R c are H.
  • R b is F
  • R a and R c are H.
  • R c is F, R a and R b are H.
  • the compound of Formula (I) is a compound of Formula (I-1) ,
  • one of M and G is O, and the other is CHR 2 .
  • the compound of Formula (I) is a compound of Formula (I-1a) , (I-1b) , or (I-1c) ,
  • the compound of Formula (I) is a compound of Formula (Ia) , (Ib) , (Ic) or (Id) ,
  • the compound of Formula (I) is a compound of Formula (Ia’ ) , (Ib’ ) , (Ic’ ) or (Id’ ) ,
  • R 5 is H, optionally substituted C1-C4 alkyl, or optionally substituted C3-C10 cycloalkyl. In some embodiments, R 5 is H, optionally substituted C1-C3 alkyl, or optionally substituted C3-C6 cycloalkyl. In some embodiments, R 5 is H or C1-C4 alkyl.
  • each of R 6 , R 7 , R 8 and R 9 is independently optionally substituted C3-C10 cycloalkyl, or optionally substituted C3-C10 heterocycloalkyl. In some embodiments, each of R 6 , R 7 , R 8 and R 9 is independently optionally substituted C5-C8 cycloalkyl, or optionally substituted C5-C8 heterocycloalkyl. In some embodiments, the ring in R 6 , R 7 , R 8 or R 9 is independently unsubstituted or substituted with one or more R 11 substituents. In some embodiments, R 11 is alkyl, cycloalkyl, alkoxy, or acyloxy, preferably C1-C4 alkyl, or C3-C6 cycloalkyl.
  • R 5 , R 6 and the N atom to which they are attached may be taken together to form a 3-8-membered ring, preferably 4-6-membered ring; wherein the ring is unsubstituted or substituted with, for example, C1-C4 alkyl.
  • X is NR 5 R 6 or OR 7 .
  • each of R 6 , R 7 , R 8 and R 9 is independently selected from wherein the ring in R 6 , R 7 , R 8 or R 9 is unsubstituted or substituted with one or more R 11 substituents.
  • R 11 is alkyl, cycloalkyl, alkoxy, or acyloxy, preferably C1-C4 alkyl, or C3-C6 cycloalkyl.
  • each of R 6 , R 7 , R 8 and R 9 is independently selected from
  • NR 5 R 6 is selected from wherein the ring in NR 5 R 6 is unsubstituted or substituted with one or more R 12 substituents.
  • R 12 is C1-C4 alkyl, C3-C6 cycloalkyl, or NR 13 2 , wherein R 13 is H or C1-C4 alkyl.
  • NR 5 R 6 is selected from
  • R 5 is methyl
  • R 6 is wherein the ring in R 6 is unsubstituted or substituted with one or more R 11 substituents, and R 11 is C1-C4 alkyl, preferably methyl or ethyl, more preferably methyl.
  • R 6 is
  • Y is optionally substituted heteroaryl or heterocyclic group.
  • Y is optionally substituted nitrogen-containing heteroaryl or heterocyclic group. In some embodiments, Y is optionally substituted 5-10-menbered nitrogen-containing heteroaryl or heterocyclic group, preferably optionally substituted 5-6-menbered nitrogen-containing heteroaryl or heterocyclic group.
  • Y is selected from the group consisting of: wherein the heteroaryl or heterocyclic group of Y is unsubstituted or substituted with one or more R 14 substituents, and R 14 is halogen, cyano, amide , OH, C1-C4 alkyl, C1-C4 hydroxylalkyl , or C1-C4 alkoxy
  • Y is selected from the group consisting of:
  • the compound of Formula (I) is a compound of Formula (I-2) or (I-3) ,
  • the compound of Formula (I) is a compound of Formula (Ie) , (If) , (Ig) , or (Ih) :
  • the compound is selected from the compounds as shown in Table 1 and Table 2.
  • the invention provides a method of preparing a compound of formula (I) .
  • the compound of formula (I) can by synthesized by substituting the bromine group of the intermediate compound (A1) or (A3) having a tricyclic core structure with compound HX, and by substituting the chlorine group of the intermediate compound (A2) with compounds Y-B (OH) 2 orHY.
  • the intermediate compound having a tricyclic core structure can be synthesized according to the following reactions:
  • the intermediate compound having a tricyclic core structure can be synthesized according to the following reaction:
  • compounds 100, 101, 102, 105, 106, 109, 110, 111, 112, 121, 122, 123, 125, 126, 133, 135, 136, 137, 144, 169, 183, 184 can be synthesized according to the following reaction scheme:
  • compounds 182, 188, 190, 197 can be synthesized according to the following reaction scheme:
  • compounds 127, 128, 145, 146, 147, 172, 186, 189, 191 can be synthesized according to the following reaction scheme:
  • compounds 116, 140, 141, 142, 143, 155, 187, 198, 200 can be synthesized according to the following reaction scheme:
  • compounds 108 can be synthesized according to the following reaction scheme:
  • compound 129 can be synthesized according to the following reaction scheme:
  • compound 130 can be synthesized according to the following reaction scheme:
  • compounds 131, 132 can be synthesized according to the following reaction scheme:
  • compounds 301, 302, 303, 306, 307, 310, 314, 315, 317, 323, 339, 344 can be synthesized according to the following reaction scheme:
  • compounds 330, 338, 341, 342, 345, 353, 354 can be synthesized according to the following reaction scheme:
  • compounds 318, 320, 321, 335, 340, 346, 347, 349, 350, 356, 357, 359, 360, 361, 364, can be synthesized according to the following reaction scheme:
  • compounds 309, 311, 313, 343 can be synthesized according to the following reaction scheme:
  • compound 324 can be synthesized according to the following reaction scheme:
  • compounds 325, 355 can be synthesized according to the following reaction scheme:
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition described herein comprises a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and a pharmaceutically acceptable excipient.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is provided in an effective amount in the pharmaceutical composition.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • the pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
  • compositions described herein can be prepared by any method known in the art of pharmacology.
  • Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • such preparatory methods include the steps of bringing the compound of Formula (I) (the “active ingredient” ) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single-or multi-dose unit.
  • compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1%and 100% (w/w) active ingredient.
  • pharmaceutically acceptable excipient refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions described herein are any of those that are well known in the art of pharmaceutical formulation and include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils.
  • compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial
  • compositions of the present invention may be administered orally, parenterally (including subcutaneous, intramuscular, intravenous and intradermal) , by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenterally including subcutaneous, intramuscular, intravenous and intradermal
  • inhalation spray topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • provided compounds or compositions are administrable intravenously and/or orally.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intraperitoneal intralesional and intracranial injection or infusion techniques.
  • the compositions are administered orally, subcutaneously, intraperitoneally, or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1, 3 -butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1, 3 -butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • Pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • provided pharmaceutically acceptable compositions may be formulated as micronized suspensions or in an ointment such as petrolatum.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
  • compositions of the present invention are typically formulated in dosage unit form, e.g., single unit dosage form, for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound (s) , mode of administration, and the like.
  • the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) .
  • an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.
  • the compounds of Formula (I) may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a compound or composition, as described herein can be administered in combination with one or more additional pharmaceutical agents.
  • the compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • additional pharmaceutical agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
  • Exemplary additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressant agents, and a pain-relieving agent.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the U.S.
  • CFR Code of Federal Regulations
  • kits e.g., pharmaceutical packs
  • inventive kits may be useful for preventing and/or treating a proliferative disease or a non-proliferative disease, e.g., as described herein.
  • the kits provided may comprise an inventive pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container) .
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of an inventive pharmaceutical composition or compound.
  • the inventive pharmaceutical composition or compound provided in the container and the second container are combined to form one-unit dosage form.
  • kits including a first container comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof.
  • the kit of the disclosure includes a first container comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof.
  • the kits are useful in preventing and/or treating a disease, disorder, or condition described herein in a subject (e.g., a proliferative disease or a non-proliferative disease) .
  • kits further include instructions for administering the compound, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof, to a subject to prevent and/or treat a proliferative disease or a non-proliferative disease.
  • Described herein are compounds useful for modulating splicing.
  • a compound described herein may be used to alter the amount, structure, or composition of a nucleic acid (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) by increasing or decreasing splicing at a splice site.
  • increasing or decreasing splicing results in modulating the level or structure of a gene product (e.g., an RNA or protein) produced.
  • a compound described herein may modulate a component of the splicing machinery, e.g., by modulating the interaction with a component of the splicing machinery with another entity (e.g., nucleic acid, protein, or a combination thereof) .
  • the splicing machinery as referred to herein comprises one or more spliceosome components.
  • Spliceosome components may comprise, for example, one or more of major spliceosome members (U1, U2, U4, U5, U6 snRNPs) , or minor spliceosome members (U11, U12, U4atac, U6atac snRNPs) and their accessory splicing factors.
  • the present disclosure features a method of modifying of a target (e.g., a precursor RNA, e.g., a pre-mRNA) through inclusion of a splice site in the target, wherein the method comprises providing a compound described herein.
  • a target e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA
  • inclusion of a splice site in a target results in addition or deletion of one or more nucleic acids to the target (e.g., a new exon, e.g. a skipped exon) .
  • Addition or deletion of one or more nucleic acids to the target may result in an increase in the levels of a gene product (e.g., RNA, e.g., mRNA, or protein) .
  • the present disclosure features a method of modifying a target (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) through exclusion of a splice site in the target, wherein the method comprises providing a compound described herein.
  • exclusion of a splice site in a target e.g., a precursor RNA, e.g., a pre-mRNA
  • results in deletion or addition of one or more nucleic acids from the target e.g., a skipped exon, e.g. a new exon
  • RNA e.g., mRNA, or protein
  • the methods of modifying a target comprise suppression of splicing at a splice site or enhancement of splicing at a splice site (e.g., by more than about 0.5%, e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) , e.g., as compared to a reference (e.g., the absence of a compound of Formula (I) , or in a healthy or diseased cell or tissue) .
  • a reference e.g., the absence of a compound of Formula (I) , or in a healthy or diseased cell or tissue
  • the present disclosure provides a method of forming a complex comprising a component of a spliceosome (e.g., a major spliceosome component or a minor spliceosome component) , a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) , and a compound described herein, or the pharmaceutical composition described herein, comprising contacting the nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) with the compound or the pharmaceutical composition.
  • a spliceosome e.g., a major spliceosome component or a minor spliceosome component
  • a nucleic acid e.g., a DNA, RNA, e.g., a pre-mRNA
  • a compound described herein e.g., a pre-mRNA
  • the present disclosure features a method of altering the conformation of a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) comprising contacting the nucleic acid with a compound described herein, or the pharmaceutical composition described herein.
  • the altering comprises forming a bulge or kink in the nucleic acid.
  • the altering comprises stabilizing a bulge or a kink in the nucleic acid.
  • the altering comprises reducing a bulge or a kink in the nucleic acid.
  • the nucleic acid comprises a splice site.
  • the compound of Formula (I) interacts with a nucleobase, ribose, or phosphate moiety of a nucleic acid (e.g., a DNA, RNA, e.g., pre-mRNA) .
  • a nucleic acid e.g., a DNA, RNA, e.g., pre-mRNA
  • Extensive posttranscriptional processing occurs before eukaryotic pre-mRNA matures and exits from the nucleus to the cytoplasm, including the addition of a 7-methylguanosine cap at the 5’ end, the cleavage and addition of a poly-Atail at the 3’ end as well as the removal of intervening sequences or introns by the spliceosome.
  • the vast majority of higher eukaryotic genes contain multiple introns that are spliced out with high precision and fidelity in order to maintain the reading frame of the exons.
  • Splicing of pre-mRNA can utilize the recognition of short consensus sequences at the boundaries and within introns and exons by an array of small nuclear ribonucleoprotein (snRNP) complexes (e.g., snRNPs U1, U2, U4, U5, U6, U11, U12m U4atc and U6 ate) and a large number of proteins, including spliceosomal proteins and positively as well as negatively acting splicing modulators.
  • snRNP small nuclear ribonucleoprotein
  • Serine-arginine-rich (SR) -domain-containing proteins generally serve to promote constitutive splicing. They can also modulate alternative splicing by binding to intronic or exonic splicing enhancer (ISE) or ESE, respectively) sequences. Other pre-mRNA binding proteins, such as hnRNPs, regulate splicing by binding to intronic or exonic splicing suppressor (ISS or ESS, respectively) sequences and can also act as general splicing modulators.
  • the SR protein family is a class of at least 10 proteins that have a characteristic serine/arginine rich domain in addition to an RNA-binding.
  • SR proteins are generally thought to enhance splicing by simultaneously binding to U170K, a core component of the U1 snRNP, at the 5’splice site, and the U2AF35 at the 3’splice site, thus bridging the two ends of the intron. While this particular function of SR proteins seems to be redundant, as any individual SR protein can commit a pre-mRNA for constitutive splicing, the role of the various SR proteins in alternative splicing of specific pre-mRNAs is distinct due in part to their ability to recognize and bind to unique consensus sequences. Phosphorylation of the RS domain of SR proteins can lead to the regulation of their protein interactions, RNA binding, localization, trafficking, and role in alternative splicing.
  • SRPKs SR protein Kinase
  • Clks Cdc2-like kinases
  • PRP4 pre-mRNA processing mutant 4
  • topoisomerase I SR protein Kinase
  • Optimal phosphorylation of SR proteins may be required for proper functioning as both hypo-and hyperphosphorylation of the RS domains may be detrimental to their role in constitutive and alternative splicing.
  • pre-mRNA splicing is the mechanism by which introns are removed from a pre-mRNA and the exons are ligated together to generate mature mRNAs and pre-miRNA that is then exported to the cytoplasm for translation into the polypeptide gene product.
  • Splicing of pre-mRNA can occur in cis, where two exons derive from two adjacent cotranscribed sequences, or in trans, when the two exons come from different pre-mRNA transcripts.
  • the ratio of the different protein products may be due to the frequency of alternative splicing events within a pre-mRNA that leads to different amounts of distinct splice variants.
  • alternative splicing of a pre-mRNA may lead to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 protein isoforms being expressed.
  • Aberrations in splicing are thought to be the cause of roughly half of all inherited diseases. Aberrant splicing due to mutations in consensus sequences involved in exon-intron boundary recognition is responsible for up to 15%of inherited diseases. In addition, defects in the splicing machinery itself due to the loss or gain of function of splicing factors and modulators are causes of a wide range of human ailments from cancer to neurodegenerative diseases. Both constitutive and alternative splicing are subject to regulation by upstream signaling pathways. This regulation can be essential during development, in tissue specific expression of certain isoforms, during the cell cycle and in response to extrinsic signaling molecules.
  • Alternative splicing allows for a single gene to express different isoforms of mRNA, thus playing a major role in contributing to the cellular complexity in higher eukaryotes without the need to expand the genome.
  • Splicing can also be subject to regulation by upstream signaling pathways.
  • an upstream signaling pathway may modulate alternative splicing and increase or decrease expression levels of different isoforms of mRNA.
  • Alternative splicing events are highly regulated by numerous splicing factors in a tissue type-, developmental stage-, and signal-dependent manner.
  • non-mutation based causes of splicing defects and defects in the splicing machinery itself, e.g., due to the loss/gain of function of splicing factors or their relative stoichiometry, cause of a wide range of human ailments, ranging from cancer to neurodegenerative diseases.
  • the disease state is caused by an alteration of the ratio of different isoforms of two or more proteins expressed from a gene.
  • the alteration in the ratio of the protein products is due to changes in the frequency of alternative splicing events within a pre-mRNA, leading to changes in the ratio of splice variants produced.
  • alternative splicing of a pre-mRNA may lead to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 protein isoforms being expressed.
  • a change in the splice variant ratio is caused by genetic mutation.
  • RNA-protein complex that occurs in unique steps and may comprise a subset of several hundred different proteins, in addition to five spliceosomal snRNAs. These factors are responsible for the accurate positioning of the spliceosome on the 5’ and 3’splice site sequences.
  • exon recognition can be affected by many pre-mRNA features such as exon length, sequence recognition, the presence of enhancer and silencer elements, the strength of upstream splicing signals, the promoter architecture, and the rate of RNA processivity, secondary and tertiary RNA structure.
  • mRNA messenger mRNA
  • mRNA messenger mRNA
  • RNA is only a small portion of the transcriptome: other transcribed RNAs also regulate cellular biology either directly by the structure and function of RNA structures (e.g., ribonucleoproteins) as well as via protein expression and action, including (but not limited to) microRNA (miRNA) , long noncoding RNA (lncRNA) , long intergenic noncoding RNA (lincRNA) , small nucleolar RNA (snoRNA) , small nuclear RNA (snRNA) , small Cajal body-specific RNA (scaRNA) , piwi-interacting RNA (piRNA) , competing endogenous (ceRNA) , and pseudo-genes.
  • miRNA microRNA
  • lncRNA long noncoding RNA
  • lincRNA long intergenic noncoding RNA
  • small nucleolar RNA small nucleolar RNA
  • snRNA small nuclear RNA
  • scaRNA small Cajal body-specific RNA
  • piRNA pi
  • Drugs that intervene at this level have the potential of modulating any and all cellular processes.
  • Existing therapeutic modalities such as antisense RNA or siRNA, in most cases, have yet to overcome significant challenges such as drug delivery, absorption, distribution to target organs, pharmacokinetics, and cell penetration.
  • small molecules have a long history of successfully surmounting these barriers and these qualities, which make them suitable as drugs, are readily optimized through a series of analogues to overcome such challenges.
  • the application of small molecules as ligands for RNA that yield therapeutic benefit has received little to no attention from the drug discovery community.
  • DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns) .
  • Introns are removed from pre-mRNAs through splicing.
  • Pre-mRNA splicing proceeds by a two-step mechanism. In the first step, the 5’splice site is cleaved, resulting in a “free” 5’ exon and a lariat intermediate. In the second step, the 5’ exon is ligated to the 3’ exon with release of the intron as the lariat product. These steps are catalyzed in a complex of small nuclear ribonucleoproteins and proteins called the spliceosome.
  • trans-splicing In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing.
  • Introns are portions of eukaryotic DNA, which intervene between the coding portions, or “exons” , of that DNA. Introns and exons are transcribed into RNA termed “primary transcript, precursor to mRNA” (or “pre-mRNA” ) . Introns can be removed from the pre-mRNA so that the native protein encoded by the exons can be produced (the term “native protein” as used herein refers to naturally occurring, wild type, or functional protein) . The removal of introns from pre-mRNA and subsequent joining of the exons is carried out in the splicing process.
  • RNA that contains both exons and intron (s)
  • mRNA mature mRNA
  • mRNA RNA in which the intron (s) have been removed and the exons joined together sequentially so that the protein may be translated therefrom by the ribosomes.
  • Introns can be defined by a set of “splice elements” that are part of the splicing machinery and may be required for splicing and which are relatively short, conserved RNA segments that bind the various splicing factors, which carry out the splicing reactions.
  • each intron is defined by a 5’splice site, a 3’splice site, and a branch point situated there between.
  • Splice elements also comprise exon splicing enhancers and silencers, situated in exons, and intron splicing enhancers and silencers situated in introns at a distance from the splice sites and branch points. In addition to splice site and branch points these elements control alternative aberrant and constitutive splicing.
  • RNA transcripts pre-mRNA of most eukaryotic genes are retained in the nucleus until non-coding intron sequences are removed by the spliceosome to produce mature messenger RNA (mRNA) .
  • the splicing that occurs can vary, so the synthesis of alternative protein products from the same primary transcript can be affected by tissue-specific or developmental signals.
  • a significant fraction of human genetic diseases, including a number of cancers, are believed to result from deviations in the normal pattern of pre-mRNA splicing.
  • the spliceosome is a complex comprising ribonucleoprotein (snRNP) particles composed of small nuclear RNAs and proteins. snRNA components of the spliceosome can promote the two transesterification reactions of splicing.
  • snRNP ribonucleoprotein
  • U2-dependent spliceosome which catalyzes the removal of U2-type introns
  • U12-dependent spliceosome which is present in only a subset of eukaryotes and splices the rare U12-type class of introns.
  • the U2-dependent spliceosome is assembled from the U1, U2, U5, and U4/U6 snRNPs and numerous non-snRNP proteins.
  • the U2 snRNP is recruited with two weakly bound protein subunits, SF3a and SF3b, during the first ATP-dependent step in spliceosome assembly.
  • SF3b is composed of seven conserved proteins, including PHF5 ⁇ , SF3b155, SF3b145, SF3b130, SF3b49, SF3b14a, and SF3b10.
  • RNA splicing typically refers to the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA) .
  • Splicing is a biochemical process which includes the removal of introns followed by exon ligation. Sequential transesterification reactions are initiated by a nucleophilic attack of the 5’splice site (5’ss) by the branch adenosine (branch point; BP) in the downstream intron resulting in the formation of an intron lariat intermediate with a 2’ , 5’ -phosphodiester linkage. This is followed by a 5’ss-mediated attack on the 3’splice site (3’ss) , leading to the removal of the intron lariat and the formation of the spliced RNA product.
  • 5’ss branch adenosine
  • Cis-acting elements are sequences of the mRNA and can include core consensus sequences and other regulatory elements. Core consensus sequences typically can refer to conserved RNA sequence motifs, including the 5’ss, 3’ss, polypyrimidine tract and BP region, which can function for spliceosome recruitment.
  • BP refers to a partially conserved sequence of pre- mRNA, generally less than 50 nucleotides upstream of the 3’ss. BP reacts with the 5’ss during the first step of the splicing reaction.
  • ESE exonic splicing enhancer
  • ESS exonic splicing silencer
  • ISE intronic splicing enhancer
  • ISS intronic splicing silencer
  • Trans-acting factors can be proteins or ribonucleoproteins which bind to cis-acting elements.
  • Splice site identification and regulated splicing can be accomplished principally by two dynamic macromolecular machines, the major (U2-dependent) and minor (U12-dependent) spliceosomes.
  • Each spliceosome contains five snRNPs: U1, U2, U4, U5 and U6 snRNPs for the major spliceosome (which processes ⁇ 95.5%of all introns) ; and U11, U12, U4atac, U5 and U6atac snRNPs for the minor spliceosome.
  • Spliceosome recognition of consensus sequence elements at the 5’ss, 3’s s and BP sites is one of the steps in the splicing pathway, and can be modulated by ESEs, ISEs, ESSs, and ISSs, which can be recognized by auxiliary splicing factors, including SR proteins and hnRNPs.
  • Polypyrimidine tract-binding protein (PTBP) can bind to the polypyrimidine tract of introns and may promote RNA looping.
  • Alternative splicing is a mechanism by which a single gene may eventually give rise to several different proteins.
  • Alternative splicing can be accomplished by the concerted action of a variety of different proteins, termed “alternative splicing regulatory proteins, ” that associate with the pre-mRNA, and cause distinct alternative exons to be included in the mature mRNA. These alternative forms of the gene's transcript can give rise to distinct isoforms of the specified protein.
  • Sequences in pre-mRNA molecules that can bind to alternative splicing regulatory proteins can be found in introns or exons, including, but not limited to, ISS, ISE, ESS, ESE, and polypyrimidine tract. Many mutations can alter splicing patterns.
  • mutations can be cis-acting elements, and can be located in core consensus sequences (e.g. 5’ss, 3’ss and BP) or the regulatory elements that modulate spliceosome recruitment, including ESE, ESS, ISE, and ISS.
  • core consensus sequences e.g. 5’ss, 3’ss and BP
  • regulatory elements that modulate spliceosome recruitment including ESE, ESS, ISE, and ISS.
  • a cryptic splice site for example, a cryptic 5’ss and a cryptic 3’ss, can refer to a splice site that is not normally recognized by the spliceosome and therefore are in the dormant state.
  • Cryptic splice site can be recognized or activated, for example, by mutations in cis-acting elements or trans-acting factors, or structural configurations, such as bulges.
  • the present invention contemplates use of small molecules with favorable drug properties that modulate the activity of splicing of a target RNA.
  • the compound bind and modulate target RNA.
  • the target RNA is mRNA.
  • the target RNA is a noncoding RNA.
  • the target RNA is a pre-mRNA.
  • the target RNA is hnRNA.
  • the small molecules modulate splicing of the target RNA.
  • a small molecule provided herein modulates splicing at a sequence of the target RNA. In some embodiments, a small molecule provided herein modulates splicing at a cryptic splice site sequence of the target RNA. In some embodiments, a small molecule provided herein binds to a target RNA. In some embodiments, a small molecule provided herein binds to a splicing complex component. In some embodiments, a small molecule provided herein binds to a target RNA and a splicing complex component.
  • splicing event in a pre-mRNA molecule
  • methods of preventing or inducing a splicing event in a pre-mRNA molecule comprising contacting the pre-mRNA molecule and/or other elements of the splicing machinery (e.g., within a cell) with a compound provided herein to prevent or induce the splicing event in the pre-mRNA molecule.
  • the splicing event that is prevented or induced can be, e.g., an aberrant splicing event, a constitutive splicing event or an alternate splicing event.
  • a method of identifying a compound capable of preventing or inducing a splicing event in a pre-mRNA molecule comprising contacting the compound with splicing elements and/or factors involved in alternative, aberrant and/or constitutive splicing as described herein (e.g., within cells) under conditions whereby a positive (prevention or induction of splicing) or negative (no prevention or induction of splicing) effect is produced and detected and identifying a compound that produces a positive effect as a compound capable of preventing or inducing a splicing event.
  • a small molecule compound described herein in a pharmaceutically acceptable carrier prevents or induces an alternative or aberrant splicing event in a pre-mRNA molecule.
  • the small molecule compounds provided herein are not antisense or antigene oligonucleotides.
  • a method of upregulating expression of a native protein in a cell containing a DNA encoding the native protein wherein the DNA contains a mutation or no mutation that causes downregulation of the native protein by aberrant and/or alternate splicing thereof.
  • the DNA can encode a pre-mRNA that has a mutation or an aberrant secondary or tertiary structure that causes downregulation of one or more isoforms of a protein.
  • the method can comprise introducing into the cell a small molecule provided herein that prevents an aberrant splicing event, whereby the native intron is removed by correct splicing and the native protein is produced by the cell.
  • a method comprises introducing into a cell a small molecule provided herein that modulates an alternate splicing event to produce a protein that has a different function than the protein that would be produced without modulation of alternate splicing.
  • a method of downregulating expression of a native protein in a cell containing a DNA encoding the native protein wherein the DNA contains a mutation or no mutation that causes upregulation of the native protein by aberrant and/or alternate splicing thereof.
  • the DNA can encode a pre-mRNA that has a mutation or an aberrant secondary or tertiary structure that causes upregulation of one or more isoforms of a protein.
  • the method can comprise introducing into the cell a small molecule provided herein that prevents an aberrant splicing event, whereby the native intron is removed by correct splicing and the native protein is produced by the cell.
  • a method comprises introducing into a cell a small molecule provided herein that modulates an alternate splicing event to produce a protein that has a different function than the protein that would be produced without modulation of alternate splicing.
  • a method can comprise preventing aberrant splicing in a pre-mRNA molecule containing a mutation or an aberrant secondary or tertiary structure and/or preventing an alternative splicing event.
  • the mutation or aberrant secondary or tertiary structure can cause a pre-mRNA to splice incorrectly and produce an aberrant mRNA or mRNA fragment different from the mRNA ordinarily resulting from a pre-mRNA without the mutation or aberrant secondary or tertiary structure.
  • s pre-mRNA molecule can contain: (i) a first set of splice elements defining a native intron which can be removed by splicing when the mutation or aberrant secondary or tertiary structure is absent to produce a first mRNA molecule encoding a native protein, and (ii) a second set of splice elements induced by the mutation or aberrant secondary or tertiary structure which defines an aberrant intron different from the native intron, which aberrant intron is removed by splicing when the mutation or aberrant secondary or tertiary structure is present to produce an aberrant second mRNA molecule different from the first mRNA molecule.
  • the method can comprise contacting the pre-mRNA molecule and/or other factors and/or elements of the splicing machinery as described herein (e.g., within a cell) with a compound described herein to prevent or promote an aberrant splicing event in a pre-mRNA molecule, whereby the native intron is removed by correct splicing and native protein production is increased in the cell.
  • Also provided herein is a method of upregulating expression of an RNA that would otherwise be downregulated by modulating an alternative splicing event in the RNA.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound described herein to modulate alternate splicing events, whereby a native splicing event is inhibited and an alternate splicing event is promoted that upregulates expression of a RNA that is otherwise downregulated when under the control of the native splicing event.
  • Also provided herein is a method of downregulating expression of an RNA that would otherwise be upregulated by modulating an alternative splicing event in the RNA.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound described herein to modulate alternate splicing events, whereby a native splicing event is inhibited and an alternate splicing event is promoted that downregulates expression of a RNA that is otherwise upregulated when under the control of the native splicing event.
  • RNA to be expressed may be any RNA encoding a protein to be produced so long as the gene contains a native intron.
  • the RNA may be mutated by any suitable means, such as site-specific mutagenesis to deliberately create an aberrant second set of splice elements which define an aberrant intron which substantially downregulates expression of the gene.
  • a sequence encoding the RNA may be inserted into a suitable expression vector and the expression vector inserted into a host cell (e.g., a eukaryotic cell such as a yeast, insect, or mammalian cell (e.g., human, rat) ) by standard recombinant techniques.
  • a host cell e.g., a eukaryotic cell such as a yeast, insect, or mammalian cell (e.g., human, rat)
  • the host cell can then be grown in culture by standard techniques.
  • a suitable compound of the present invention in a suitable formulation, can be added to the culture medium so that expression of the gene is upregulated.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound or compounds described herein to modulate alternative splicing events.
  • the compound or compounds of this invention can be used to act upon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 alternative splicing events that may occur within a pre-mRNA.
  • a first splice variant may be downregulated or inhibited and/or a second splice variant may be upregulated, resulting in an altered ratio of splice variants of the two or more RNA.
  • a first splice variant may be upregulated while a second splice variant may be unaffected, thereby altering the ratio of the RNA. In some embodiments, a first splice variant may be downregulated while a second splicing event may be unaffected thereby altering the ratio of the RNA.
  • RNAs encoded by genes e.g., those developmentally and/or tissue regulated (e.g., alternate splicing events) .
  • a method of treating a subject having a condition or disorder associated with an alternative or aberrant splicing event in a pre-mRNA molecule comprises administering to the subject a therapeutically effective amount of a compound described herein to modulate an alternative splicing event or prevent an aberrant splicing event, thereby treating the subject.
  • the method can, e.g., restore a correct splicing event in a pre-mRNA molecule.
  • the method can, e.g., utilize a small molecule compound described herein in a pharmaceutically acceptable carrier.
  • Formulations containing the small molecules described herein can comprise a physiologically or pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a physiologically or pharmaceutically acceptable carrier such as an aqueous carrier.
  • formulations for use in the methods described herein include, but are not limited to, those suitable for oral administration, parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous and intra-arterial administration, as well as topical administration (e.g., administration of an aerosolized formulation of respirable particles to the lungs of a patient afflicted with cystic fibrosis or lung cancer or a cream or lotion formulation for transdermal administration of patients with psoriasis) .
  • topical administration e.g., administration of an aerosolized formulation of respirable particles to the lungs of a patient afflicted with cystic fibrosis or lung cancer or a cream or lotion formulation for transdermal administration of patients with psoriasis
  • the formulations may conveniently be presented in unit dosage form
  • the medicament upregulates gene expression.
  • the medicament downregulates gene expression.
  • the compound in the manufacture of at medicament according to the invention, can be admixed with, inter alia, a pharmaceutically acceptable carrier.
  • the carrier may be a solid or a liquid.
  • One or more compounds may be incorporated in any combination in the formulations described herein, which may be prepared by any of the well-known techniques of pharmacy, such as admixing the components, and/or including one or more accessory therapeutic ingredients.
  • the present inventors identify herein low molecular weight compounds (sometimes referred to herein as small molecules) , which block mRNA splicing and/or enhance (facilitate, augment) mRNA splicing.
  • the splicing that can be regulated by the methods described herein include alternative splicing, e.g., exon skipping, intron retention, pseudoexons skipping, exon exclusion, partial intron exclusion and others.
  • modulation of splicing can be accomplished in the presence of, or in the absence of, antisense oligonucleotides (AOs) that are specific for splicing sequences of interest.
  • AOs antisense oligonucleotides
  • a small molecule and an AO act synergistically.
  • a method comprises contacting a splice modulating compound (e.g., the compound described herein) to a pre-mRNA that modulates splicing of the pre-mRNA to favor expression of a transcript that promotes cell proliferation.
  • a splice modulating compound e.g., the compound described herein
  • compound or composition described herein can increase one or more isoforms of a transcript that promotes cell proliferation.
  • compound or composition described herein can decrease expression one or more isoforms of a transcript that prevents or inhibits cell proliferation.
  • a method comprises contacting a splice modulating compound (e.g., the compound described herein) to a pre-mRNA that modulates splicing of the pre-mRNA to favor expression of a transcript that prevents or inhibits cell proliferation.
  • a splice modulating compound e.g., the compound described herein
  • compound or composition described herein can increase one or more isoforms of a transcript that prevents or inhibits cell proliferation.
  • compound or composition described herein can decrease expression one or more isoforms of a transcript that promotes cell proliferation.
  • a method of modulating splicing of pre-mRNA comprises using the compound described herein to decrease expression or functionality of one or more isoforms of a transcript in a subject.
  • the method can comprise administering the compound described herein, or a composition comprising the compound described herein, to a subject, wherein the compound described herein binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to favor expression of one or more isoforms of a transcript.
  • the method can comprise administering the compound described herein, or a composition comprising the compound described herein, to a subject, wherein the compound described herein binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to disfavor expression of one or more isoforms of a transcript.
  • the present invention provides a method of treating a subject afflicted with a disease or condition associated with aberrant splicing of a pre-mRNA.
  • the method can comprise administering the compound described herein, or a composition comprising the compound described herein, to a subject, wherein the compound described herein binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to inhibit expression of one or more isoforms of a transcript.
  • the method can comprise administering the compound described herein, or a composition comprising the compound described herein, to a subject, wherein the compound described herein binds to a pre-mRNA or a splicing complex component and modulates the splicing of the pre-mRNA to increase expression of one or more isoforms of a transcript.
  • a number of diseases are associated with expression of an aberrant gene product (e.g., an RNA transcript or protein) of a gene.
  • aberrant amounts of an RNA transcript may lead to disease due to corresponding changes in protein expression.
  • Changes in the amount of a particular RNA transcript may be the result of several factors. First, changes in the amount of RNA transcripts may be due to an aberrant level of transcription of a particular gene, such as by the perturbation of a transcription factor or a portion of the transcription process, resulting in a change in the expression level of a particular RNA transcript.
  • changes in the splicing of particular RNA transcripts can change the levels of a particular RNA transcript.
  • Changes to the stability of a particular RNA transcript or to components that maintain RNA transcript stability such as the process of poly-Atail incorporation or an effect on certain factors or proteins that bind to and stabilize RNA transcripts, may lead to changes in the levels of a particular RNA transcript.
  • the level of translation of particular RNA transcripts can also affect the amount of those transcripts, affecting or upregulating RNA transcript decay processes.
  • aberrant RNA transport or RNA sequestration may also lead to changes in functional levels of RNA transcripts, and may have an effect on the stability, further processing, or translation of the RNA transcripts.
  • RNA transcripts encoded by a pre-mRNA comprising contacting a cell with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
  • the cell is contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a cell culture.
  • the cell is contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a subject (e.g., a non-human animal subject or a human subject) .
  • provided herein are methods for treatment, prevention and/or delay of progression of a disease or condition comprising administering an effective amount of a small molecule splicing modulator as described herein to a subject, in particular to a mammal.
  • compositions and methods for treating a disease or condition including steric modulator compounds or pharmaceutically acceptable salts thereof that promote prevention or correction of exon skipping of a pre-mRNA.
  • the invention further provides compositions and methods for increasing production of mature mRNA and, in turn, protein, in cells of a subject in need thereof, for example, a subject that can benefit from increased production of protein.
  • the invention further provides compositions and methods for decreasing production of mature mRNA and, in turn, protein, in cells of a subject in need thereof, for example, a subject that can benefit from decreased production of protein.
  • the described methods may be used to treat subjects having a disease or condition caused by a mutation in a gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, which result in deficient protein production.
  • the described methods may be used to treat subjects having a disease or condition not caused by gene mutation.
  • the compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from increased production of protein.
  • the compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from increased production of protein.
  • the compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from decreased production of a protein.
  • provided herein are methods of treating a disease or condition in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a mutation that causes, e.g., exon skipping or intron inclusion, or a portion thereof, of pre-mRNA, wherein the pre-mRNA encodes the target protein or functional RNA.
  • the method can comprise contacting cells of a subject with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof that targets the pre-mRNA encoding the target protein or functional RNA or splicing complex component, whereby splicing of an exon from a pre-mRNA encoding a target protein or functional RNA is prevented or inhibited, thereby increasing a level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject.
  • a method of increasing expression of a target protein by cells having a mutation or aberrant secondary or tertiary RNA structure that causes exon skipping of pre-mRNA the pre-mRNA comprising a mutation or aberrant secondary or tertiary RNA structure that causes exon skipping.
  • the method can comprise contacting the cells with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof that targets a pre-mRNA encoding a target protein or functional RNA, whereby splicing of an exon from a pre-mRNA encoding a target protein or functional RNA is prevented or inhibited, thereby increasing the level of mRNA encoding functional protein, and increasing the expression of protein in the cells.
  • the target protein is a tumor suppressor.
  • the target protein is a tumor promoter.
  • the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.
  • the cells are in or from a subject having a condition caused by a deficient amount or activity of the protein.
  • the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof binds to a targeted portion of a pre-mRNA transcribed from the first allele.
  • the target protein is produced in a form that is fully-functional compared to the equivalent protein produced from mRNA in which an exon has been skipped or is missing.
  • the pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to a pre-mRNA.
  • a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof increases the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
  • a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof increases the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, or about 200%to about 250%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • the total amount of target protein produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the total amount of target protein produced by a control cell.
  • the total amount of target protein produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, or about 200%to about 250%, compared to the total amount of target protein produced by a control cell.
  • a total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • a total amount of an mRNA encoding the target protein or functional RNA produced in a cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, or about 4 to about 9-fold, compared to a total amount of the mRNA encoding the target protein or
  • a total amount of target protein produced by a cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.
  • the total amount of target protein produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8- fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, or about 4 to about 9-fold, compared to a total amount of target protein produced by a control cell.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 10%to about 100%, about 20%to about 100%, about 30%to about 100%, about 40%to about 100%, about 50%to about 100%, about 60%to about 100%, about 70%to about 100%, about 80%to about 100%about 90%to about 100%, about 20%to about 30%, about 20%to about 40%, about 20%to about 50%, about 20%to about 60%, about 20%to about 70%, about 20%to about 80%, about 20%to about 90%, about 30%to about 40%, about 30%to about 50%, about 30%to about 60%, about 30%to about 70%, about 30%to about 80%, about 30%to about 90%, about 40%to about 50%, about 40%to about 60%, about 40%to about 70%, about 40%to about 80%, about 40%to about 90%, about 50%to about 60%, about 50%to about 70%, about 50%to about 80%, about 50%to about 90%, about 60%to about
  • the total amount of target protein produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to the total amount of target protein produced by a control cell.
  • the total amount of target protein produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 10%to about 100%, about 20%to about 100%, about 30%to about 100%, about 40%to about 100%, about 50%to about 100%, about 60%to about 100%, about 70%to about 100%, about 80%to about 100%about 90%to about 100%, about 20%to about 30%, about 20%to about 40%, about 20%to about 50%, about 20%to about 60%, about 20%to about 70%, about 20%to about 80%, about 20%to about 90%, about 30%to about 40%, about 30%to about 50%, about 30%to about 60%, about 30%to about 70%, about 30%to about 80%, about 30%to about 90%, about 40%to about 50%, about 40%to about 60%, about 40%to about 70%, about 40%to about 80%, about 40%to about 90%, about 50%to about 60%, about 50%to about 70%, about 50%to about 80%, about 50%to about 90%, about 60%to about 70%, about 60%to about 80%, about 60%to about
  • the difference in amount between a first splice variant and a second splice variant encoding a target protein or functional RNA isoform produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, about 200%to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared
  • the difference in amount between a first protein isoform expressed from a first splice variant and a second protein isoform expressed from a second splice variant produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, about 200%to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 30
  • the difference in amount between a first splice variant and a second splice variant encoding a target protein or functional RNA isoform produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 to about 10-
  • the difference in amount between a first protein isoform expressed from a first splice variant and a second protein isoform expressed from a second splice variant produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, about 4 to about
  • a difference in amount between a first splice variant and a second splice variant encoding a target protein or functional RNA isoform produced in a cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, about 200%to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%,
  • a difference in amount between a first protein isoform expressed from a first splice variant and a second protein isoform expressed from a second splice variant produced by a cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 20%to about 300%, about 50%to about 300%, about 100%to about 300%, about 150%to about 300%, about 20%to about 50%, about 20%to about 100%, about 20%to about 150%, about 20%to about 200%, about 20%to about 250%, about 50%to about 100%, about 50%to about 150%, about 50%to about 200%, about 50%to about 250%, about 100%to about 150%, about 100%to about 200%, about 100%to about 250%, about 150%to about 200%, about 150%to about 250%, about 200%to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least
  • the difference in amount between a first splice variant and a second splice variant encoding a target protein or functional RNA isoform produced in the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 to about 10-
  • the difference in amount between a first protein isoform expressed from a first splice variant and a second protein isoform expressed from a second splice variant produced by the cell contacted with a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is decreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, about 4 to about
  • the ratio of a first isoform and a second isoform may contribute to a number of conditions or diseases.
  • a subject without a condition or disease has a first isoform to second isoform ratio of 1: 1.
  • a subject with a condition or disease described herein has a first isoform to second isoform ratio of about 1: 1.2, 1: 1.4, 1: 1.6, 1: 1.8, 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5 or 1: 5.
  • a subject with a condition or disease described herein has a first isoform to second isoform ratio from about 1: 1 to about 1: 1.1, about 1:1 to about 1: 1.2, about 1: 1 to about 1: 1.3, about 1: 1 to about 1: 1.4, about 1: 1 to about 1: 1.5, about 1: 1 to about 1:1.6, about 1: 1 to about 1: 1.8, about 1: 1 to about 1: 2, about 1: 1 to about 1: 3, about 1: 1 to about 1: 3.5, about 1: 1 to about 1: 4, about 1: 1 to about 1: 4.5, about 1: 1 to about 1: 5, 1: 2 to about 1: 3, about 1: 2 to about 1: 4, about 1: 2 to about 1: 5, about 1: 3 to about 1: 4, about 1: 3 to about 1: 5, or about 1: 4 to about 1: 5.
  • binding of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof to pre-mRNA prevents splicing out of one or more exons and/or introns and/or proteins thereof, from the population of pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the cell comprises a population of pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of pre-mRNAs comprises a mutation that causes the splicing out of one or more exons, and wherein a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof binds to the mutation that causes the splicing out of the one or more exons in the population of pre-mRNAs.
  • the binding of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof to the mutation that causes the splicing out of the one or more exons prevents splicing out of the one or more exons from the population of pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the condition is a disease or disorder.
  • the method further comprises assessing protein expression.
  • a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof binds to a targeted portion of a pre-mRNA.
  • the binding of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof catalyzes the inclusion of a missing exon or removal of an undesired retained intron or portions thereof, resulting in healthy mRNA and proteins.
  • the binding of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof has minimal to no effect on non-diseased cells.
  • the compound described herein modulates splicing at a splice site sequence of a polynucleotide of the primary cells. In some embodiments, the compound described herein modulates proliferation or survival of the primary cells. In some embodiments, the primary cells are primary diseased cells. In some embodiments, the primary diseased cells are primary cancer cells. In some embodiments, the compound described herein is present at a concentration of at least about 1 nM, 10 nM, 100 nM, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M, 1 mM, 10 mM, 100 mM, or 1 M.
  • At least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%of the primary diseased cells are killed. In some embodiments, at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%of the primary diseased cells undergo apoptosis. In some embodiments, at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%of the primary diseased cells undergo necrosis.
  • proliferation is reduced or inhibited in at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%of the primary diseased cells.
  • the primary diseased cells are non-transformed cells.
  • Aberrant splicing of mRNA can result in a defective protein and can cause a disease or a disorder in a subject.
  • the compositions and methods described herein can reduce this aberrant splicing of mRNA, such as pre-mRNA, and treat a disease or a disorder caused by this aberrant splicing.
  • RNA transcripts Diseases associated with changes to RNA transcript amount are often treated with a focus on the aberrant protein expression.
  • the processes responsible for the aberrant changes in RNA levels such as components of the splicing process or associated transcription factors or associated stability factors, could be targeted by treatment with a small molecule, it would be possible to restore protein expression levels such that the unwanted effects of the expression of aberrant levels of RNA transcripts or associated proteins. Therefore, there is a need for methods of modulating the amount of RNA transcripts encoded by certain genes as a way to prevent or treat diseases associated with aberrant expression of the RNA transcripts or associated proteins.
  • Mutations and/or aberrant secondary or tertiary RNA structures in cis-acting elements can induce three-dimensional structural change in pre-mRNA. Mutations and/or aberrant secondary RNA structures in cis-acting elements can induce three-dimensional structural change in pre-mRNA when the pre-mRNA is, for example, bound to at least one snRNA, or at least one snRNP, or at least one other auxiliary splicing factor.
  • non-canonical base pairing of a non-canonical splice site sequence to a snRNA can form a bulge.
  • a bulge can be formed when the 5’ss is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be induced to form when 5’ss containing at least one mutation is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be formed when the cryptic 5’ss is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be induced to form when cryptic 5’ss containing at least one mutation is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be formed when the 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be induced to form when the 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be formed when the cryptic 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be induced to form when the cryptic 3’ss is bound to U2 snRNA or a portion thereof.
  • the protein components of U1 and U2 may or may not present to form the bulge.
  • a small molecule can bind to a bulge.
  • a bulge is naturally occurring.
  • a bulge is formed by non-canonical base-pairing between the splice site and the small nuclear RNA.
  • a bulge can be formed by non-canonical base-pairing between the 5’ss and U1-U12 snRNA.
  • the bulge can comprise 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides.
  • 3-dimensional structural changes can be induced by a mutation without bulge formation.
  • a bulge may be formed without any mutation in a splice site.
  • a recognition portion can be formed by a mutation in any of the cis-acting elements.
  • a small molecule can bind to a recognition portion that is induced by a mutation.
  • a mutation and/or aberrant secondary or tertiary RNA structure at an authentic 5’splice site can result in splicing at a cryptic 5’splice site.
  • a mutation and/or aberrant secondary or tertiary RNA structure can be in one of the regulatory elements including ESEs, ESSs, ISEs, and ISSs.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide in an exon.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide upstream (5’ ) of the splice site of the splice site sequence.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -1 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNN*nnnnn, wherein N*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NN*Nnnnnn, wherein N*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -3 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of N*NNnnnnn, wherein N*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide in an intron. In some embodiments, a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide downstream (3’ ) of the splice site of the splice site sequence.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +1 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNn*nnnn, wherin n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +2 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnn*nnnn, wherin n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +3 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnn*nnn, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +4 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnnn*nn, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +5 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnnnn*n, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +6 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnnnnn*, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +7 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNnnnnn*, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the -1, -2, -3, +1, +2, +3, +4, +5, +6 and/or +7 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNN*nnnnn . NN*Nnnnnn. N*NNnnnnnn.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the -1, -2, and/or -3 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNN*nnnnn, NN*Nnnnnn, or N*NNnnnnnn, wherin N*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the +1, +2, +3, +4, +5, +6 and/or +7 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NNNn*nnnnn, NNNnn*nnnn, NNNnnn*nn, NNNnnnn*n, NNNnnnn*n, NNNnnnnn*, or NNNnnnnnn*, wherein n*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -1 position relative to the splice site of the splice site sequence and a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of NN*N*nnnnn, wherein N*represents a bulged nucleotide.
  • a target of a compound described herein is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence and a bulged nucleotide at the -3 position relative to the splice site of the splice site sequence.
  • a target of a compound described herein can be a pre-mRNA comprising a splice site sequence of N*N*Nnnnnn, wherein N*represents a bulged nucleotide.
  • a compound described herein interacts with a bulged nucleotide of an RNA duplex comprising a splice site.
  • the RNA duplex comprises pre-mRNA.
  • a compound described herein binds to an RNA duplex and interacts with an unpaired bulged nucleobase of an RNA duplex comprising a splice site.
  • a first portion of the compound described herein interacts with the bulged nucleotide on a first RNA strand of the RNA duplex.
  • a second portion of the compound described herein interacts with one or more nucleotides of a second RNA strand of the RNA duplex, wherein the first RNA strand is not the second RNA strand.
  • the compound described herein forms one or more intermolecular interactions with the duplex RNA, for example, an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • the compound described herein forms one or more intermolecular interactions with the bulged nucleotide, for example, an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • the duplex RNA comprises an alpha helix. In some embodiments, the bulged nucleotide is located on an external portion of a helix of the duplex RNA. In some embodiments, the bulged nucleotide is located within an internal portion of the helix of the duplex RNA.
  • a rate of exchange of the bulged nucleotide from within the interior of a helix of the duplex RNA to an exterior portion of the helix is reduced.
  • the compound described herein modulates a distance of the bulged nucleotide from a second nucleotide of the duplex RNA. In some embodiments, the compound described herein reduces the distance of the bulged nucleotide from a second nucleotide of the duplex RNA. In some embodiments, the compound described herein increases the distance of the bulged nucleotide from a second nucleotide of the duplex RNA.
  • the bulged nucleotide is located within the interior of a helix of the duplex RNA of the complex. In some embodiments, the bulged nucleotide has modulated base stacking within an RNA strand of the RNA duplex. In some embodiments, the bulged nucleotide has increased base stacking within an RNA strand of the RNA duplex. In some embodiments, the bulged nucleotide has decreased base stacking within an RNA strand of the RNA duplex.
  • the compound described herein modulates splicing at the splice site of the RNA duplex. In some embodiments, the compound described herein increases splicing at the splice site of the RNA duplex. In some embodiments, the compound described herein reduces splicing at the splice site of the RNA duplex. In some embodiments, the compound described herein reduces a size of a bulge of the RNA duplex. In some embodiments, the compound described herein removes a bulge of the RNA duplex. In some embodiments, the compound described herein stabilizes a bulge of the RNA duplex. In some embodiments, the compound described herein destabilizes a bulge of the RNA duplex.
  • the unpaired bulged nucleotide is free to rotate around a phosphate backbone of an RNA strand of the RNA duplex in the absence of the compound described herein. In some embodiments, the compound described herein reduces a rate of rotation of the unpaired bulged nucleotide. In some embodiments, the compound described herein reduces a rate of rotation of the unpaired bulged nucleotide around a phosphate backbone of an RNA strand of the RNA duplex.
  • a method of modulating splicing comprising contacting the compound or the composition described herein to a cell; wherein the compound described herein interacts with an unpaired bulged nucleotide of an RNA duplex in the cell; wherein the duplex RNA comprises a splice site; and wherein the compound described herein modulates splicing of the RNA duplex.
  • a method for modulating the relative position of a first nucleotide relative to a second nucleotide, wherein the first nucleotide and the second nucleotide are within a duplex RNA comprising contacting the compound or the composition described herein to the duplex RNA, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein the first nucleotide is a bulged nucleotide of the RNA duplex; wherein the duplex RNA comprises a splice site.
  • the duplex RNA comprises a helix.
  • the bulged nucleotide is located on an external portion of a helix of the duplex RNA prior to contacting the compound described herein.
  • the compound described herein forms one or more intermolecular interactions with the duplex RNA.
  • the compound described herein forms one or more intermolecular interactions with an unpaired bulged nucleotide.
  • the intermolecular interaction is selected from the group comprising an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • a rate of exchange of the unpaired bulged nucleotide from within the interior of a helix of the duplex RNA to an exterior portion of the helix is reduced.
  • a rate of rotation of the unpaired bulged nucleotide is reduced.
  • a rate of rotation of the unpaired bulged nucleotide around a phosphate backbone of an RNA strand of the RNA duplex is reduced.
  • a distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is modulated after contacting the compound described herein.
  • the distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is reduced.
  • unpaired bulged nucleotide is located within the interior of the helix of the duplex RNA.
  • a size of a bulge of the RNA duplex is reduced.
  • a bulge of the RNA duplex is removed or maintained.
  • splicing at the splice site of the RNA duplex is promoted.
  • base stacking of the unpaired bulged nucleotide within an RNA strand of the RNA duplex is increased after contacting the compound described herein.
  • the distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is increased or maintained.
  • a bulge of the RNA duplex is stabilized after contacting the compound described herein.
  • the unpaired bulged nucleotide is located on an exterior portion of a helix of the duplex RNA.
  • a size of a bulge of the RNA duplex is increased.
  • splicing at the splice site of the RNA duplex is inhibited. In some embodiments, splicing is inhibited at the splice site. In some embodiments, base stacking of the unpaired bulged nucleotide within an RNA strand of the RNA duplex is reduced after contacting the compound described herein.
  • Exemplary sites targeted by the compounds described herein described herein include 5’splice sites, 3’splice sites, polypyrimidine tracts, branch sites, splicing enhancers and silencer elements.
  • Mutations or aberrant secondary or tertiary RNA structures at hot spots can create mRNA sites or scaffold sequences that can be targeted. For example, many exons are flanked by the intronic dinucleotides GT and AG at the 5’and 3’splice sites, respectively.
  • mutations or aberrant secondary or tertiary RNA structures at these sites can cause, e.g., exclusion of an adjacent exon or inclusion of an adjacent intron.
  • exemplary sites targeted by the compounds described herein described herein include secondary and sometimes tertiary structures of RNA.
  • exemplary sites targeted by the compounds described herein described herein include a stem loop, hairpin, branch point sequence (BPS) , polypyrimidine tract (PPT) , 5’splice site (5’ss) and 3’splice site (3’ss) , duplex snRNA and splice sites and trans acting protein binding to RNA.
  • the target pre-mRNA can comprise a defective sequence, such as a sequence that produces a deficient protein, such as a protein with altered function such as enzyme activity, or expression, such as lack of expression.
  • a defective sequence impacts the structure of the RNA.
  • the defect sequence impacts recognition by snRNP.
  • ESE/ISE exonic/intronic splicing enhancers
  • ESS/ISS silencer elements
  • a mutation in native DNA and/or pre-mRNA, or an aberrant secondary or tertiary structure of RNA creates a new splice site sequence.
  • a mutation or aberrant RNA structure may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • Such splice sites and elements can be referred to as “cryptic” .
  • a native intron may become divided into two aberrant introns, with a new exon situated there between.
  • a mutation may create a new splice site between a native 5’splice site and a native branch point.
  • a mutation may activate a cryptic branch point sequence between a native splice site and a native branch point.
  • a mutation may create a new splice site between a native branch point and a native splice site and may further activate a cryptic splice site and a cryptic branch point sequentially upstream from the aberrant mutated splice site.
  • a mutation or misexpression of trans-acting proteins that regulate splicing activity may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • a mutation or misexpression of an SR protein may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • a mutation in native DNA and/or pre-mRNA inhibits splicing at a splice site.
  • a mutation may result in a new splice site upstream from (i.e., 5’ to) a native splice site sequence and downstream from (i.e., 3’ to) a native branch point sequence.
  • the native splice site sequence and the native branch point sequence may serve as members of both the native set of splice site sequences and the aberrant set of splice site sequences.
  • a native splice element (e.g., a branch point) is also a member of the set of aberrant splice elements.
  • the compounds described herein provided herein can block the native element and activate a cryptic element (e.g., a cryptic 5’ss, a cryptic 3’ss or a cryptic branch point) , which may recruit remaining members of the native set of splice elements to promote correct splicing over incorrect splicing.
  • a cryptic element e.g., a cryptic 5’ss, a cryptic 3’ss or a cryptic branch point
  • an activated cryptic splice element is in an intron.
  • an activated cryptic splice element is in an exon.
  • the compounds and methods provided herein can be used to block or activate a variety of different splice elements, depending on the type of aberrant splice element (e.g., mutated splice element or non-mutated splice element) and/or depending on regulation of a splice element (e.g., regulation by upstream signaling pathways) .
  • the compounds and methods provided herein can block a mutated element, a non-mutated element, a cryptic element, or a native element; it may block a 5’splice site, a 3’splice site, or a branch point.
  • an alternate splicing event can be modulated by employing the compounds provided herein.
  • a compound provided herein can be introduced into a cell in which a gene is present that encodes a pre-mRNA that comprises alternate splice sites.
  • a first splicing event occurs to produce a gene product having a particular function.
  • the first splicing event can be inhibited.
  • the first splicing event in the presence of the compound provided herein, the first splicing event can be inhibited and a second or alternate splicing event occurs, resulting in expression of the same gene to produce a gene product having a different function.
  • a first inhibited splicing event (e.g., a splicing event inhibited by a mutation, a mutation-induced bulge or a non-mutation induced bulge) , is promoted or enhanced in the presence of a compound provided herein.
  • the inhibition of the first splicing event e.g., a splicing event inhibited by a mutation, a mutation-induced bulge or a non-mutation induced bulge
  • the inhibition of the first splicing event can be restored to a corresponding first splicing event that is uninhibited, in the presence of a compound provided herein; or the inhibition of the first splicing event can be decreased, in the presence of a compound provided herein.
  • a second or alternate splicing event occurs, resulting in expression of the same gene to produce a gene product having a different function.
  • the compounds described herein can modulate splicing of gene products.
  • the compounds described herein are use in the treatment, prevention and/or delay of progression of diseases or conditions (e.g., cancer and neurodegenerative diseases) .
  • the compounds described herein can modulate splicing and induce a transcriptionally inactive variant or transcript of a gene product.
  • the compounds described herein modulate splicing and repress a transcriptionally active variant or transcript of a gene product.
  • Modulation of splicing by the compounds described herein includes, but is not limited to, modulation of naturally occurring splicing, splicing of an RNA expressed in a diseased cell, splicing of cryptic splice site sequences of an RNA or alternative splicing. Modulation of splicing by the compounds described herein can restore or promote correct splicing or a desired splicing event. Modulation of splicing by the compounds described herein includes, but is not limited to, prevention of aberrant splicing events, e.g., splicing events caused by mutations or aberrant secondary or tertiary structures of RNA that are associated with conditions and diseases.
  • the compounds described herein prevent or inhibit splicing at a splice site sequence. In some embodiments, the compounds described herein promote or increase splicing at a splice site sequence. In some embodiments, the compounds described herein modulate splicing at a specific splice site sequence.
  • RNA e.g., DNA or RNA, e.g., pre-mRNA
  • a target sequence e.g., a target sequence comprising DNA or RNA, e.g., pre-mRNA
  • genes encoding a target sequence include, inter alia, ABCA4, ABCA9, ABCB1, ABCB5, ABCC9, ABCD1, ACADL, ACADM, ACADSB, ACSS2, ACTB, ACTG2, ADA, ADAL, ADAM10, ADAM15, ADAM22, ADAM32, ADAMTS12, ADAMTS13, ADAMTS20, ADAMTS42, ADAMTS6, ADAMTS9, ADAR, ADCY10, ADCY3, ADCY8, ADNP, ADRBK2, AFP, AGL, AGP, AGT, AGT, AHCTF1, AHR, AKAP10, AKAP3, AKNA, ALAS1, ALB
  • Additional exemplary genes encoding a target sequence include genes include A1CF, A4GALT, AAR2, ABAT, ABCA11P, ZNF721, ABCA5, ABHD10, ABHD13, ABHD2, ABHD6, AC000120.3, KRIT1, AC004076.1, ZNF772, AC004076.9, ZNF772, AC004223.3, RAD51D, AC004381.6, AC006486.1, ERF, AC007390.5, AC007780.1, PRKAR1A, AC007998.2, INO80C, AC009070.1, CMC2, AC009879.2, AC009879.3, ADHFE1, AC010487.3, ZNF816-ZNF321P, ZNF816, AC010328.3, AC010522.1, ZNF587B, AC010547.4, ZNF19, AC012313.3, ZNF4
  • the gene encoding a target sequence comprises the HTT gene. In some embodiments, the gene encoding a target sequence comprises the MYB gene. In some embodiments, the gene encoding a target sequence comprises the SMN2 gene.
  • Exemplary genes that may be modulated by the compounds of Formula (I) described herein may also include, inter alia, AC005258.1, AC005943.1, AC007849.1, AC008770.2, AC010487.3, AC011477.4, AC012651.1, AC012531.3, AC034102.2, AC073896.4, AC104472.3, AL109811.3, AL133342.1, AL137782.1, AL157871.5, AF241726.2, AL355336.1, AL358113.1, AL360181.3, AL445423.2, AL691482.3, AP001267.5, RF01169, and RF02271.
  • the compounds described herein may further be used to modulate a sequence comprising a particular splice site sequence, e.g., an RNA sequence (e.g., a pre-mRNA sequence) .
  • a particular splice site sequence e.g., an RNA sequence (e.g., a pre-mRNA sequence) .
  • the splice site sequence comprises a 5’splice site sequence.
  • the splice site sequence comprises a 3’splice site sequence.
  • Exemplary gene sequences and splice site sequences include the RNA sequences of SEQ ID NOs: 1-5547, AAAataagt, AAAgtacat, AAAgtaga, AAAgtatg, AAAgtatgt, AACgtaagt, AACgtgact, AACgtgatt, AAGatgagc, AAGatttgt, AAGgatgag, AAGgcaaaa, AAGggaaaa, AAGgtaag, AAGgtaaat, AAGgtaca, AAGgtaact, AAGgtacag, AAGgtacgg, AAGgtactg, AAGgtagag, AAGgtagcg, AAGgtagta, AAGgtagtg, AAGgtatac, AAGgtatat, AAGgtatca, AAGgtatcg, AAGgtatct
  • the splice site sequence (e.g., 5’splice site sequence) comprises AGA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AAA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AAC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AAU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AAG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AC A.
  • the splice site sequence (e.g., 5’splice site sequence) comprises AUA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AUU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AUG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AUC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CAA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CAU.
  • the splice site sequence (e.g., 5’splice site sequence) comprises CAC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CAG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GAA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GAC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GAU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GAG.
  • the splice site sequence (e.g., 5’splice site sequence) comprises GGA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GCA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GGG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GGC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GUU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GGU.
  • the splice site sequence (e.g., 5’splice site sequence) comprises GUC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GUA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GUG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UCU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UCC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UCA.
  • the splice site sequence (e.g., 5’splice site sequence) comprises UCG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UUU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UUC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UUA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UUG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UGU.
  • the splice site sequence (e.g., 5’splice site sequence) comprises UAU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises GGA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CUU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CUC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CUA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CUG.
  • the splice site sequence (e.g., 5’splice site sequence) comprises CCU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CCC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CCA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CCG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises ACU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises ACC.
  • the splice site sequence (e.g., 5’splice site sequence) comprises ACG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AGC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AGU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises AGG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CGU. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UAC.
  • the splice site sequence (e.g., 5’splice site sequence) comprises UAA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises UAG. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CGC. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CGA. In some embodiments, the splice site sequence (e.g., 5’splice site sequence) comprises CGG. In some embodiments, the splice site sequence comprises AGAguaaggg. In some embodiments, the splice site sequence comprises UGAguaagca.
  • Mutations and/or aberrant secondary or tertiary RNA structures in cis-acting elements of splicing can alter splicing patterns. Mutations and/or aberrant secondary or tertiary RNA structures can be found in core consensus sequences, including 5’ss, 3’ss, and BP regions, or other regulatory elements, including ESEs, ESSs, ISEs, and ISSs. Mutations in cis-acting elements can result in multiple diseases. Exemplary diseases are described below.
  • the present disclosure provides splice modulating compounds and methods that target pre-mRNA containing one or more mutations and/or aberrant secondary or tertiary RNA structures in cis-acting elements.
  • the present disclosure provides methods and small molecule binding agents that target pre-mRNA containing one or more mutations and/or aberrant secondary or tertiary RNA structures in splice sites or BP regions. In some embodiments, the present disclosure provides methods and small molecule binding agents that target pre-mRNA containing one or more mutations and/or aberrant secondary or tertiary RNA structures in other regulatory elements, for example, ESEs, ESSs, ISEs, and ISSs.
  • splicing at a splice site sequence of a polynucleotide of primary cells is modulated.
  • splicing at a splice site sequence of a polynucleotide of cells of a tumor is modulated.
  • the compound described herein modulates splicing at a cryptic splice site sequence.
  • a compound described herein modulates splicing of splice site of a polynucleotide.
  • wherein the polynucleotide is transcribed from the gene.
  • compound described herein modulates exon inclusion in the polynucleotide and splicing of the splice site sequence. In some embodiments, the compound described herein modulates pseudoexons inclusion in the polynucleotide and splicing of the splice site sequence. In some embodiments, the compound described herein modulates splicing at a cryptic splice site sequence of a polynucleotide.
  • a compound described herein modulates splicing by preventing, inhibiting or reducing splicing of the polynucleotide. In some embodiments, a compound described herein modulates splicing by preventing, inhibiting or reducing splicing at the splice site sequence. In some embodiments, a compound described herein decreases affinity of a splicing complex component to the polynucleotide. In some embodiments, a compound described herein decreases affinity of a splicing complex component to the polynucleotide at the splice site sequence, upstream of the splice site sequence or downstream of the splice site sequence.
  • a compound described herein inhibits or reduces a rate of catalysis of splicing of the polynucleotide. In some embodiments, a compound described herein inhibits or reduces a rate of catalysis of splicing of the polynucleotide at the splice site sequence. In some embodiments, a compound described herein increases steric hindrance between a splicing complex component and the polynucleotide. In some embodiments, a compound described herein increases steric hindrance between a splicing complex component and the polynucleotide at the splice site sequence, upstream of the splice site sequence or downstream of the splice site sequence.
  • a compound described herein increases steric hindrance between a first splicing complex component and a second splicing complex component. In some embodiments, a compound described herein prevents, inhibits, disrupts or reduces binding of a first splicing complex component and a second splicing complex component.
  • a compound described herein decreases affinity of a first splicing complex component to a second splicing complex component. In some embodiments, a compound described herein prevents, inhibits, disrupts or reduces binding of a splicing complex component to the polynucleotide. In some embodiments, a compound described herein prevents, inhibits, disrupts or reduces binding of a splicing complex component to the polynucleotide at the splice site sequence, upstream of the splice site sequence or downstream of the splice site sequence.
  • a compound described herein modulates splicing by promoting or increasing splicing of the polynucleotide. In some embodiments, a compound described herein modulates splicing by promoting or increasing splicing the splice site sequence. In some embodiments, a compound described herein increases affinity of a splicing complex component to the polynucleotide. In some embodiments, a compound described herein increases affinity of a splicing complex component to the polynucleotide at the splice site sequence, upstream of the splice site sequence or downstream of the splice site sequence.
  • a compound described herein increases a rate of catalysis of splicing of the polynucleotide. In some embodiments, a compound described herein increases a rate of catalysis of splicing of the polynucleotide at the splice site sequence. In some embodiments, a compound described herein decreases or reduces steric hindrance between a splicing complex component and the polynucleotide.
  • a compound described herein decreases steric hindrance between a splicing complex component and the polynucleotide at the splice site sequence, 1-1000 nucleobases bases upstream of the splice site sequence or 1-1000 nucleobases downstream of the splice site sequence. In some embodiments, a compound described herein decreases or reduces steric hindrance between a first splicing complex component and a second splicing complex component. In some embodiments, a compound described herein promotes or increases binding of a first splicing complex component and a second splicing complex component.
  • a compound described herein increases affinity of a first splicing complex component to a second splicing complex component. In some embodiments, a compound described herein promotes or increases binding of a splicing complex component to the polynucleotide. In some embodiments, a compound described herein promotes or increases binding of a splicing complex component to the polynucleotide at the splice site sequence, 1-1000 nucleobases upstream of the splice site sequence or 1-1000 nucleobases downstream of the splice site sequence. In some embodiments, a compound described herein binds to a splicing complex component, the polynucleotide, or a combination thereof.
  • a compound described herein binds to the polynucleotide at the splice site sequence, 1-1000 nucleobases upstream of the splice site sequence or 1-1000 nucleobases downstream of the splice site sequence.
  • a compound described herein structurally modulates a splicing complex component, the polynucleotide, or both.
  • a compound described herein promotes or increases steric hindrance, steric shielding, steric attraction, chain crossing, steric repulsions, steric inhibition of resonance, steric inhibition of protonation, or a combination thereof of the polynucleotide, a splicing complex component or a combination thereof.
  • binding of a compound described herein to a polynucleotide or a splicing complex component decreases conformational stability of a splice site sequence. In some embodiments, binding of a compound described herein to a polynucleotide increases conformational stability of a splice site sequence.
  • a compound described herein modulates exon skipping of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can inhibit exon skipping of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can promote exon skipping of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with exon skipping of the polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with aberrant exon skipping of the polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates exon inclusion of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can inhibit exon inclusion of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can promote exon inclusion of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with exon inclusion of the polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with aberrant exon inclusion of the polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates nonsense mediated degradation (NMD) of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can inhibit nonsense mediated degradation (NMD) of a target polynucleotide, such as a pre-mRNA or an mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with NMD of the polynucleotide, such as a pre-mRNA or an mRNA.
  • a compound described herein modulates intron inclusion of a target polynucleotide.
  • a compound described herein can inhibit intron inclusion of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein can promote intron inclusion of a target polynucleotide, such as a pre-mRNA.
  • a compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with intron inclusion of the polynucleotide.
  • the compound described herein modulates splicing at a splice site sequence of a polynucleotide in a cell of a subject with a disease or condition associated with intron inclusion of the polynucleotide.
  • a compound described herein modulates splicing at splice site sequence of a polynucleotide, such as a pre-mRNA, wherein the splice site sequence comprises a sequence selected from the group consisting of NGAgunvm, NHAdddddn, NNBnnnnn, and NHAddmhvk; wherein N or n is A, U, G or C; B is C, G, or U; H or h is A, C, or U; d is a, g, or u; m is a or c; r is a or g; v is a, c or g; k is g or u.
  • a compound described herein modulates splicing of a splice site sequence comprising a sequence NNBgunnnn, NNBhunnnn, or NNBgvnnnn. In some embodiments, a compound described herein modulates splicing of a splice site sequence comprising a sequence NNBgurrm, NNBguwwdn, NNBguvmvn, NNBguvbbn, NNBgukddn, NNBgubnbd, NNBhunngn, NNBhurmhd, or NNBgvdnvn; wherein N or n is A, U, G or C; B is C, G, or U; H or h is A, C, or U; d is a, g, or u; m is a or c; r is a or g; v is a, c or g; k is g or u.
  • a gene sequence or splice site sequence provided herein is related to a proliferative disease, disorder, or condition (e.g., cancer, benign neoplasm, or inflammatory disease) .
  • a gene sequence or splice site sequence provided herein is related to a non-proliferative disease, disorder, or condition.
  • a gene sequence or splice site sequence provided herein is related to a neurological disease or disorder; autoimmune disease or disorder; immunodeficiency disease or disorder; lysosomal storage disease or disorder; cardiovascular condition, disease or disorder; metabolic disease or disorder; respiratory condition, disease, or disorder; renal disease or disorder; or infectious disease in a subject.
  • a gene sequence or splice site sequence provided herein is related to a neurological disease or disorder (e.g., Huntington’s disease) .
  • a gene sequence or splice site sequence provided herein is related to an immunodeficiency disease or disorder.
  • a gene sequence or splice site sequence provided herein is related to a lysosomal storage disease or disorder.
  • a gene sequence or splice site sequence provided herein is related to a cardiovascular condition, disease or disorder.
  • a gene sequence or splice site sequence provided herein is related to a metabolic disease or disorder.
  • a gene sequence or splice site sequence provided herein is related to a respiratory condition, disease, or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a renal disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to an infectious disease.
  • a compound of Formula (I) described herein interacts with (e.g., binds to) a splicing complex component (e.g., a nucleic acid (e.g., an RNA) or a protein) .
  • a splicing complex component e.g., a nucleic acid (e.g., an RNA) or a protein
  • the splicing complex component is selected from 9G8, Al hnRNP, A2 hnRNP, ASD-1, ASD-2b, ASF, BRR2, Bl hnRNP, Cl hnRNP, C2 hnRNP, CBP20, CBP80, CELF, F hnRNP, FBP11, Fox-1, Fox-2, G hnRNP, H hnRNP, hnRNP 1, hnRNP 3, hnRNP C, hnRNP G, hnRNP K, hnRNP M, hnRNP U, Hu, HUR, I hnRNP, K hnRNP, KH-type splicing regulatory protein (KSRP) , L hnRNP, LUC7L, M hnRNP, mBBP, muscle-blind like (MBNL) , NF45, NF AR, Nova-1, Nova-2, nPTB, P54/SFRS11
  • the splicing complex component comprises RNA (e.g., snRNA) .
  • a compound described herein binds to a splicing complex component comprising snRNA.
  • the snRNA may be selected from, e.g., U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, U11 snRNA, U12 snRNA, U4atac snRNA, and any combination thereof.
  • the splicing complex component comprises a protein, e.g., a protein associated with an snRNA.
  • the protein comprises SC35, SRp55, SRp40, SRm300, SFRS10, TASR-1, TASR-2, SF2/ASF, 9G8, SRp75, SRp30c, SRp20 and P54/SFRS11.
  • the splicing complex component comprises a U2 snRNA auxiliary factor (e.g., U2AF65, U2AF35) , Urp/U2AFl-RS2, SF1/BBP, CBP80, CBP 20, SF1 or PTB/hnRNPl.
  • the hnRNP protein comprises Al, A2/B1, L, M, K, U, F, H, G, R, I or C1/C2.
  • Human genes encoding hnRNPs include HNRNPAO, HNRNPA1, HNRNPA1L1, HNRNPA1L2, HNRNPA3, HNRNPA2B1, HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNRPLL, HNRNPM, HNRNPR, HNRNPU, HNRNPUL1, HNRNPUL2, HNRNPUL3, and FMRI.
  • the compounds of Formula (I) and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers, and compositions thereof may modulate (e.g., increase or decrease) a splicing event of a target nucleic acid sequence (e.g., DNA, RNA, or a pre-mRNA) , for example, a nucleic acid encoding a gene described herein, or a nucleic acid encoding a protein described herein, or a nucleic acid comprising a splice site described herein.
  • the splicing event is an alternative splicing event.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof increases splicing at splice site on a target nucleic acid (e.g., an RNA, e.g., a pre-mRNA) , by about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, e.g., as determined by a known method in the art, e.g., qPCR.
  • a target nucleic acid e.g., an RNA, e.g., a pre-mRNA
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof decreases splicing at splice site on a target nucleic acid (e.g., an RNA, e.g., a pre-mRNA) , by about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, e.g., as determined by a known method in the art, e.g., qPCR.
  • a target nucleic acid e.g., an RNA, e.g., a pre-mRNA
  • the present disclosure features a method of forming a complex comprising a component of a spliceosome (e.g., a major spliceosome component or a minor spliceosome component) , a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) , and a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof, comprising contacting the nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) with said compound of Formula (I) .
  • a spliceosome e.g., a major spliceosome component or a minor spliceosome component
  • a nucleic acid e.g., a DNA, RNA, e.g., a pre-mRNA
  • the component of a spliceosome is selected from the Ul, U2, U4, U5, U6, U11, U12, U4atac, U6atac small nuclear ribonucleoproteins (snRNPs) , or a related accessory factor.
  • the component of a spliceosome is recruited to the nucleic acid in the presence of the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof.
  • the present disclosure features a method of altering the conformation of a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) comprising contacting the nucleic acid with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof.
  • the altering comprises forming a bulge or kink in the nucleic acid.
  • the altering comprises stabilizing a bulge or a kink in the nucleic acid.
  • the altering comprises reducing a bulge or a kink in the nucleic acid.
  • the nucleic acid comprises a splice site.
  • the compound of Formula (I) interacts with a nucleobase, ribose, or phosphate moiety of a nucleic acid (e.g., a DNA, RNA, e.g., pre-mRNA) .
  • the present disclosure also provides methods for the treatment or prevention of a disease, disorder, or condition.
  • the disease, disorder or condition is related to (e.g., caused by) a splicing event, such as an unwanted, aberrant, or alternative splicing event.
  • the disease, disorder or condition comprises a proliferative disease (e.g., cancer, benign neoplasm, or inflammatory disease) or non-proliferative disease.
  • the disease, disorder, or condition comprises a neurological disease, autoimmune disorder, immunodeficiency disorder, cardiovascular condition, metabolic disorder, lysosomal storage disease, respiratory condition, renal disease, or infectious disease in a subject.
  • the disease, disorder, or condition comprises a haploinsufficiency disease, an autosomal recessive disease (e.g., with residual function) , or a paralogue activation disorder.
  • the disease, disorder, or condition comprises an autosomal dominant disorder (e.g., with residual function) .
  • Such methods comprise the step of administering to the subject in need thereof an effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer thereof, or a pharmaceutical composition thereof.
  • the methods described herein include administering to a subject an effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof.
  • the subject being treated is a mammal.
  • the subject is a human.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal such as a dog or cat.
  • the subject is a livestock animal such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal such as a rodent, dog, or non-human primate.
  • the subject is a non-human transgenic animal such as a transgenic mouse or transgenic pig.
  • a proliferative disease may also be associated with inhibition of apoptosis of a cell in a biological sample or subject. All types of biological samples described herein or known in the art are contemplated as being within the scope of the disclosure.
  • the compounds of Formula (I) and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers, and compositions thereof, may induce apoptosis, and therefore, be useful in treating and/or preventing proliferative diseases.
  • the proliferative disease to be treated or prevented using the compounds of Formula (I) is cancer.
  • cancer refers to a malignant neoplasm (Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams &Wilkins: Philadelphia, 1990) . All types of cancers disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma) ; appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma) ; bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast) ; brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma) , medulloblastoma) ; bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adeno
  • liver cancer e.g., hepatocellular cancer (HCC) , malignant hepatoma
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC) , non-small cell lung cancer (NSCLC) , adenocarcinoma of the lung
  • leiomyosarcoma I-MS
  • mastocytosis e.g., systemic mastocytosis
  • muscle cancer myelodysplastic syndrome (MDS)
  • MDS myelodysplastic syndrome
  • MDS myeloproliferative disorder
  • MPD e.g., polycythemia vera (PV) , essential thrombocytosis (ET) , agnogenic myeloid metaplasia (AMM) a.
  • MF myelofibrosis
  • CML chronic myelocytic leukemia
  • CNL chronic neutrophilic leukemia
  • HES hypereosinophilic syndrome
  • neuroblastoma neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis)
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET) , carcinoid tumor
  • osteosarcoma e.g., bone cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous
  • the cancer is selected from adenoid cystic carcinoma (ACC) , acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML) , chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML) , non-Hodgkin lymphoma (NHL) , Burkitt lymphoma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma) , prostate cancer (e.g., prostate adenocarcinoma) , ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma) , and myelodysplastic syndrome (MDS) .
  • ACC adenoid cystic carcinoma
  • AML acute myelocytic leukemia
  • CML chronic myelocytic leukemia
  • the proliferative disease is associated with a benign neoplasm.
  • a benign neoplasm may include adenoma, fibroma, hemangioma, tuberous sclerosis, and lipoma. All types of benign neoplasms disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the proliferative disease is associated with angiogenesis. All types of angiogenesis disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a non-proliferative disease.
  • non-proliferative diseases include a neurological disease, autoimmune disorder, immunodeficiency disorder, lysosomal storage disease, cardiovascular condition, metabolic disorder, respiratory condition, inflammatory disease, renal disease, or infectious disease.
  • the non-proliferative disease is a neurological disease.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound, is used to prevent or treat a neurological disease, disorder, or condition.
  • a neurological disease, disorder, or condition may include a neurodegenerative disease, a psychiatric condition, or a musculoskeletal disease.
  • a neurological disease may further include a repeat expansion disease, e.g., which may be characterized by the expansion of a nucleic acid sequence in the genome.
  • a repeat expansion disease includes myotonic dystrophy, amyotrophic lateral sclerosis, Huntington’s disease, a trinucleotide repeat disease, or a polyglutamine disorder (e.g., ataxia, fragile X syndrome) .
  • the neurological disease comprises a repeat expansion disease, e.g., Huntington’s disease.
  • Additional neurological diseases, disorders, and conditions include Alzheimer’s disease, Huntington’s chorea, a prion disease (e.g., Creutzfeld-Jacob disease, bovine spongiform encephalopathy, Kuru, or scrapie) , a mental retardation disorder (e.g., a disorder caused by a SETD5 gene mutation, e.g., intellectual disability-facial dysmorphism syndrome, autism spectrum disorder) , Lewy Body disease, diffuse Lewy body disease (DLBD) , dementia, progressive supranuclear palsy (PSP) , progressive bulbar palsy (PBP) , psuedobulbar palsy, spinal and bulbar muscular atrophy (SBMA) , primary lateral sclerosis, Pick’s disease, primary progressive aphasia, corticobasal dementia, Parkinson’s disease, Down’s syndrome, multiple system atrophy, spinal muscular atrophy (SMA) , progressive spinobulbar muscular atrophy (e.g.
  • the neurological disease comprises Friedrich’s ataxia or Sturge Weber syndrome. In some embodiments, the neurological disease comprises Huntington’s disease. In some embodiments, the neurological disease comprises spinal muscular atrophy. All types of neurological diseases disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the non-proliferative disease is an autoimmune disorder or an immunodeficiency disorder.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat an autoimmune disease, disorder, or condition, or an immunodeficiency disease, disorder, or condition.
  • autoimmune and immunodeficiency diseases, disorders, and conditions include arthritis (e.g., rheumatoid arthritis, osteoarthritis, gout) , Chagas disease, chronic obstructive pulmonary disease (COPD) , dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture’s syndrome, Graves’ disease, Guillain-Barre syndrome (GBS) , Hashiomoto’s disease, Hi dradenitis suppurativa, Kawasaki disease, ankylosing spondylitis, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet’s syndrome, infective colitis, indeterminate colitisinterstitial cystitis, lupus (e.g., systemic lupus
  • the non-proliferative disease is a cardiovascular condition.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a cardiovascular disease, disorder, or condition.
  • a cardiovascular disease, disorder, or condition may include a condition relating to the heart or vascular system, such as the arteries, veins, or blood.
  • Exemplary cardiovascular diseases, disorders, or conditions include angina, arrhythmias (atrial or ventricular or both) , heart failure, arteriosclerosis, atheroma, atherosclerosis, cardiac hypertrophy, cardiac or vascular aneurysm, cardiac myocyte dysfunction, carotid obstructive disease, endothelial damage after PTCA (percutaneous transluminal coronary angioplasty) , hypertension including essential hypertension, pulmonary hypertension and secondary hypertension (renovascular hypertension, chronic glomerulonephritis) , myocardial infarction, myocardial ischemia, peripheral obstructive arteriopathy of a limb, an organ, or a tissue; peripheral artery occlusive disease (PAOD) , reperfusion injury following ischemia of the brain, heart or other organ or tissue, restenosis, stroke, thrombosis, transient ischemic attack (TIA) , vascular occlusion, vasculitis, and vasoconstriction.
  • the non-proliferative disease is a metabolic disorder.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a metabolic disease, disorder, or condition.
  • a metabolic disease, disorder, or condition may include a disorder or condition that is characterized by abnormal metabolism, such as those disorders relating to the consumption of food and water, digestion, nutrient processing, and waste removal.
  • a metabolic disease, disorder, or condition may include an acid-base imbalance, a mitochondrial disease, a wasting syndrome, a malabsorption disorder, an iron metabolism disorder, a calcium metabolism disorder, a DNA repair deficiency disorder, a glucose metabolism disorder, hyperlactatemia, a disorder of the gut microbiota.
  • Exemplary metabolic conditions include obesity, diabetes (Type I or Type II) , insulin resistance, glucose intolerance, lactose intolerance, eczema, hypertension, Hunter syndrome, Krabbe disease, sickle cell anemia, maple syrup urine disease, Pompe disease, and metachromatic leukodystrophy. All types of metabolic diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the non-proliferative disease is a respiratory condition.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a respiratory disease, disorder, or condition.
  • a respiratory disease, disorder, or condition can include a disorder or condition relating to any part of the respiratory system, such as the lungs, alveoli, trachea, bronchi, nasal passages, or nose.
  • Exemplary respiratory diseases, disorders, or conditions include asthma, allergies, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease (COPD) , lung cancer, oxygen toxicity, emphysema, chronic bronchitis, and acute respiratory distress syndrome. All types of respiratory diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the non-proliferative disease is a renal disease.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a renal disease, disorder, or condition.
  • a renal disease, disorder, or condition can include a disease, disorder, or condition relating to any part of the waste production, storage, and removal system, including the kidneys, ureter, bladder, urethra, adrenal gland, and pelvis.
  • Exemplary renal diseases include acute kidney failure, amyloidosis, Alport syndrome, adenovirus nephritis, acute lobar nephronia, tubular necrosis, glomerulonephritis, kidney stones, urinary tract infections, chronic kidney disease, polycystic kidney disease, and focal segmental glomerulosclerosis (FSGS) .
  • the renal disease, disorder, or condition comprises HIV-associated nephropathy or hypertensive nephropathy. All types of renal diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.
  • the non-proliferative disease is an infectious disease.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat an infectious disease, disorder, or condition.
  • An infectious disease may be caused by a pathogen such as a virus or bacteria.
  • infectious diseases include human immunodeficiency syndrome (HIV) , acquired immunodeficiency syndrome (AIDS) , meningitis, African sleeping sickness, actinomycosis, pneumonia, botulism, chlamydia, Chagas disease, Colorado tick fever, cholera, typhus, giardiasis, food poisoning, ebola hemorrhagic fever, diphtheria, Dengue fever, gonorrhea, streptococcal infection (e.g., Group A or Group B) , hepatitis A, hepatitis B, hepatitis C, herpes simplex, hookworm infection, influenza, Epstein-Barr infection, Kawasaki disease, kuru, leprosy, leishmaniasis, measles, mumps, norovirus, meningococcal disease, malaria, Lyme disease, listeriosis, rabies, rhinovirus, rubella, tetanus, shingles, scarlet fever,
  • the disease, disorder, or condition is a haploinsufficiency disease.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound is used to prevent or treat a haploinsufficiency disease, disorder, or condition.
  • a haploinsufficiency disease, disorder, or condition may refer to a monogenic disease in which an allele of a gene has a loss-of-function lesion, e.g., a total loss of function lesion.
  • the loss-of-function lesion is present in an autosomal dominant inheritance pattern or is derived from a sporadic event.
  • the reduction of gene product function due to the altered allele drives the disease phenotype despite the remaining functional allele (i.e. said disease is haploinsufficient with regard to the gene in question) .
  • a compound of Formula (I) increases expression of the haploinsufficient gene locus.
  • a compound of Formula (I) increases one or both alleles at the haploinsufficient gene locus.
  • haploinsufficiency diseases, disorders, and conditions include Robinow syndrome, cardiomyopathy, cerebellar ataxia, pheochromocytoma, Charcot-Mari e-Tooth disease, neuropathy, Takenouchi-Kosaki syndrome, Coffin-Siris syndrome 2, chromosome lp35 deletion syndrome, spinocerebellar ataxia 47, deafness, seizures, dystonia 9, GLUT1 deficiency syndrome 1, GLUT1 deficiency syndrome 2, stomatin-deficient cryohydrocytosis, basal cell carcinoma, basal cell nevus syndrome, medulloblastoma, somatic, brain malformations, macular degeneration, cone-rod dystrophy, Dejerine-Sottas disease, hypomyelinating neuropathy, Roussy -Levy syndrome, glaucoma, autoimmune lymphoproliferative syndrome, pituitary hormone deficiency, epileptic encephalopathy, early infantile, popliteal
  • the disease, disorder, or condition is an autosomal recessive disease, e.g., with residual function.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound, is used to prevent or treat an autosomal recessive disease, disorder, or condition.
  • An autosomal recessive disease with residual function may refer to a monogenic disease with either homozygous recessive or compound heterozygous heritability. These diseases may also be characterized by insufficient gene product activity (e.g., a level of gene product greater than 0%) .
  • a compound of Formula (I) may increase the expression of a target (e.g., a gene) related to an autosomal recessive disease with residual function.
  • a target e.g., a gene
  • autosomal recessive diseases with residual function include Friedreich’s ataxia, Stargardt disease, Usher syndrome, chlorioderma, fragile X syndrome, achromatopsia 3, Hurler syndrome, hemophilia B, alpha-1-antitrypsin deficiency, Gaucher disease, X-linked retinoschisis, Wiskott-Aldrich syndrome, mucopolysaccharidosis (Sanfilippo B) , DDC deficiency, epidermolysis bullosa dystrophica, Fabry disease, metachromatic leukodystrophy, and odontochondrodysplasia.
  • the disease, disorder, or condition is an autosomal dominant disease.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound, is used to prevent or treat an autosomal dominant disease, disorder, or condition.
  • An autosomal dominant disease may refer to a monogenic disease in which the mutated gene is a dominant gene. These diseases may also be characterized by insufficient gene product activity (e.g., a level of gene product greater than 0%) .
  • a compound of Formula (I) may increase the expression of a target (e.g., a gene) related to an autosomal dominant disease.
  • Exemplary autosomal dominant diseases include Huntington’s disease, achondroplasia, antithrombin III deficiency, Gilbert’s disease, Ehlers-Danlos syndrome, hereditary hemorrhagic telangiectasia, intestinal polyposis, hereditary elliptosis, hereditary spherocytosis, marble bone disease, Marfan’s syndrome, protein C deficiency, Treacher Collins syndrome, Von Willebrand’s disease, tuberous sclerosis, osteogenesis imperfecta, polycystic kidney disease, neurofibromatosis, and idiopathic hypoparathyroidi sm .
  • the disease, disorder, or condition is a paralogue activation disorder.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or compositions comprising such compound, is used to prevent or treat a paralogue activation disease, disorder, or condition.
  • a paralogue activation disorder may comprise a homozygous mutation of genetic locus leading to loss-of-function for the gene product. In these disorders, there may exist a separate genetic locus encoding a protein with overlapping function (e.g. developmental paralogue) , which is otherwise not expressed sufficiently to compensate for the mutated gene.
  • a compound of Formula (I) activates a gene connected with a paralogue activation disorder (e.g., a paralogue gene) .
  • the cell described herein may be an abnormal cell.
  • the cell may be in vitro or in vivo.
  • the cell is a proliferative cell.
  • the cell is a cancer cell.
  • the cell is a non-proliferative cell.
  • the cell is a blood cell.
  • the cell is a lymphocyte.
  • the cell is a benign neoplastic cell.
  • the cell is an endothelial cell.
  • the cell is an immune cell.
  • the cell is a neuronal cell.
  • the cell is a glial cell.
  • the cell is a brain cell.
  • the cell is a fibroblast.
  • the cell is a primary cell, e.g., a cell isolated from a subject (e.g., a human subject) .
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., as described herein, increases the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof decreases the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample.
  • a target protein e.g., HTT or MYB
  • the effect of an exemplary compound of Formula (I) on protein abundance may be measured using a standard assay for measuring protein abundance, such as Western Blot.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., as described herein, has a protein abundance response less than 50 ⁇ M.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, e.g., as described herein has a protein abundance response less than 30 ⁇ M, or less than 20 ⁇ M, between 0.1-10 ⁇ M or 0.5-5 ⁇ M, or less than 0.5 ⁇ M, for example, 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 5 ⁇ M, 8 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M or 50 ⁇ M.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., as described herein, modulates the protein abundance of a target protein by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, 99%or more, e.g., compared with a reference compound.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the abundance expression of mRNA (e.g., HTT or MYB) in a reference cell or sample.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., as described herein, increases the expression of a mRNA (e.g., HTT or MYB) in a reference cell or sample.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof decreases the expression of a mRNA (e.g., HTT or MYB) in a reference cell or sample.
  • a mRNA e.g., HTT or MYB
  • the effect of an exemplary compound of Formula (I) on mRNA may be measured using a standard assay for measuring mRNA abundance, such as RT-qPCR.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof e.g., as described herein, has a mRNA abundance response less than 50 ⁇ M.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, e.g., as described herein has a mRNA abundance response less than 30 ⁇ M, or less than 20 ⁇ M, between 0.1-10 ⁇ M or 0.5-5 ⁇ M, or less than 0.5 ⁇ M, for example, 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 5 ⁇ M, 8 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M or 50 ⁇ M.
  • the methods described herein comprise the additional step of administering one or more additional pharmaceutical agents in combination with the compound of Formula (I) , a pharmaceutically acceptable salt thereof, or compositions comprising such compound.
  • additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti -cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressant agents, and a pain-relieving agent.
  • the additional pharmaceutical agent (s) may synergistically augment the modulation of splicing induced by the inventive compounds or compositions of this disclosure in the biological sample or subject.
  • the combination of the inventive compounds or compositions and the additional pharmaceutical agent (s) may be useful in treating, for example, a cancer or other disease, disorder, or condition resistant to a treatment using the additional pharmaceutical agent (s) without the inventive compounds or compositions.
  • the compound or composition utilized in the methods described herein can be, e.g., administered at dosages that may be varied depending upon the requirements of the subject the severity of the condition being treated and/or imaged, and/or the compound or composition being employed. For example, dosages can be empirically determined considering the type and stage of disease diagnosed in a particular subject and/or the type of imaging modality being used in conjunction with the compound or composition.
  • the dose administered to a subject, in the context of the present invention should be sufficient to affect a beneficial diagnostic or therapeutic response in the subject.
  • the size of the dose also can be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a compound or composition in a particular subject.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms described herein are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50%of the population) and the ED 50 (the dose therapeutically effective in 50%of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • Therapeutic index data obtained from cell culture assays and/or animal studies can be used in predicting the therapeutic index in vivo and formulating a range of dosages for use in subjects, such as human subjects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound which achieves a half-maximal inhibition of symptoms as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Various animal models and clinical assays for evaluating effectiveness of a particular compound or composition in preventing or reducing a disease or condition are known in the art may be used in the present invention. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • the compound or composition provided have a therapeutic index (LD 50 /ED 50 ) of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000 or more. In some aspects, the compound or composition provided have a therapeutic index (LD 50 /ED 50 ) of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000 or more as determined in cell culture.
  • LD 50 /ED 50 therapeutic index of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000 or
  • the compound or composition provided have an IC 50 viability/EC 50 splicing value of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000 or more. In some aspects, the compound or composition provided have an IC 50 viability/EC 50 splicing value of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000 or more as determined in cell culture.
  • a dosage of using a compound or composition when administered may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 g/m 2 in humans, or a dosage in another subject comparable to that in humans.
  • a dosage ( “dosage X” ) of a compound or composition in a subject other than a human is comparable to a dosage ( “dosage Y” ) of the compound or composition in humans if the serum concentration of the scavenger in the subject post administration of the compound or composition at dosage X is equal to the serum concentration of the compound or composition in humans post administration of the compound at dosage Y.
  • the effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof for use in the manufacture of a medicament, the preparation of a pharmaceutical kit or in a method for preventing and/or treating a disease in a human subject in need thereof is intended to include an amount in a range of from about 1 ⁇ g to about 50 g.
  • compositions of the present invention can be administered as frequently as necessary, including hourly, daily, weekly or monthly.
  • any of the aforementioned aspects are further embodiments comprising single administrations of an effective amount of a compound or composition described herein, including further embodiments in which (i) the compound is administered once; (ii) the compound is administered to the mammal multiple times over the span of one day; (iii) continually; or (iv) continuously.
  • any of the aforementioned aspects are further embodiments comprising multiple administrations of the effective amount of a compound or composition described herein, including further embodiments in which (i) the compound is administered continuously or intermittently: as in a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the compound is administered to the mammal every 8 hours; (iv) the compound is administered to the mammal every 12 hours; (v) the compound is administered to the mammal every 24 hours.
  • the method comprises a drug holiday, wherein the administration of a compound or composition described herein is temporarily suspended or the dose of the compound being administered is temporarily reduced; at the end of the drug holiday, dosing of the compound is resumed.
  • the length of the drug holiday varies from 2 days to 1 year.
  • a compound compound or composition described herein can be co-administered with a second therapeutic agent, wherein compound or composition and the second therapeutic agent modulate different aspects of the disease, disorder or condition being treated, thereby providing a greater overall benefit than administration of either therapeutic agent alone.
  • a compound or composition described herein can be used in combination with an anti-cancer therapy.
  • a steric modulator is used in combination with conventional chemotherapy, radiotherapy, hormonal therapy, and/or immunotherapy.
  • a compound or composition described herein can be used in combination with conventional chemotherapeutic agents including alkylating agents (e.g., temozolomide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.
  • alkylating agents e.g., temozolomide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.
  • anti-metabolites e.g., 5-fluorouracil, azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.
  • plant alkaloids e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.
  • topoisomerase inhibitors e.g., irinotecan, topotecan, amsacrine, etoposide (VP16) , etoposide phosphate, teniposide, etc.
  • antitumor antibiotics e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.
  • platinum-based compounds e.g. cisplatin, oxaloplatin, carboplatin, etc.
  • EGFR inhibitors e.g., gefitinib, erlotinib, etc.
  • a compound or composition may be administered in combination with one or more other compound or composition.
  • a compound or composition may be administered to a subject in need thereof prior to, concurrent with, or following the administration of chemotherapeutic agents.
  • compound or composition may be administered to a subject at least 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, or 30 minutes before the starting time of the administration of chemotherapeutic agent (s) .
  • they may be administered concurrent with the administration of chemotherapeutic agent (s) .
  • compound or composition are administrated at the same time when the administration of chemotherapeutic agent (s) starts.
  • compound or composition may be administered following the starting time of administration of chemotherapeutic agent (s) (e.g., at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours after the starting time of administration of chemotherapeutic agents) .
  • compound or composition may be administered at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours after the completion of administration of chemotherapeutic agents.
  • these compounds or compositions are administered for a sufficient period of time so that the disease or condition is prevented or reduced. Such sufficient period of time may be identical to, or different from, the period during which chemotherapeutic agent (s) are administered.
  • multiple doses of compound or composition are administered for each administration of a chemotherapeutic agent or a combination of multiple chemotherapeutic agents.
  • an appropriate dosage of a compound or composition is combined with a specific timing and/or a particular route to achieve the optimum effect in preventing or reducing the disease or condition.
  • a compound or composition may be administered to a human orally at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours 8 hours, 9 hours, 10 hours, 11 hours or 12 hours; or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days; or at least 1 week, 2 weeks, 3 weeks or 4 weeks; or at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months; prior to or after the beginning or the completion, of the administration of a chemotherapeutic agent or a combination of chemotherapeutic agents.
  • the subjects that can be treated with the compound or composition and methods described herein can be any subject that produces mRNA that is subject to alternative splicing, e.g., the subject may be a eukaryotic subject, such as a plant or an animal.
  • the subject is a mammal, e.g., human.
  • the subject is a human.
  • the subject is a non-human animal.
  • the subject is a fetus, an embryo, or a child.
  • the subject is a non-human primate such as chimpanzee, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non-human primate such as chimpanzee, and other apes and monkey species
  • farm animals such as cattle, horses, sheep, goats, swine
  • domestic animals such as rabbits, dogs, and cats
  • laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • the subject is prenatal (e.g., a fetus) , a child (e.g., a neonate, an infant, a toddler, a preadolescent) , an adolescent, a pubescent, or an adult (e.g., an early adult, a middle aged adult, a senior citizen) .
  • the human subject can be between about 0 months and about 120 years old, or older.
  • the human subject can be between about 0 and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old.
  • the human subject can be between about 0 and 12 years old; for example, between about 0 and 30 days old; between about 1 month and 12 months old; between about 1 year and 3 years old; between about 4 years and 5 years old; between about 4 years and 12 years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old.
  • the human subject can be between about 13 years and 19 years old; for example, about 13, 14, 15, 16, 17, 18, or 19 years old.
  • the human subject can be between about 20 and about 39 years old; for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old.
  • the human subject can be between about 40 to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old.
  • the human subject can be greater than 59 years old; for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old.
  • the human subjects can include living subjects or deceased subjects.
  • the human subjects can include male subjects and/or female subjects.
  • Gene expression experiments often involve measuring the relative amount of gene expression products, such as mRNA, expressed in two or more experimental conditions. This is because altered levels of a specific sequence of a gene expression product can suggest a changed need for the protein coded for by the gene expression product, perhaps indicating a homeostatic response or a pathological condition.
  • a method can comprise measuring, assaying or obtaining expression levels of one or more genes. In some cases, the method provides a number or a range of numbers, of genes that the expression levels of the genes can be used to diagnose, characterize or categorize a biological sample. In some embodiments, the gene expression data corresponds to data of an expression level of one or more biomarkers that are related to a disease or condition.
  • the number of genes used can be between about 1 and about 500; for example about 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-25, 1-10, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-25, 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, 400-500, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
  • At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 38, 40, 43, 45, 48, 50, 53, 58, 63, 65, 68, 100, 120, 140, 142, 145, 147, 150, 152, 157, 160, 162, 167, 175, 180, 185, 190, 195, 200, 300, 400, 500 or more total genes can be used.
  • the number of genes used can be less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 38, 40, 43, 45, 48, 50, 53, 58, 63, 65, 68, 100, 120, 140, 142, 145, 147, 150, 152, 157, 160, 162, 167, 175, 180, 185, 190, 195, 200, 300, 400, 500, or more.
  • relative gene expression as compared to normal cells and/or tissues of the same organ, can be determined by measuring the relative rates of transcription of RNA, such as by production of corresponding cDNAs and then analyzing the resulting DNA using probes developed from the gene sequences as corresponding to a genetic marker.
  • the levels of cDNA produced by use of reverse transcriptase with the full RNA complement of a cell suspected of being cancerous produces a corresponding amount of cDNA that can then be amplified using polymerase chain reaction, or some other means, such as linear amplification, isothermal amplification, NASB, or rolling circle amplification, to determine the relative levels of resulting cDNA and, thereby, the relative levels of gene expression.
  • Gene expression product levels may include but are not limited to one or more of the following: additional cytological assays, assays for specific proteins or enzyme activities, assays for specific expression products including protein or RNA or specific RNA splice variants, in situ hybridization, whole or partial genome expression analysis, microarray hybridization assays, SAGE, enzyme linked immuno-absorbance assays, mass-spectrometry, immuno-histochemistry, blotting, microarray, RT-PCR, quantitative PCR, sequencing, RNA sequencing, DNA sequencing (e.g., sequencing of cDNA obtained from RNA) ; Next-Gen sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing.
  • Gene expression product levels may be normalized to an internal standard such as total mRNA or the expression level of a particular gene including but not limited to glyceraldehyde 3-phosphate dehydrogenase, or tubulin.
  • Gene expression data generally comprises the measurement of the activity (or the expression) of a plurality of genes, to create a picture of cellular function. Gene expression data can be used, for example, to distinguish between cells that are actively dividing, or to show how the cells react to a particular treatment. Microarray technology can be used to measure the relative activity of previously identified target genes and other expressed sequences. Sequence based techniques, like serial analysis of gene expression (SAGE, SuperSAGE) are also used for assaying, measuring or obtaining gene expression data. SuperSAGE is especially accurate and can measure any active gene, not just a predefined set. In an RNA, mRNA or gene expression profiling microarray, the expression levels of thousands of genes can be simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on gene expression.
  • the expression level of a gene, marker, gene expression product, mRNA, pre-mRNA, or a combination thereof may be determined using northern blotting and employing the sequences as identified herein to develop probes for this purpose.
  • probes may be composed of DNA or RNA or synthetic nucleotides or a combination of these and may advantageously be comprised of a contiguous stretch of nucleotide residues matching, or complementary to, a sequence corresponding to a genetic marker identified in FIG. 4.
  • Such probes will most usefully comprise a contiguous stretch of at least 15-200 residues or more including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, or 200 nucleotides or more.
  • a single probe binds multiple times to the transcriptome of experimental cells, whereas binding of the same probe to a similar amount of transcriptome derived from the genome of control cells of the same organ or tissue results in observably more or less binding, this is indicative of differential expression of a gene, marker, gene expression product, mRNA, or pre-mRNA comprising, or corresponding to, sequences corresponding to a genetic marker from which the probe sequence was derived.
  • gene expression may be determined by microarray analysis using, for example, Affymetrix arrays, cDNA microarrays, oligonucleotide microarrays, spotted microarrays, or other microarray products from Biorad, Agilent, or Eppendorf.
  • Microarrays provide particular advantages because they may contain a large number of genes or alternative splice variants that may be assayed in a single experiment.
  • the microarray device may contain the entire human genome or transcriptome or a substantial fraction thereof allowing a comprehensive evaluation of gene expression patterns, genomic sequence, or alternative splicing. Markers may be found using standard molecular biology and microarray analysis techniques as described in Sam brook Molecular Cloning a Laboratory Manual 2001 and Baldi, P., and Hatfield, W. G., DNA Microarrays and Gene Expression 2002.
  • Microarray analysis generally begins with extracting and purifying nucleic acid from a biological sample, (e.g. a biopsy or fine needle aspirate) using methods known to the art.
  • a biological sample e.g. a biopsy or fine needle aspirate
  • RNA samples with RIN ⁇ 5.0 are typically not used for multi-gene microarray analysis, and may instead be used only for single-gene RT-PCR and/or TaqMan assays.
  • Microarray, RT-PCR and TaqMan assays are standard molecular techniques well known in the relevant art. TaqMan probe-based assays are widely used in real-time PCR including gene expression assays, DNA quantification and SNP genotyping.
  • Ambion WT-expression kit can be used.
  • Ambion WT-expression kit allows amplification of total RNA directly without a separate ribosomal RNA (rRNA) depletion step.
  • rRNA ribosomal RNA
  • WT Expression Kit samples as small as 50 ng of total RNA can be analyzed on Human, Mouse, and Rat Exon and Gene 1.0 ST Arrays.
  • the WT Expression Kit provides a significant increase in sensitivity.
  • a greater number of probe sets detected above background can be obtained at the exon level with the WT Expression Kit as a result of an increased signal-to-noise ratio.
  • Ambion WT-expression kit may be used in combination with additional Affymetrix labeling kit.
  • AmpTec Trinucleotide Nano mRNA Amplification kit (6299-A15) can be used in the subject methods.
  • the TRinucleotide mRNA amplification Nano kit is suitable for a wide range, from 1 ng to 700 ng of input total RNA. According to the amount of input total RNA and the required yields of a RNA, it can be used for 1-round (input>300 ng total RNA) or 2-rounds (minimal input amount 1 ng total RNA) , with a RNA yields in the range of >10 ⁇ g.
  • AmpTec's proprietary TRinucleotide priming technology results in preferential amplification of mRNAs (independent of the universal eukaryotic 3’ -poly (A) -sequence) , combined with selection against rRNAs.
  • This kit can be used in combination with cDNA conversion kit and Affymetrix labeling kit.
  • gene expression levels can be obtained or measured in an individual without first obtaining a sample.
  • gene expression levels may be determined in vivo, that is in the individual.
  • Methods for determining gene expression levels in vivo include imaging techniques such as CAT, MRI; NMR; PET; and optical, fluorescence, or biophotonic imaging of protein or RNA levels using antibodies or molecular beacons. Such methods are described in US 2008/0044824, US 2008/0131892, herein incorporated by reference. Additional methods for in vivo molecular profiling are contemplated to be within the scope of the present invention.
  • RNA transcripts e.g., pre-mRNA or mRNA transcripts or isoforms thereof
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising: (a) contacting a cell with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and (b) determining the amount of the RNA transcript produced by the cell, wherein an alteration in the amount of the RNA transcript in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof relative to the amount of the RNA transcript in the absence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising: (a) contacting a first cell with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, (b) contacting a second cell with a negative control (e.g., a vehicle control, such as PBS or DMSO) ; and (c) determining the amount of the RNA transcript produced by the first cell and the second cell; and (d) comparing the amount of the RNA transcript produced by the first cell to the amount of the RNA transcript expressed by the second cell, wherein an alteration in the amount of the RNA transcript produced by the first cell relative to the amount of the RNA transcript produced by the second cell indicates that a compound of Formula (I) or
  • the contacting of the cell with the compound occurs in cell culture. In other embodiments, the contacting of the cell with the compound occurs in a subject, such as a non-human animal subject.
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the splicing of an RNA transcript comprising: (a) culturing a cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; and (b) determining the amount of the two or more RNA transcripts splice variants produced by the cell, wherein an alteration in the amount of the two or more RNA transcripts in the presence of the compound relative to the amount of the two or more RNA transcripts splice variants in the absence of the compound or the
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the splicing of an RNA transcript comprising: (a) culturing a cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; (b) isolating two or more RNA transcript splice variants from the cell after a certain period of time; and (c) determining the amount of the two or more RNA transcript splice variants produced by the cell, wherein an alteration in the amount of the two or more RNA transcript in the presence of the compound relative to the amount of the two or more RNA transcript splice variants in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the splicing of an RNA transcript comprising (a) culturing a first cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; (b) culturing a second cell in the presence of a negative control (e.g., a vehicle control, such as PBS or DMSO) ; (c) isolating two or more RNA transcript splice variants produced by the first cell and isolating two or more RNA transcript splice variants produced by the second cell; (d) determining the amount of the two or more RNA transcript splice variants produced by the first cell and the second cell; and (e) comparing the amount
  • a negative control
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising: (a) contacting a cell-free system with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and (b) determining the amount of the RNA transcript produced by the cell-free system, wherein an alteration in the amount of the RNA transcript in the presence of the compound relative to the amount of the RNA transcript in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of the RNA transcript.
  • a negative control e.g
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising: (a) contacting a first cell-free system with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, (b) contacting a second cell-free system with a negative control (e.g., a vehicle control, such as PBS or DMSO) ; and (c) determining the amount of the RNA transcript produced by the first cell-free system and the second cell-free system; and (d) comparing the amount of the RNA transcript produced by the first cell-free system to the amount of the RNA transcript expressed by the second cell-free system, wherein an alteration in the amount of the RNA transcript produced by the first cell-free system relative to the amount
  • the cell-free system comprises purely synthetic RNA, synthetic or recombinant (purified) enzymes, and protein factors. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template, synthetic or recombinant (purified) enzymes, and protein factors. In other embodiments, the cell-free system comprises purely synthetic RNA and nuclear extract. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template and nuclear extract. In other embodiments, the cell-free system comprises purely synthetic RNA and whole cell extract. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template and whole cell extract. In some embodiments, the cell-free system additionally comprises regulatory RNAs (e.g., microRNAs) .
  • regulatory RNAs e.g., microRNAs
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the splicing of an RNA transcript comprising: (a) contacting a cell-free system with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; and (b) determining the amount of two or more RNA transcript splice variants produced by the cell-free system, wherein an alteration in the amount of the two or more RNA transcript splice variants in the presence of the compound relative to the amount of the two or more RNA transcript splice variants in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of Formula (I) or a pharmaceutically acceptable salt, solvate
  • a negative control
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the splicing of an RNA transcript comprising: (a) contacting a first cell-free system with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; (b) contacting a second cell-free system with a negative control (e.g., a vehicle control, such as PBS or DMSO) ; and (c) determining the amount of two or more RNA transcript splice variants produced by the first cell-free system and the second cell-free system; and (d) comparing the amount of the two or more RNA transcript splice variants produced by the first cell-free system to the amount of the RNA transcript expressed by the second cell-free system, wherein an alteration
  • the cell-free system comprises purely synthetic RNA, synthetic or recombinant (purified) enzymes, and protein factors. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template, synthetic or recombinant (purified) enzymes, and protein factors. In other embodiments, the cell-free system comprises purely synthetic RNA and nuclear extract. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template and nuclear extract. In other embodiments, the cell-free system comprises purely synthetic RNA and whole cell extract. In other embodiments, the cell-free system comprises RNA transcribed from a synthetic DNA template and whole cell extract. In some embodiments, the cell-free system additionally comprises regulatory RNAs (e.g., microRNAs) .
  • regulatory RNAs e.g., microRNAs
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising: (a) culturing a cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, (b) isolating the RNA transcript from the cell after a certain period of time; and (c) determining the amount of the RNA transcript produced by the cell, wherein an alteration in the amount of the RNA transcript in the presence of the compound relative to the amount of the RNA transcript in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer
  • a negative control
  • RNA transcript e.g., an mRNA transcript
  • a method for determining whether a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of an RNA transcript comprising (a) culturing a first cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, (b) culturing a second cell in the presence of a negative control (e.g., a vehicle control, such as PBS or DMSO) ; (c) isolating the RNA transcript produced by the first cell and isolating the RNA transcript produced by the second cell; (d) determining the amount of the RNA transcript produced by the first cell and the second cell; and (e) comparing the amount of the RNA transcript produced by the first cell to the amount of the RNA transcript produced by the second cell, wherein an alter
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a primary cell from a subject. In some embodiments, the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a primary cell from a subject with a disease.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a primary cell from a subject with a disease associated with an aberrant amount of an RNA transcript for a particular gene.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a primary cell from a subject with a disease associated with an aberrant amount of an isoform of a particular gene.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a fibroblast, an immune cell, or a muscle cell.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a diseased cell.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is from a cell line.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a cell line derived from a subject with a disease.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is from a cell line known to have aberrant RNA transcript levels for a particular gene.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is from a cell line derived from a subject with a disease known to have aberrant RNA transcript levels for a particular gene.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is a diseased cell line.
  • the cell contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is from a cell line derived from a subject with a disease known to have an aberrant amount of an RNA isoform and/or protein isoform of a particular gene.
  • cell lines include 293, 3T3, 4T1, 721, 9L, A2780, A172, A20, A253, A431, A-549, A-673, ALC, B 16, B35, BCP-1, BEAS-2B, bEnd.
  • a dose-response assay comprises: (a) contacting a cell with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; (b) determining the amount of the RNA transcript produced by the cell, wherein an alteration in the amount of the RNA transcript in the presence of the compound relative to the amount of the RNA transcript in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of the RNA transcript; (c) repeating steps (a) and (b) , wherein the only experimental variable changed is the concentration of the compound or a form thereof; and (d) comparing the amount of the RNA transcript produced at the different concentrations
  • a negative control e.g., a vehicle control
  • the dose response assay comprises: (a) culturing a cell in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, (b) isolating the RNA transcript from the cell after a certain period of time; (c) determining the amount of the RNA transcript produced by the cell, wherein an alteration in the amount of the RNA transcript in the presence of the compound relative to the amount of the RNA transcript in the absence of the compound or the presence of a negative control (e.g., a vehicle control such as PBS or DMSO) indicates that a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof modulates the amount of the RNA transcript; (d) repeating steps (a) , (b) , and (c) , wherein the only experimental variable changed is the concentration of the compound or a form thereof; and (e) comparing the
  • the dose-response assay comprises: (a) contacting each well of a microtiter plate containing cells with a different concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof; (b) determining the amount of an RNA transcript produced by cells in each well; and (c) assessing the change of the amount of the RNA transcript at the different concentrations of the compound or form thereof.
  • the cell is contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a negative control for a period of 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours or more.
  • the cell is contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a negative control for a period of 15 minutes to 1 hour, 1 to 2 hours, 2 to 4 hours, 6 to 12 hours, 12 to 18 hours, 12 to 24 hours, 28 to 24 hours, 24 to 48 hours, 48 to 72 hours.
  • the cell is contacted or cultured with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein the concentration is 0.01 ⁇ M, 0.05 ⁇ M, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, or 150 ⁇ M.
  • the cell is contacted or cultured with concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein the concentration is 175 ⁇ M, 200 ⁇ M, 250 ⁇ M, 275 ⁇ M, 300 ⁇ M, 350 ⁇ M, 400 ⁇ M, 450 ⁇ M, 500 ⁇ M, 550 ⁇ M, 600 ⁇ M, 650 ⁇ M, 700 ⁇ M, 750 ⁇ M, 800 ⁇ M, 850 ⁇ M, 900 ⁇ M, 950 ⁇ M or 1 mM.
  • the cell is contacted or cultured with concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein the concentration is 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, or 950 nM.
  • the cell is contacted or cultured with concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a tissue sample is contacted with a concentration of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein the concentration is between 0.01 ⁇ M to 0.1 ⁇ M, 0.1 ⁇ M to 1 ⁇ M, 1 ⁇ M to 50 ⁇ M, 50 ⁇ M to 100 ⁇ M, 100 ⁇ M to 500 ⁇ M, 500 ⁇ M to 1 nM, 1 nM to 10 nM, 10 nM to 50 nM, 50 nM to 100 nM, 100 nM to 500 nM, 500 nM to 1000 nM.
  • the amount of one, two, three or more RNA transcripts is measured using deep sequencing, such as RNASeq, next generation sequencing (NGS) , ION TORRENT TM RNA next generation sequencing, 454 TM pyrosequencing, or Sequencing by Oligo Ligation Detection (SOLID TM ) .
  • deep sequencing such as RNASeq, next generation sequencing (NGS) , ION TORRENT TM RNA next generation sequencing, 454 TM pyrosequencing, or Sequencing by Oligo Ligation Detection (SOLID TM ) .
  • the amount of multiple RNA transcripts is measured using an exon array, such as the human exon array.
  • the amount of one, two, three or more RNA transcripts is determined by RT-PCR.
  • the amount of one, two, three or more RNA transcripts is measured by RT-qPCR. Techniques for conducting these assays are known to one skilled in the art.
  • a statistical analysis or other analysis is performed on data from the assay utilized to measure an RNA transcript.
  • a student t-test statistical analysis is performed on data from the assay utilized to measure an RNA transcript to determine those RNA transcripts that have an alternation in amount in the presence of the compound relative to the amount in the absence of the compound or presence of a negative control.
  • the student t-test value of those RNA transcripts with the alternation is 10%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1%.
  • p value of those RNA transcripts with the alternation is 10%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1%.
  • the student t-test and p values of those RNA transcripts with the alteration are 10%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1%and 10%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1%) , respectively.
  • a further analysis is performed to determine how a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof is changing the amount of an RNA transcript.
  • a further analysis is performed to determine if an alternation in the amount of an RNA transcript in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof relative the amount of the RNA transcript in the absence of the compound or a form thereof, or the presence of a negative control is due to changes in transcription, splicing, and/or stability of the RNA transcript.
  • RNA transcript e.g., the transcription, splicing and/or stability of an RNA transcript.
  • the stability of one or more RNA transcripts is determined by serial analysis of gene expression (SAGE) , differential display analysis (DD) , RNA arbitrarily primer (RAP) -PCR, restriction endonuclease-lytic analysis of differentially expressed sequences (READS) , amplified restriction fragment-length polymorphism (ALFP) , total gene expression analysis (TOGA) , RT-PCR, RT-qPCR, high-density cDNA filter hybridization analysis (HDFCA) , suppression subtractive hybridization (SSH) , differential screening (DS) , cDNA arrays, oligonucleotide chips, or tissue microarrays.
  • the stability of one or more RNA transcripts is determined by Northern blots, RNase protection, or slot blots.
  • the transcription in a cell or tissue sample is inhibited before (e.g., 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours before) or after (e.g., 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after) the cell or the tissue sample is contacted or cultured with an inhibitor of transcription, such as a-amanitin, DRB, flavopiridol, triptolide, or actinomycin-D.
  • an inhibitor of transcription such as a-amanitin, DRB, flavopiridol, triptolide, or actinomycin-D.
  • the transcription in a cell or tissue sample is inhibited with an inhibitor of transcription, such as ⁇ -amanitin, DRB, flavopiridol, triptolide, or actinomycin-D, while the cell or tissue sample is contacted or cultured with a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
  • an inhibitor of transcription such as ⁇ -amanitin, DRB, flavopiridol, triptolide, or actinomycin-D
  • the level of transcription of one or more RNA transcripts is determined by nuclear run-on assay or an in vitro transcription initiation and elongation assay. In some embodiments, the detection of transcription is based on measuring radioactivity or fluorescence. In some embodiments, a PCR-based amplification step is used.
  • the amount of alternatively spliced forms of the RNA transcripts of a particular gene are measured to see if there is an alteration in the amount of one, two or more alternatively spliced forms of the RNA transcripts of the gene.
  • the amount of an isoform encoded by a particular gene is measured to see if there is an alteration in the amount of the isoform.
  • the levels of spliced forms of RNA are quantified by RT-PCR, RT-qPCR, or northern blotting.
  • sequence-specific techniques may be used to detect the levels of an individual splice form.
  • splicing is measured in vitro using nuclear extracts.
  • detection is based on measuring radioactivity or fluorescence.
  • Techniques known to one skilled in the art may be used to measure alterations in the amount of alternatively spliced forms of an RNA transcript of a gene and alterations in the amount of an isoform encoded by a gene.
  • a sample e.g., a biological sample can be taken from a subject and examined to determine whether the subject produces mRNA that is subject to alternative splicing.
  • a biological sample can comprise a plurality of biological samples.
  • the plurality of biological samples can contain two or more biological samples; for examples, about 2-1000, 2-500, 2-250, 2-100, 2-75, 2-50, 2-25, 2-10, 10-1000, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-500, 50-250, 50-100, 50-75, 60-70, 100-1000, 100-500, 100-250, 250-1000, 250-500, 500-1000, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
  • the biological samples can be obtained from a plurality of subjects, giving a plurality of sets of a plurality of samples.
  • the biological samples can be obtained from about 2 to about 1000 subjects, or more; for example, about 2-1000, 2-500, 2-250, 2-100, 2-50, 2-25, 2-20, 2-10, 10-1000, 10-500, 10-250, 10-100, 10-50, 10-25, 10-20, 15-20, 25-1000, 25-500, 25-250, 25-100, 25-50, 50-1000, 50-500, 50-250, 50-100, 100-1000, 100-500, 100-250, 250-1000, 250-500, 500-1000, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 68, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
  • the biological samples can be obtained from human subjects.
  • the biological samples can be obtained from human subjects at different ages.
  • the human subject can be prenatal (e.g., a fetus) , a child (e.g., a neonate, an infant, a toddler, a preadolescent) , an adolescent, a pubescent, or an adult (e.g., an early adult, a middle aged adult, a senior citizen) .
  • the human subject can be between about 0 months and about 120 years old, or older.
  • the human subject can be between about 0 and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old.
  • the human subject can be between about 0 and 12 years old; for example, between about 0 and 30 days old; between about 1 month and 12 months old; between about 1 year and 3 years old; between about 4 years and 5 years old; between about 4 years and 12 years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old.
  • the human subject can be between about 13 years and 19 years old; for example, about 13, 14, 15, 16, 17, 18, or 19 years old.
  • the human subject can be between about 20 and about 39 years old; for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old.
  • the human subject can be between about 40 to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old.
  • the human subject can be greater than 59 years old; for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old.
  • the human subjects can include living subjects or deceased subjects.
  • the human subjects can include male subjects and/or female subjects.
  • Bio samples can be obtained from any suitable source that allows determination of expression levels of genes, e.g., from cells, tissues, bodily fluids or secretions, or a gene expression product derived therefrom (e.g., nucleic acids, such as DNA or RNA; polypeptides, such as protein or protein fragments) .
  • the nature of the biological sample can depend upon the nature of the subject. If a biological sample is from a subject that is a unicellular organism or a multicellular organism with undifferentiated tissue, the biological sample can comprise cells, such as a sample of a cell culture, an excision of the organism, or the entire organism. If a biological sample is from a multicellular organism, the biological sample can be a tissue sample, a fluid sample, or a secretion.
  • tissue is meant to include ensembles of cells that are of a common developmental origin and have similar or identical function.
  • tissue is also meant to encompass organs, which can be a functional grouping and organization of cells that can have different origins.
  • the biological sample can be obtained from any tissue. Suitable tissues from a plant can include, but are not limited to, epidermal tissue such as the outer surface of leaves; vascular tissue such as the xylem and phloem, and ground tissue. Suitable plant tissues can also include leaves, roots, root tips, stems, flowers, seeds, cones, shoots, stobili, pollen, or a portion or combination thereof.
  • Suitable tissues can include connective tissues, muscle tissues, nervous tissues, epithelial tissues or a portion or combination thereof. Suitable tissues can also include all or a portion of a lung, a heart, a blood vessel (e.g., artery, vein, capillary) , a salivary gland, a esophagus, a stomach, a liver, a gallbladder, a pancreas, a colon, a rectum, an anus, a hypothalamus, a pituitary gland, a pineal gland, a thyroid, a parathyroid, an adrenal gland, a kidney, a ureter, a bladder, a urethra, a lymph node, a tonsil, an adenoid, a thymus, a spleen, skin, muscle, a brain, a spinal cord, a nerve, an ovary, a fall
  • a blood vessel e.g., artery, vein, capillary
  • a biological sample from a human or non-human animal can also include a bodily fluid, secretion, or excretion; for example, a biological sample can be a sample of aqueous humour, vitreous humour, bile, blood, blood serum, breast milk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, or a combination thereof.
  • the biological sample can be from healthy tissue, diseased tissue, tissue suspected of being diseased, or a combination thereof.
  • the biological sample is a fluid sample, for example a sample of blood, serum, sputum, urine, semen, or other biological fluid.
  • the sample is a blood sample.
  • the biological sample is a tissue sample, such as a tissue sample taken to determine the presence or absence of disease in the tissue.
  • the sample is a sample of thyroid tissue.
  • the biological samples can be obtained from subjects in different stages of disease progression or different conditions. Different stages of disease progression or different conditions can include healthy, at the onset of primary symptom, at the onset of secondary symptom, at the onset of tertiary symptom, during the course of primary symptom, during the course of secondary symptom, during the course of tertiary symptom, at the end of the primary symptom, at the end of the secondary symptom, at the end of tertiary symptom, after the end of the primary symptom, after the end of the secondary symptom, after the end of the tertiary symptom, or a combination thereof.
  • Different stages of disease progression can be a period of time after being diagnosed or suspected to have a disease; for example, at least about, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 years after being diagnosed or suspected to have a disease.
  • Different stages of disease progression or different conditions can include before, during or after an action or state; for example, treatment with drugs, treatment with a surgery, treatment with a procedure, performance of a standard of care procedure, resting, sleeping, eating, fasting, walking, running, performing a cognitive task, sexual activity, thinking, jumping, urinating, relaxing, being immobilized, being emotionally traumatized, being shock, and the like.
  • the methods of the present disclosure provide for analysis of a biological sample from a subject or a set of subjects.
  • the subject (s) may be, e.g., any animal (e.g., a mammal) , including but not limited to humans, non-human primates, rodents, dogs, cats, pigs, fish, and the like.
  • the present methods and compositions can apply to biological samples from humans, as described herein.
  • a biological sample can be obtained by methods known in the art such as the biopsy methods provided herein, swabbing, scraping, phlebotomy, or any other suitable method.
  • the biological sample can be obtained, stored, or transported using components of a kit of the present disclosure.
  • multiple biological samples such as multiple thyroid samples, can be obtained for analysis, characterization, or diagnosis according to the methods of the present disclosure.
  • multiple biological samples such as one or more samples from one tissue type (e.g., thyroid) and one or more samples from another tissue type (e.g., buccal) can be obtained for diagnosis or characterization by the methods of the present disclosure.
  • multiple samples such as one or more samples from one tissue type (e.g., thyroid) and one or more samples from another tissue (e.g., buccal) can be obtained at the same or different times.
  • the samples obtained at different times are stored and/or analyzed by different methods.
  • a sample can be obtained and analyzed by cytological analysis (e.g., using routine staining) .
  • a further sample can be obtained from a subject based on the results of a cytological analysis.
  • the diagnosis of cancer or other condition can include an examination of a subject by a physician, nurse or other medical professional.
  • the examination can be part of a routine examination, or the examination can be due to a specific complaint including, but not limited to, one of the following: pain, illness, anticipation of illness, presence of a suspicious lump or mass, a disease, or a condition.
  • the subject may or may not be aware of the disease or condition.
  • the medical professional can obtain a biological sample for testing. In some cases, the medical professional can refer the subject to a testing center or laboratory for submission of the biological sample.
  • the methods of obtaining provided herein include methods of biopsy including fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In some cases, the methods and compositions provided herein are applied to data only from biological samples obtained by FNA.
  • the methods and compositions provided herein are applied to data only from biological samples obtained by FNA or surgical biopsy. In some cases, the methods and compositions provided herein are applied to data only from biological samples obtained by surgical biopsy.
  • a biological sample can be obtained by non-invasive methods, such methods including, but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.
  • the biological sample can be obtained by an invasive procedure, such procedures including, but not limited to: biopsy, alveolar or pulmonary lavage, needle aspiration, or phlebotomy.
  • the method of biopsy can further include incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, or skin biopsy.
  • the method of needle aspiration can further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy.
  • Multiple biological samples can be obtained by the methods herein to ensure a sufficient amount of biological material.
  • Methods of obtaining suitable samples of thyroid are known in the art and are further described in the ATA Guidelines for thyroid nodule management (Cooper et al. thyroid Vol. 16 No. 2 2006) , herein incorporated by reference in its entirety.
  • Generic methods for obtaining biological samples are also known in the art and further described in for example Ramzy, Wheat Clinical Cytopathology and Aspiration Biopsy 2001 which is herein incorporated by reference in its entirety.
  • the biological sample can be a fine needle aspirate of a thyroid nodule or a suspected thyroid tumor.
  • the fine needle aspirate sampling procedure can be guided by the use of an ultrasound, X-ray, or other imaging device.
  • the subject can be referred to a specialist such as an oncologist, surgeon, or endocrinologist for further diagnosis.
  • the specialist can likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample.
  • the biological sample can be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.
  • the medical professional can indicate the appropriate test or assay to perform on the sample, or the molecular profiling business of the present disclosure can consult on which assays or tests are most appropriately indicated.
  • the molecular profiling business can bill the individual or medical or insurance provider thereof for consulting work, for sample acquisition and or storage, for materials, or for all products and services rendered.
  • kits can contain a means for obtaining said sample as described herein, a means for storing the sample for inspection, and instructions for proper use of the kit.
  • molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.
  • a biological sample suitable for use by the molecular profiling business can be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, and/or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.
  • the biological sample can include, but is not limited to, tissue, cells, and/or biological material from cells or derived from cells of an individual.
  • the sample can be a heterogeneous or homogeneous population of cells or tissues.
  • the biological sample can be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.
  • kits can be provided containing materials for obtaining, storing, and/or shipping biological samples.
  • the kit can contain, for example, materials and/or instruments for the collection of the biological sample (e.g., sterile swabs, sterile cotton, disinfectant, needles, syringes, scalpels, anesthetic swabs, knives, curette blade, liquid nitrogen, etc. ) .
  • the kit can contain, for example, materials and/or instruments for the storage and/or preservation of biological samples (e.g., containers; materials for temperature control such as ice, ice packs, cold packs, dry ice, liquid nitrogen; chemical preservatives or buffers such as formaldehyde, formalin, paraformaldehyde, glutaraldehyde, alcohols such as ethanol or methanol, acetone, acetic acid, HOPE fixative (Hepes-glutamic acid buffer-mediated organic solvent protection effect) , heparin, saline, phosphate buffered saline, TAPS, bicine, Tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cadodylate, SSC, MES, phosphate buffer; protease inhibitors such as aprotinin, bestatin, calpain inhibitor I and II, chymostatin, E-64, leupeptin, alpha-2-macroglobulin
  • the kit can contain instructions for use.
  • the kit can be provided as, or contain, a suitable container for shipping.
  • the shipping container can be an insulated container.
  • the shipping container can be self-addressed to a collection agent (e.g., laboratory, medical center, genetic testing company, etc. ) .
  • the kit can be provided to a subject for home use or use by a medical professional. Alternatively, the kit can be provided directly to a medical professional.
  • One or more biological samples can be obtained from a given subject. In some cases, between about 1 and about 50 biological samples are obtained from the given subject; for example, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-7, 1-5, 5-50, 5-40, 5-30, 5-25, 5-15, 5-10, 10-50, 10-40, 10-25, 10-20, 25-50, 25-40, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 biological samples can be obtained from the given subject.
  • Multiple biological samples from the given subject can be obtained from the same source (e.g., the same tissue) , e.g., multiple blood samples, or multiple tissue samples, or from multiple sources (e.g., multiple tissues) .
  • Multiple biological samples from the given subject can be obtained at the same time or at different times.
  • Multiple biological samples from the given subject can be obtained at the same condition or different condition.
  • Multiple biological samples from the given subject can be obtained at the same disease progression or different disease progression of the subject. If multiple biological samples are collected from the same source (e.g., the same tissue) from the particular subject, the samples can be combined into a single sample. Combining samples in this way can ensure that enough material is obtained for testing and/or analysis.
  • Reactions can be purified or analyzed according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance (NMR) spectroscopy (e.g., 1 H or 13 C) , infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible) , mass spectrometry (MS) , or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) .
  • NMR nuclear magnetic resonance
  • IR infrared
  • spectrophotometry e.g., UV-visible
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the aqueous phase was extracted with ethyl acetate (10.0 mL *3) .
  • the combined organic phase was concentrated in vacuum.
  • 7-Chloro-2- ( (2, 2, 6, 6-tetramethylpiperidin-4-yl) oxy) -5H-isochromeno [3, 4-d] thiazole (C5, 400 mg, 51.6%yield) was obtained as brown solid.
  • the mixture was stirred at 110 °C for 1 hr.
  • the mixture was cooled to 20 °C and poured into water (10.0 mL) .
  • the aqueous phase was extracted with ethyl acetate (10.0 mL *3) .
  • the combined organic phase was washed with brine (10.0 mL *1) , dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuum.
  • the mixture was stirred at 110 °C for 1 hr.
  • the mixture was cooled to 20 °C and poured into aq. NH 4 Cl (20.0 mL) .
  • the aqueous phase was extracted with ethyl acetate (10.0 mL *3) .
  • the combined organic phase was washed with brine (10.0 mL *1) , dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuum.
  • TBAB 205 mg, 637 ⁇ mol, 0.50 eq
  • XPhos Pd G3 107 mg, 127 ⁇ mol, 0.10 eq
  • the mixture was stirred at 120 °C for 2 hrs under N 2 .
  • the reaction mixture was filtered and concentrated under reduced pressure to give a residue.
  • N- (tert-butyl) -1- (7-chloro-5H-isochromeno [3, 4-d] thiazol-2-yl) pyrrolidin-3-amine (C40, 100 mg, 274 ⁇ mol, 1.00 eq) in DMF (1.50 mL) and H 2 O (300 ⁇ L) was added 4-pyrazoleboronic acid pinacol ester (79.9 mg, 412 ⁇ mol, 1.50 eq) , K 3 PO 4 (174 mg, 824 ⁇ mol, 3.00 eq) , TBAB (101 mg, 274 ⁇ mol, 1.00 eq) and dichloropalladium; dicyclohexyl (cyclopentyl) phosphane; iron (41.5 mg, 54.9 ⁇ mol, 0.20 eq) at 20 °C.

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Abstract

L'invention concerne des composés et des compositions associées qui modulent l'épissage d'acide nucléique, ainsi que des procédés d'utilisation desdits composés pour moduler l'épissage et traiter des maladies et affections.
PCT/CN2024/110654 2023-08-15 2024-08-08 Composés et procédés de modulation de l'épissage de l'arn Pending WO2025036241A1 (fr)

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CNPCT/CN2023/113103 2023-08-15

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015049629A1 (fr) * 2013-10-01 2015-04-09 Piramal Enterprises Limited Composés d'imidazoquinoline à utiliser en tant qu'inhibiteurs de bromodomaine
WO2016186453A1 (fr) * 2015-05-20 2016-11-24 Kainos Medicine, Inc. Dérivés de quinoléine à utiliser en tant qu'inhibiteurs de bromodomaine
US20190218193A1 (en) * 2016-09-02 2019-07-18 Bristol-Myers Squibb Company Substituted tricyclic heterocyclic compounds

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015049629A1 (fr) * 2013-10-01 2015-04-09 Piramal Enterprises Limited Composés d'imidazoquinoline à utiliser en tant qu'inhibiteurs de bromodomaine
WO2016186453A1 (fr) * 2015-05-20 2016-11-24 Kainos Medicine, Inc. Dérivés de quinoléine à utiliser en tant qu'inhibiteurs de bromodomaine
US20190218193A1 (en) * 2016-09-02 2019-07-18 Bristol-Myers Squibb Company Substituted tricyclic heterocyclic compounds

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