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WO2016022839A1 - Modulateurs d'ire1 - Google Patents

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Publication number
WO2016022839A1
WO2016022839A1 PCT/US2015/044073 US2015044073W WO2016022839A1 WO 2016022839 A1 WO2016022839 A1 WO 2016022839A1 US 2015044073 W US2015044073 W US 2015044073W WO 2016022839 A1 WO2016022839 A1 WO 2016022839A1
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unsubstituted
substituted
independently
compound
disease
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Kevan M. Shokat
Aaron S. MENDEZ
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • ER-resident unfolded protein sensors continuously monitor the folding status in the ER lumen.
  • the UPR is induced when the protein folding capacity of the ER is surpassed, triggering the activation of three transmembrane sensors/signal transducers, IRE1 (inositol-requiring enzyme 1), PERK (protein kinase RNA (PKR)-like ER kinase), and ATF6 (activating transcription factor-6) 1 .
  • IRE1 inositol-requiring enzyme 1
  • PERK protein kinase RNA (PKR)-like ER kinase)
  • ATF6 activating transcription factor-6 1 .
  • Two of these sensors, IRE1 and PERK are protein kinases that are amenable to modulation by small molecule ATP- mimetics.
  • IREl is the most conserved of these proteins. It contains an ER-lumenal sensor domain that is activated by binding directly to unfolded polypeptides 2 ' 3 . As a result, IREl oligomerizes, activating its cytosolic kinase and endoribonuclease domains 4 ' 5 . IREl 's RNase domain initiates a non-conventional splicing reaction that results in the excision of an intron from the mRNA encoding the transcription factor XBP1 6"8 . XBP1 produced from the spliced mRNA drives transcription of UPR target genes to remedy ER stress.
  • PERK activation is presumably also driven through interactions of its lumenal domain with unfolded polypeptides in the ER 3 .
  • Active PERK phosphorylates the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2a) 9 , leading to trapping eIF2a in its GDP-bound state, which blocks eIF2a recycling 10 .
  • eIF2a eukaryotic translation initiation factor 2
  • ATF4 complements XBP1 in driving transcription of appropriate UPR target genes 11 .
  • R 1 is halogen, -CN, -NO, -N0 2 , -NR 3 R 4 , -OR 5 , -COOR 5 , -SR 5 , -COR 5 , , -CX 3 , -NHNH 2 ,
  • R 2 is
  • R 3 , R 4 , and R 5 are independently hydrogen, R 2 -substituted or unsubstituted C1-C5 alkyl, or R 2 -substituted or unsubstituted 2 to 5 membered heteroalkyl.
  • the symbol X is independently halogen.
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound described herein, or a pharmaceutically acceptable salt thereof.
  • FIGS. 1A-1D Design and characterization of IPA activators.
  • FIG. 1 A The core scaffold of APY29 (aminopyrazole pyrimidine-base indicated).
  • FIG. IB Structure-activity analysis of activating compounds. Compounds were assayed at 1 ⁇ in a RNA cleavage assay containing IREla-KR43 (200 nM) and 5' [ 32 P]-labeled RNA substrate HP21 (see Examples). IP Ax is methylated at the N[l] position in the pyrazole ring indicated as shown.
  • FIG. 1C The effects of activating compounds (20 ⁇ ) as a function of IREla-KR43 concentration.
  • APY24 contains the aminopyrozole pyrimidine-base, which is similar to APY29 (FIG. 7)
  • FIGS. 2A-2E IPA activates the IRE1 branch of the UPR in HEK293T cells.
  • FIG.2 A HEK293T cells were treated with increasing concentrations of IPA as a function of time. Tunicamycin ("Tm", 2 ⁇ g/ml) was used as positive control to induce ER stress. The resulting XBPl mRNA spliced products detected by RT-PCR ("u”: unspliced and "s”: spliced are indicated. Control cells were treated with DMSO only. The asterisk identifies a hybrid amplicon resulting from spliced and unspliced XBPl mRNA.
  • FIG.2 D IRE1-GFP foci formation in T-REx293 cells. IREl-GFP was visualized by confocal microscopy.
  • FIGS. 3A-3F Effects of IPA on the ATF6 and PERK branches of the UPR.
  • FIG. 3 A The ATF6 transcriptional target mRNAs HERPUDl ("HERPl") and DERLIN3 (“DERL3") were observed by RT-PCR in HEK293T cells treated with IPA at 1 ⁇ . GAPDH served as a loading control.
  • FIG. 3B The ATF6 transcriptional target mRNAs HERPUDl (“HERPl”) and DERLIN3 (“DERL3”) were observed by RT-PCR in HEK293T cells treated with IPA at 4 ⁇ . GAPDH served as a loading control.
  • FIG. 3C Phosphorylation of PERK and eIF2a in
  • FIG. 3D HEK293T cells were treated for 4 h with IPA (1 ⁇ ), IP Ax (1 ⁇ ), or Tm (2 ⁇ g/ml). PERK shift and eIF2a phosphorylation were detected as in C. (FIG.
  • Wild-type (“WT”), IreV 1' , and Perk 7" mouse embryonic fibroblast (“MEFs”) cells were treated with IPA (1 ⁇ ) or Tm (5 ⁇ g/ml) for 4 h. Induction oiXBPl mR A splicing by IPA was observed by RT-PCR.
  • FIG. 3F Cells were treated as above. PERK gel mobility shift and eIF2a phosphorylation of were detected by immunoblotting.
  • FIGS. 4A-4B In vitro effects of IPA on GST-PERK kinase activity.
  • FIG. 4B HEK293T cells were pre-treated with IPA for 30 min. Cells were then either co-incubated with IPA and Tm (2 ⁇ g/ml) for an additional 4 h (lanes 4, 5, and 6) or washed (IPA "wash-out”) and treated with Tm alone (lanes 7, 8, and 9).
  • FIGS. 5A-5E Inhibition of IPA-mediated PERK activation by GSK2606414.
  • GSK (1 ⁇ ) was used to also inhibit PERK branch activation by thapsigargin ("Tg"; 100 nM; lane 3).
  • Tg thapsigargin
  • HEK293T cells were incubated with [ 35 S] methionine to monitor protein translation upon addition of IPA (1 ⁇ ) or a combination of IPA (1 ⁇ ) and GSK (1 ⁇ ). The UPR was induced with Tg (100 nM) or DMSO as indicated.
  • FIG. 5D Cotreatment of HEK293T cells with IPA+GSK. HEK293T cell viability was measured as a dose-response of IPA in combination with 1 ⁇ of GSK (pink circles). The presence of 1 ⁇ GSK shifted the IPA LD 50 from 0.82 ⁇ to 6.21 ⁇ .
  • FIG. 8 AD60 inhibition of .XBW-luciferase-splicing reporter activation in
  • FIG. 9 Effects of GSK on HEK293T cell viability. GSK at the indicated concentrations was incubated with HEK293T cells for 24 h.
  • IREl 's kinase domain is in its active conformational state, characterized by the inward positioning of the aC helix and the DFG-loop in the kinase active site (DFG-in conformation) 13"15 .
  • ATP- mimetic ligands that trap IREl 's kinase domain in the inactive, DFG-out conformation act as inhibitors, rather than activators, of IRE1 oligomerization and signaling via its RNase domain 15 .
  • IRE1 is unique in that it is possible to monitor the functional consequences of conformational changes in the kinase domain induced by ligand occupancy of the ATP-binding site without concerns of losing the kinase activity 14 ' .
  • the model depicting IRE1 's kinase domain as a switch that becomes trapped in two states (DFG-in and DFG-out) depending on the ligand bound to its active site is an over- simplification. Different ligands yield different plateaus of maximal oligomerization and RNase activation, even when saturating the active site.
  • IPA a novel small molecule
  • IPA activates PERK only at low concentrations typically below 2 ⁇ .
  • PERK activation results from ligand induced conformational changes that drive PERK hyperphosphorylation resulting in PERK pathway activation.
  • the unfolded protein response responds to an accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER) to adjust the protein folding capacity of the organelle according to need.
  • ER endoplasmic reticulum
  • Two ER stress sensors, IREl and PERK contain kinase domains.
  • IREl also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction.
  • IPA for IREl and PERK activator
  • IPA also activates PERK at low concentrations, while it inhibits it at higher concentrations.
  • Treatment of cells with IPA in combination with other UPR modulators allowed us to elicit signaling through different UPR branches.
  • the ability to pharmacologically dial the outputs of IREl and PERK provides a valuable new tool for exploring the therapeutic value of ER stress modulators in models of human diseases.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., Ci-Cio means one to ten carbons).
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec -butyl,
  • an unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3- butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-).
  • An alkyl moiety may be an alkenyl moiety.
  • An alkyl moiety may be an alkynyl moiety.
  • An alkyl moiety may be fully saturated.
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited
  • alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched non-cyclic 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 quaternized.
  • the heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • a heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • heteroalkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy,
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(0)R', -C(0)NR, -NR'R", -OR', -SR', and/or -S0 2 R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive.
  • heteroalkyl should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.
  • cycloalkyl and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, non-aromatic cyclic versions of “alkyl” and
  • heteroalkyl respectively, wherein the carbons making up the ring or rings do not necessarily need to be bonded to a hydrogen due to all carbon valencies participating in bonds with non- hydrogen atoms. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3 -cyclohexenyl, cycloheptyl, 3-hydroxy-cyclobut-3-enyl-l,2, dione, lH-l,2,4-triazolyl-5(4H)- one, 4H-l,2,4-triazolyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3 - yl, 1-piperazinyl, 2-piperazinyl, and the like.
  • a heterocycloalkyl moiety may include one ring heteroatom (e.g., O, N, S, Si, or P).
  • a heterocycloalkyl moiety may include two optionally different ring heteroatoms (e.g., O, N, S, Si, or P).
  • a heterocycloalkyl moiety may include three optionally different ring heteroatoms (e.g., O, N, S, Si, or P).
  • a heterocycloalkyl moiety may include four optionally different ring heteroatoms (e.g., O, N, S, Si, or P).
  • a heterocycloalkyl moiety may include five optionally different ring heteroatoms (e.g., O, N, S, Si, or P).
  • a heterocycloalkyl moiety may include up to 8 optionally different ring heteroatoms (e.g., O, N, S, Si, or P).
  • halo or halogen
  • haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C4)alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • acyl means, unless otherwise stated, -C(0)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
  • a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring).
  • a 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,5- fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non- limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4- biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indolyl, 1-isoquino
  • aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
  • Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl,
  • a heteroaryl moiety may include one ring heteroatom (e.g., O, N, or S).
  • a heteroaryl moiety may include two optionally different ring heteroatoms (e.g., O, N, or S).
  • a heteroaryl moiety may include three optionally different ring heteroatoms (e.g., O, N, or S).
  • a heteroaryl moiety may include four optionally different ring heteroatoms (e.g., O, N, or S).
  • a heteroaryl moiety may include five optionally different ring heteroatoms (e.g., O, N, or S).
  • An aryl moiety may have a single ring.
  • An aryl moiety may have two optionally different rings.
  • An aryl moiety may have three optionally different rings.
  • An aryl moiety may have four optionally different rings.
  • a heteroaryl moiety may have one ring.
  • a heteroaryl moiety may have two optionally different rings.
  • a heteroaryl moiety may have three optionally different rings.
  • a heteroaryl moiety may have four optionally different rings.
  • a heteroaryl moiety may have five optionally different rings.
  • a fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl.
  • heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.
  • heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl.
  • Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl- cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
  • alkylsulfonyl means a moiety having the formula -S(02)- ', where R' is a substituted or unsubstituted alkyl group as defined above. R may have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").
  • alkyl and heteroalkyl radicals including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
  • -NR'C (0)NR"NR"R"", -CN, -N0 2 , in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical.
  • R, R, R", R'", and R"" each preferably
  • each of the R groups is independently selected as are each R, R", R'", and R"" group when more than one of these groups is present.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring.
  • -NRR includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like).
  • substituents for the aryl and heteroaryl groups are varied and are selected from, for
  • R, R", R'", and R" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • R groups are independently selected as are each R', R", R", and R"" groups when more than one of these groups is
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl 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.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(0)-(CRR') q -U-, wherein T and U are
  • q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -0-, -NR-, -S-, -S(O) -, -S(0) 2 -, -S(0) 2 NR-, or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the
  • the substituents R, R, R", and R m are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • a substituent group as used herein, may be a group selected from the following moieties:
  • NHC (O)H, -NHC(0)-OH, -NHOH, -OCF 3 , -OCHF 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
  • heterocycloalkyl unsubstituted aryl, unsubstituted heteroaryl, and
  • heterocycloalkyl unsubstituted aryl, unsubstituted heteroaryl.
  • a "size-limited substituent” or " size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a "substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 2 o alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C 8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C1 0 aryl, and each substituted or unsubstituted heteroary
  • a "lower substituent” or " lower substituent group,” as used herein, means a group selected from all of the substituents described above for a "substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-Cs alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C1 0 aryl, and each substituted or unsubstituted heteroaryl is a substituted or un
  • each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group.
  • each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 8 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -Ci 0 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted Q-Cg alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted Ci-C 8 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • the compound is a chemical species set forth in the Examples section, figures, or tables below.
  • salts are 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.
  • pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)).
  • 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.
  • Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • the compounds of the present invention may exist as salts, such as with pharmaceutically acceptable acids.
  • the present invention includes such salts.
  • salts examples include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid.
  • These salts may be prepared by methods known to those skilled in the art.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • agents e.g. compounds, drugs, therapeutic agents
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under select physiological conditions to provide the final agents (e.g. compounds, drugs, therapeutic agents). Additionally, prodrugs can be converted to agents (e.g. compounds, drugs, therapeutic agents) by chemical or biochemical methods in an ex vivo environment.
  • Prodrugs described herein include compounds that readily undergo chemical changes under select physiological conditions to provide agents (e.g. compounds, drugs, therapeutic agents) to a biological system (e.g. in a subject, in a cell, in the extracellular space near a cell).
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • salt refers to acid or base salts of the compounds used in the methods of the present invention.
  • acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute
  • stereochemistry as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present invention.
  • the compounds of the present invention do not include those which are known in art to be too unstable to synthesize and/or isolate.
  • the present invention is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefmic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • the term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. It will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine- 125 ( I), or carbon-14 ( C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
  • a group such as an alkyl or heteroaryl group
  • the group may contain one or more unsubstituted C1-C2 0 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
  • R substituent the group may be referred to as "R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.
  • treating refers to any indicia of success in the treatment or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. For example, certain methods herein treat diseases associated with Irel activity.
  • Certain methods described herein may treat diseases associated with Irel activity by inhibiting Irel activity. Certain methods described herein may treat diseases associated with Irel activity by increasing Irel activity. Certain methods described herein may treat diseases associated with PERK activity by inhibiting PERK activity. Certain methods described herein may treat diseases associated with PERK activity by increasing PERK activity. For example, certain methods herein treat cancer. For example certain methods herein treat cancer by decreasing a symptom of cancer. Symptoms of cancer would be known or may be determined by a person of ordinary skill in the art. For example, certain methods herein treat a neurodegenerative disease. For example certain methods herein treat a neurodegenerative disease by decreasing a symptom of the neurodegenerative disease. Symptoms of a
  • neurodegenerative disease would be known or may be determined by a person of ordinary skill in the art.
  • the term “treating” and conjugations thereof, include prevention of an injury, pathology, condition, or disease.
  • an “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce protein function, reduce one or more symptoms of a disease or condition).
  • An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a "therapeutically effective amount.”
  • a “reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • a “prophylactically effective amount" of a drug or prodrug is an amount of a drug or prodrug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman,
  • a disease associated with Irel activity may be treated with an agent (e.g. compound as described herein) effective for increasing the level of Irel activity.
  • a disease associated with PERK activity may be treated with an agent (e.g. compound as described herein) effective for reducing the level of PERK activity.
  • Control or "control experiment” or “standard control” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects.
  • Contacting is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including
  • contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme.
  • inhibition means negatively affecting (e.g. decreasing) the level of activity or function of the protein relative to the level of activity or function of the protein in the absence of the inhibitor.
  • inhibition refers to reduction of a disease or symptoms of disease.
  • inhibition may include, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating,
  • activation means positively affecting (e.g.
  • activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease.
  • activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up- regulating signal transduction or enzymatic activity or the amount of a protein.
  • modulator refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule.
  • a modulator is an anticancer agent. In embodiments, a modulator is an anti-neurodegenerative disease agent. In embodiments, a modulator is a modulator of the unfolded protein response. In embodiments, a modulator is an Irel activity inhibitor. In embodiments, a modulator is an Irel activity activator. In embodiments, a modulator is a PERK activity inhibitor. In embodiments, a modulator is a PERK activity activator.
  • Anti-cancer agent or "anti-cancer drug” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells.
  • an anti-cancer agent is a chemotherapeutic.
  • an anticancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, anti-androgens (e.g., Casodex, Flutamide, MDV3100, or AR -509), MEK (e.g.
  • MEK1, MEK2, or MEKl and MEK2 inhibitors e.g. XL518, CI-1040, PD035901, selumetinib/ AZD6244, GSKl 120212/ trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766
  • alkylating agents e.g.,
  • cyclophosphamide ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine,
  • cyclophosphamide chlorambucil, meiphalan
  • ethylenimine and methylmelamines e.g., hexamethlymelamine, thiotepa
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g., carmustine, lomusitne, semustine, streptozocin
  • triazenes decarbazine
  • anti-metabolites e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), pyrimidine analogs (e.g., fluorouracil, floxouridine,
  • Cytarabine purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP 16), 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
  • anthracenedione e.g., mitoxantrone
  • substituted urea e.g., hydroxyurea
  • methyl hydrazine derivative e.g., procarbazine
  • adrenocortical suppressant e.g., mitotane, aminoglutethimide
  • epipodophyllotoxins e.g., etoposide
  • antibiotics e.g., daunorubicin, doxorubicin, bleomycin
  • enzymes e.g., L-asparaginase
  • inhibitors of mitogen-activated protein kinase signaling e.g.
  • BCR/ABL antagonists beta lactam derivatives; bFGF inhibitor; bicalutamide; camptothecin derivatives; casein kinase inhibitors (ICOS); clomifene analogues; cytarabine dacliximab;
  • dexamethasone dexamethasone; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; finasteride; fludarabine; fluorodaunorunicin hydrochloride; gadolinium texaphyrin; gallium nitrate; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
  • leuprolide+estrogen+progesterone leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitor; mifepristone; mismatched double stranded RNA; monoclonal antibody,; mycobacterial cell wall extract; nitric oxide modulators; oxaliplatin; panomifene; pentrozole; phosphatase inhibitors; plasminogen activator inhibitor; platinum complex; platinum compounds; prednisone; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
  • ribozymes signal transduction inhibitors; signal transduction modulators; single chain antigen- binding protein; stem cell inhibitor; stem-cell division inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroid stimulating hormone; translation inhibitors; tyrosine kinase inhibitors; urokinase receptor antagonists;
  • steroids e.g., dexamethasone
  • finasteride aromatase inhibitors
  • gonadotropin-releasing hormone agonists GnRH
  • adrenocorticosteroids e.g., prednisone
  • progestins e.g., hydroxyprogesterone caproate, megestrol acetate
  • estrogens e.g., diethlystilbestrol, ethinyl estradiol
  • antiestrogen e.g., tamoxifen
  • androgens e.g., testosterone propionate, fluoxymesterone
  • antiandrogen e.g., flutamide
  • immunostimulants e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.
  • monoclonal antibodies e.g., anti-CD20, anti-HER2, anti-CD52, anti- HLA-DR, and anti-VEGF monoclonal antibodies
  • immunotoxins e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.
  • radioimmunotherapy e.g., anti-CD20 monoclonal antibody conjugated
  • gefitinib IressaTM
  • erlotinib TarcevaTM
  • cetuximab ErbituxTM
  • lapatinib TykerbTM
  • panitumumab VectibixTM
  • vandetanib CaprelsaTM
  • afatinib/BIBW2992 CI- 1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY- 380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib,
  • tomaymycin carboplatin
  • CC-1065 and CC-1065 analogs including amino-CBIs, nitrogen mustards (such as chlorambucil and melphalan), dolastatin and dolastatin analogs (including auristatins: eg. monomethyl auristatin E), anthracycline antibiotics (such as doxorubicin, daunorubicin, etc.), duocarmycins and duocarmycin analogs, enediynes (such as neocarzinostatin and calicheamicins), leptomycin derivaties, maytansinoids and maytansinoid analogs (e.g.
  • “Chemotherapeutic” or “chemotherapeutic agent” is used in accordance with its plain ordinary meaning and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit the growth or proliferation of cells.
  • Patient or “subject in need thereof or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound or pharmaceutical composition or by a method, as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • a subject is human.
  • Disease or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein.
  • the disease is a disease having the symptom of cell
  • the disease is a disease having the symptom of an aberrant level of Ire 1 activity. In some embodiments, the disease is a disease having the symptom of an aberrant level of PERK activity. In some embodiments, the disease is a cancer. In some embodiments, the disease is a neurodegenerative disease.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas), Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.
  • the disease is multiple myeloma.
  • the disease is breast cancer.
  • the disease is triple negative breast cancer.
  • the disease is a neurode
  • Alzheimer's disease Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado- Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff s disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to
  • the disease is cystic fibrosis, retinitis pigmentosa, or diabetes.
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemia, carcinomas and sarcomas.
  • Exemplary cancers that may be treated with a compound or method provided herein include cancer of the prostate, thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, Medulloblastoma, colorectal cancer, pancreatic cancer.
  • Additional examples may include, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.
  • leukemia refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia,
  • hemocytoblastic leukemia histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia,
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sar
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
  • neurodegenerative disease refers to a disease or condition in which the function of a subject's nervous system becomes impaired. Examples of
  • neurodegenerative diseases that may be treated with a compound, pharmaceutical composition, or method described herein include Alexander's disease, Alper's disease, Alzheimer's disease,
  • Amyotrophic lateral sclerosis also known as Spielmeyer-
  • Vogt-Sjogren-Batten disease Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, Lewy body dementia, Machado- Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff s disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia,
  • Schizophrenia Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, or Tabes dorsalis.
  • inflammatory disease refers to a disease or condition characterized by aberrant inflammation (e.g. an increased level of inflammation compared to a control such as a healthy person not suffering from a disease).
  • inflammatory diseases include traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's
  • vasculitis vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.
  • signaling pathway refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.
  • extra-cellular components e.g. proteins, nucleic acids, small molecules, ions, lipids
  • aberrant refers to different from normal. When used to describe enzymatic activity, aberrant refers to activity that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non- disease-associated amount (e.g. by administering a compound or using a method as described herein), results in reduction of the disease or one or more disease symptoms.
  • "Nucleic acid” or "oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together.
  • nucleic acid includes single-, double-, or multiple-stranded DNA, RNA and analogs (derivatives) thereof. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids.
  • a particular nucleic acid sequence also encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate
  • polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 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% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 10 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well- known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence with a higher affinity, e.g. , under more stringent conditions, than to other nucleotide sequences (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.
  • amino acids are commonly found in proteins. Those amino acids can be grouped into nine classes or groups based on the chemical properties of their side chains. Substitution of one amino acid residue for another within the same class or group is referred to herein as a "conservative" substitution. Conservative amino acid substitutions can frequently be made in a protein without significantly altering the conformation or function of the protein. Substitution of one amino acid residue for another from a different class or group is referred to herein as a "non- conservative" substitution. In contrast, non-conservative amino acid substitutions tend to modify conformation and function of a protein.
  • the conservative amino acid substitution comprises substituting any of glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L) for any other of these aliphatic amino acids; serine (S) for threonine (T) and vice versa; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; lysine (K) for arginine (R) and vice versa; phenylalanine (F), tyrosine (Y) and tryptophan (W) for any other of these aromatic amino acids; and methionine (M) for cysteine (C) and vice versa.
  • G glycine
  • A alanine
  • I isoleucine
  • V valine
  • L leucine
  • substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein.
  • G glycine
  • A alanine
  • V valine
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine.
  • Lysine (K) and arginine (R) are frequently
  • Polypeptide “peptide,” and “protein” are used herein interchangeably and mean any pepti de-linked chain of amino acids, regardless of length or post-translational modification.
  • the polypeptides described herein can be, e.g., wild-type proteins, biologically- active fragments of the wild-type proteins, or variants of the wild- type proteins or fragments.
  • Variants, in accordance with the disclosure can contain amino acid substitutions, deletions, or insertions. The substitutions can be conservative or non-conservative.
  • proteins can be isolated.
  • purified or “isolated” as applied to any of the proteins described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a cell expressing the proteins.
  • a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.
  • amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue.
  • a selected residue in a selected protein corresponds to a residue when the selected residue occupies the same essential spatial or other structural relationship as a specified residue relative the rest of the protein.
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleted ously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleted ously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleted ously react with the compounds of the invention.
  • preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow- release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g. anti-cancer agent, anti -neurodegenerative disease agent, antiinflammatory disease agent).
  • additional therapies e.g. anti-cancer agent, anti -neurodegenerative disease agent, antiinflammatory disease agent.
  • the compound of the invention can be administered alone or can be coadministered to the patient.
  • Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation, to increase degradation of a prodrug and release of the drug, detectable agent).
  • the compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • the compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates.
  • compositions of the present invention can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623- 645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res.
  • the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis.
  • liposomes particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo.
  • compositions of the present invention can also be delivered as nanoparticles.
  • Pharmaceutical compositions provided by the present invention include compositions wherein the active ingredient (e.g. compounds described herein, including embodiments or examples) is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated.
  • compositions When administered in methods to treat a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., reducing, eliminating, or slowing the progression of disease symptoms (e.g. symptoms of cancer or neurodegenerative disease). Determination of a therapeutically effective amount of a compound of the invention is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.
  • the dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g.
  • the therapeutically effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
  • therapeutically effective amounts for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.
  • Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent.
  • the compounds described herein can be used in combination with one another, with other active agents known to be useful in treating cancer, or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.
  • co-administration includes administering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a second active agent.
  • Co-administration includes administering two active agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order.
  • co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including both active agents.
  • the active agents can be formulated separately.
  • the active and/or adjunctive agents may be linked or conjugated to one another.
  • the compounds described herein may be combined with treatments for cancer such as radiation or surgery.
  • Irel or “Ire la” or “ERNl” refers to the protein "Serine/threonine-protein kinase/endoribonuc lease IREl"a.k.a. "Endoplasmic reticulum to nucleus signaling 1".
  • Irel or “Ire la” or “ER 1” refers to the human protein. Included in the term “Irel” or “Ire la” or “ER 1" are the wildtype and mutant forms of the protein.
  • Irel or “Ire la” or “ERNl” refers to the protein associated with Entrez Gene 2081, OMIM 604033, UniProt 075460, and/or RefSeq (protein) NP_001424 (SEQ ID NO: 1).
  • the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.
  • “Irel” or “Ire la” or “ER 1” refers to the wildtype human protein.
  • “Irel” or “Ire la” or “ERN1” refers to the wildtype human nucleic acid.
  • the Irel is a mutant Irel.
  • the mutant Irel is associated with a disease that is not associated with wildtype Irel .
  • the Irel includes at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations) compared to wildtype Irel.
  • "Irel” or “Ire la” or “ERN1” refers to the protein or nucleic acid corresponding to GI: 153946420 (SEQ ID NO: 14).
  • "Irel” or “Irela” or “ERNl” refers to the protein or nucleic acid corresponding to NM_001433.3.
  • “Irel” or “Ire la” or “ER 1” refers to the protein or nucleic acid corresponding to GL 153946421 (SEQ ID NO: 15). In embodiments, “Irel” or “Ire la” or “ERN1” refers to the protein or nucleic acid corresponding to NP_001424.3.
  • PERK or "EIF2AK3” or “PEK” refers to the protein "Eukaryotic translation initiation factor 2-alpha kinase 3"a.k.a. "PRKR-like endoplasmic reticulum kinase” or “protein kinase R (PKR)-like endoplasmic reticulum kinase”.
  • PKA protein kinase R
  • PERK or “EIF2AK3” or “PEK” refers to the protein associated with Entrez Gene 9451, OMIM 604032, UniProt Q9NZJ5, and/or RefSeq (protein) NP_004827 (SEQ ID NO:2).
  • the reference numbers immediately above refer to the protein, and associated nucleic acids, known as of the date of filing of this application.
  • "PERK” or “EIF2AK3” or “PEK” refers to the wildtype human protein.
  • PERK or “EIF2AK3” or “PEK” refers to the wildtype human nucleic acid.
  • the PERK is a mutant PERK.
  • the mutant PERK is associated with a disease that is not associated with wildtype PERK.
  • the PERK includes at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations) compared to wildtype PERK.
  • PERK" or "EIF2AK3" or “PEK” refers to the protein or nucleic acid corresponding to GL284005480 (SEQ ID NO: 16).
  • "PERK” or “EIF2AK3” or “PEK” refers to the protein or nucleic acid
  • PERK or “EIF2AK3” or “PEK” refers to the protein or nucleic acid corresponding to GI: 134304838 (SEQ ID NO: 17). In embodiments, PERK” or “EIF2AK3” or “PEK” refers to the protein or nucleic acid corresponding to
  • Antibody refers to a polypeptide including a framework region from an
  • immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • antibodies or fragments of antibodies may be derived from different organisms, including humans, mice, rats, hamsters, camels, etc.
  • Antibodies of the invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g. glycosylation, expression, antigen recognition, effector functions, antigen binding, specificity, etc.).
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VR-CRI by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • the term antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al , Nature 348:552-554 (1990)).
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby,
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
  • Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBO J. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121 :210 (1986)).
  • Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO
  • Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments.
  • Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • the symbol X 1 is independently halogen.
  • R 3 is independently hydrogen, R 36 -substituted or unsubstituted C1-C5 alkyl, or R 36 -substituted or unsubstituted 2 to 5 membered heteroalkyl.
  • R 4 is independently hydrogen, R 39 -substituted or unsubstituted C1-C5 alkyl, or R 39 -substituted or unsubstituted 2 to 5 membered heteroalkyl.
  • R 5 is independently hydrogen, R 42 -substituted or unsubstituted C1-C5 alkyl, or R 42 -substituted or unsubstituted 2 to 5 membered heteroalkyl.
  • the symbol z is an integer from 0 to 2.
  • Ring A is an unsubstituted cycloalkyl (e.g. unsubstituted C3-C8 cycloalkyl).
  • R 30 may independently be oxo,
  • X 30 is independently halogen.
  • R 33 may independently be oxo,
  • R 36 may independently be oxo
  • X 36 is independently halogen.
  • R 39 may independently be oxo,
  • X 39 is independently halogen.
  • R 42 may independently be oxo,
  • the compound has the following formula (A): -ONH 2 , -C(0)NH 2 , -NHC(0)H, -NHC(0)OH, -NHOH, -OCX 42 3 , -OCHX 42 2 , unsubstituted d- alkyl, or unsubstituted 2 to 5 membered heteroalkyl.
  • the symbol X 42 is independently halogen. 17] In embodiments, the compound has the
  • R 1 is as described herein.
  • the compound has the formula:
  • R 1 is as described herein.
  • the compound has the formula:
  • R 1 is as described herein.
  • the compound has the formula: (V). R 1 is as described herein.
  • the compound has the formula: R 1 is as described herein.
  • the compound has the formula:
  • R 1 is as described herein.
  • the compound has the formula:
  • R 1 is as described herein.
  • the compound has the formula:
  • the compounds having a formula I-X may include an embodiment described in a paragraph of this COMPOSITIONS section or may include any combination of an embodiment of any paragraph in this COMPOSITIONS section with an embodiment of any one or more additional paragraphs of this COMPOSITIONS section. [0119] In embodiments of the compounds of formulae I-X, R 1 is
  • R 1 is
  • R 1 may be halogen, -OR 5 , -SR 5 , -CX , -OCX 1 3, -OCHX ⁇ , -OCH2X 1 , -SCX , -SCHX ⁇ , -SCH2X 1 , R 30 - substituted or unsubstituted C1-C3 alkyl, or R 30 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 1 may be
  • R 1 is halogen, -SR 5 , or unsubstituted C1-C2 alkyl.
  • two adjacent R 1 substituents are
  • two adjacent R 1 substituents are joined to form an unsubstituted 2,3-dihydro-l,4-
  • Ring A is an unsubstituted C3-C5 cycloalkyl. In embodiments, Ring A is an unsubstituted saturated C3-C5 cycloalkyl. In embodiments, Ring A is an unsubstituted unsaturated C3-C5 cycloalkyl. In embodiments, Ring A is an unsubstituted C3-C5 cycloalkenyl. In embodiments, Ring A is an unsubstituted C3-C5 cycloalkynyl. In embodiments, Ring A is an unsubstituted cylopropyl.
  • Ring A is an unsubstituted cyclobutyl. In embodiments, Ring A is an unsubstituted cyclopentyl. In embodiments, Ring A is an unsubstituted cyclohexyl. In embodiments, Ring A is an
  • Ring A is an unsubstituted cylopropenyl. In embodiments, Ring A is an unsubstituted cyclobutenyl. In embodiments, Ring A is an unsubstituted cyclopentenyl. In embodiments, Ring A is an unsubstituted cyclohexenyl.
  • R 3 is independently hydrogen, R 36 - substituted or unsubstituted C1-C3 alkyl, or R 36 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 3 is independently hydrogen, R 36 -substituted or unsubstituted Ci- C2 alkyl, or R 36 -substituted or unsubstituted 2 membered heteroalkyl.
  • R 3 is independently hydrogen, unsubstituted C1-C2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 3 is independently hydrogen or unsubstituted C1-C2 alkyl.
  • R 3 is independently hydrogen or unsubstituted methyl.
  • R 3 is independently hydrogen.
  • R 4 is independently hydrogen, R 39 - substituted or unsubstituted C1-C3 alkyl, or R 39 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 4 is independently hydrogen, R 39 -substituted or unsubstituted Ci- C 2 alkyl, or R 39 -substituted or unsubstituted 2 membered heteroalkyl.
  • R 4 is independently hydrogen, unsubstituted C1-C2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 4 is independently hydrogen or unsubstituted C1-C2 alkyl.
  • R 4 is independently hydrogen or unsubstituted methyl.
  • R 4 is independently hydrogen.
  • R 5 is independently hydrogen, R 42 - substituted or unsubstituted C1-C3 alkyl, or R 42 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 5 is independently hydrogen, R 42 -substituted or unsubstituted Ci- C2 alkyl, or R 42 -substituted or unsubstituted 2 membered heteroalkyl.
  • R 5 is independently hydrogen, unsubstituted C1-C2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 5 is independently hydrogen or unsubstituted C1-C2 alkyl. In embodiments, R 5 is independently unsubstituted methyl. In embodiments, R 5 is independently hydrogen or unsubstituted methyl. In embodiments, R 5 is independently hydrogen.
  • R 3 is independently
  • R 3 is independently halogen, -NH 2 , -OH, or -SH.
  • X 3 is independently -F. In embodiments, X 3 is independently -CI. In
  • X 3 is independently -Br. In embodiments, X 3 is independently -I. [0125] In embodiments of the compounds of formulae I-X, R 4 is independently
  • R 4 is independently halogen, -NH 2 , -OH, or -SH.
  • X 4 is independently -F. In embodiments, X 4 is independently -CI. In
  • X 4 is independently -Br. In embodiments, X 4 is independently -I. [0126] In embodiments of the compounds of formulae I-X, R 5 is independently
  • R 5 is independently halogen, -NH 2 , -OH, or -SH.
  • X 5 is independently -F. In embodiments, X 5 is independently -CI. In
  • X 5 is independently -Br. In embodiments, X 5 is independently -I.
  • R 1 is independently halogen. In embodiments, R 1 is independently -NR 3 R 4 . In embodiments, R 1 is independently -OR 5 . In embodiments, R 1 is independently -COOR 5 . In embodiments, R 1 is independently -SR 5 . In embodiments, R 1 is independently -COR 5 . In embodiments, R 1 is independently -CX - In embodiments, R 1 is independently -ONR 3 R 4 . In embodiments, R 1 is
  • R 1 is independently -NR 3 C(0)R 5 .
  • R 1 is independently -OCXS- In embodiments, R 1 is independently -OCHX ⁇ . In embodiments, R 1 is independently -OCH 2 X 1 . In embodiments, R 1 is independently -SCX - In embodiments, R 1 is independently -SCHX ⁇ . In embodiments, R 1 is independently -SCH 2 X 1 . In embodiments, R 1 is independently R 30 -substituted or unsubstituted C1-C3 alkyl. In embodiments, R 1 is independently R 30 -substituted or unsubstituted 2 to 3 membered heteroalkyl. In
  • R 1 is independently -F. In embodiments, R 1 is independently -CI. In
  • R 1 is independently -Br. In embodiments, R 1 is independently -I. In
  • R 1 is independently -CH 3 . In embodiments, R 1 is independently -CH 2 CH 3 . In embodiments, R 1 is independently -OCH 3 . In embodiments, R 1 is independently -OCH 2 CH 3 . In embodiments, R 1 is independently -SCH 3 . In embodiments, R 1 is independently -SCH 2 CH 3 . In embodiments, R 1 is independently -CH(CH 3 ) 2 . In embodiments, R 1 is independently -C(CH 3 ) 3 .
  • R 1 is independently -SCH 2 F. In embodiments, R 1 is independently -SCHF 2 . In embodiments, R 1 is independently -SCF 3 . In embodiments, R 1 is independently -OCH 2 F. In embodiments, R 1 is independently -OCHF 2 . In embodiments, R 1 is independently -OCF 3 . In embodiments, R 1 is independently -SCH 2 C1. In embodiments, R 1 is independently -SCHC1 2 . In embodiments, R 1 is independently -SCC1 3 . In embodiments, R 1 is independently -OCH 2 Cl. In embodiments, R 1 is independently -OCHCl 2 .
  • R 1 is independently -OCCl 3 . In embodiments, R 1 is independently -SCH 2 Br. In embodiments, R 1 is independently -SCHBr 2 . In embodiments, R 1 is independently -SCBr 3 . In embodiments, R 1 is independently -OCH 2 Br. In embodiments, R 1 is independently - OCHBr 2 . In embodiments, R 1 is independently -OCBr 3 . In embodiments, R 1 is independently - SCH 2 I. In embodiments, R 1 is independently -SCHI 2 . In embodiments, R 1 is independently - SCI 3 . In embodiments, R 1 is independently -OCH 2 I. In embodiments, R 1 is independently - OCHI 2 .
  • R 1 is independently -OCI 3 . In embodiments, R 1 is independently - SCC1 2 F. In embodiments, R 1 is independently -SCC1F 2 . In embodiments, R 1 is independently - 0CC1 2 F. In embodiments, R 1 is independently -0CC1F 2 . In embodiments, R 1 is independently -SCBr 2 F. In embodiments, R 1 is independently -SCBrF 2 . In embodiments, R 1 is independently -OCBr 2 F. In embodiments, R 1 is independently -OCBrF 2 . In embodiments, R 1 is independently -SCI 2 F. In embodiments, R 1 is independently -SCIF 2 .
  • R 1 is independently - OCI 2 F. In embodiments, R 1 is independently -OCIF 2 . In embodiments, R 1 is independently - SCBr 2 Cl. In embodiments, R 1 is independently -SCBrCl 2 . In embodiments, R 1 is independently -OCBr 2 Cl. In embodiments, R 1 is independently -OCBrCl 2 . In embodiments, R 1 is
  • R 1 is independently -SCI 2 C1. In embodiments, R 1 is independently -SCIC1 2 . In embodiments, R 1 is independently -0CI 2 C1. In embodiments, R 1 is independently -0CIC1 2 . In embodiments, R 1 is independently -SCBr 2 I. In embodiments, R 1 is independently -SCBrI 2 . In embodiments, R 1 is independently -OCBr 2 I. In embodiments, R 1 is independently -OCBrI 2 .
  • R 1 is independently -NHCH 3 .
  • R 1 is independently -NH 2 .
  • R 1 is independently -N(CH 3 ) 2 .
  • R 1 is independently -NHCH 2 CH 3 .
  • R 1 is independently - N(CH 3 )(CH 2 CH 3 ).
  • R 1 is independently -N(CH 2 CH 3 ) 2 .
  • R 1 is independently -COOH.
  • R 1 is independently -COOCH 3 .
  • R 1 is independently -COOCH 2 CH 3 .
  • R 1 is independently -COOCF 3 .
  • R 1 is independently -COOCCl 3 .
  • R 1 is independently - COOCBr 3 .
  • R 1 is independently -COOCI 3 .
  • R 1 is
  • R 1 is independently -COOCHF 2 .
  • R 1 is independently -COOCH 2 F.
  • R 1 is independently -COOCHCl 2 .
  • R 1 is independently -COOCH 2 Cl.
  • R 1 is independently -COOCHBr 2 .
  • R 1 is independently - COOCH 2 Br.
  • R 1 is independently -COOCHI 2 .
  • R 1 is independently -COOCH 2 I. [0130]
  • R 1 is independently -COH.
  • R 1 is independently -COCH 3 .
  • R 1 is independently -COCH 2 CH 3 .
  • R 1 is independently -COCF 3 ). In embodiments, R 1 is independently -C0CC1 3 . In embodiments, R 1 is independently -COCBr 3 . In embodiments, R 1 is independently -COCI 3 . In embodiments, R 1 is independently -COCHF 2 . In embodiments, R 1 is independently - COCH 2 F. In embodiments, R 1 is independently -COCHCI 2 . In embodiments, R 1 is
  • R 1 is independently -C0CH 2 C1. In embodiments, R 1 is independently -COCHBr 2 . In embodiments, R 1 is independently -COCH 2 Br. In embodiments, R 1 is independently -COCHI 2 . In embodiments, R 1 is independently -COCH 2 I.
  • R 1 is independently -ONHCH 3 . In embodiments, R 1 is independently -ONH 2 . In embodiments, R 1 is independently -ON(CH 3 ) 2 . In embodiments, R 1 is independently -ONHCH 2 CH 3 . In embodiments, R 1 is independently - ON(CH 3 )(CH 2 CH 3 ). In embodiments, R 1 is independently -ON(CH 2 CH 3 ) 2 .
  • R 1 is independently -C(0)NHCH 3 . In embodiments, R 1 is independently -C(0)NH 2 . In embodiments, R 1 is
  • R 1 is independently -C(0)N(CH 3 ) 2 . In embodiments, R 1 is independently -C(0)NHCH 2 CH 3 . In embodiments, R 1 is independently -C(0)N(CH 3 )(CH 2 CH 3 ). In embodiments, R 1 is
  • R 1 is independently -NHCOH. In embodiments, R 1 is independently -NHCOCH 3 . In embodiments, R 1 is independently - NHCOCH 2 CH 3 . In embodiments, R 1 is independently -NHCOCF 3 . In embodiments, R 1 is independently -NHCOCCl 3 . In embodiments, R 1 is independently -NHCOCBr 3 . In
  • R 1 is independently -NHCOCI 3 . In embodiments, R 1 is independently - NHCOCHF 2 . In embodiments, R 1 is independently -NHCOCH 2 F. In embodiments, R 1 is independently -NHCOCHCl 2 . In embodiments, R 1 is independently -NHCOCH 2 Cl. In embodiments, R 1 is independently -NHCOCHBr 2 . In embodiments, R 1 is independently - NHCOCH 2 Br. In embodiments, R 1 is independently -NHCOCHI 2 . In embodiments, R 1 is independently -NHCOCH 2 I.
  • R 1 is independently -N(CH 3 )COH. In embodiments, R 1 is independently -N(CH 3 )COCH 3 . In embodiments, R 1 is independently - N(CH 3 )COCH 2 CH 3 . In embodiments, R 1 is independently -N(CH 3 )COCF 3 . In embodiments, R 1 is independently -N(CH 3 )C0CC1 3 . In embodiments, R 1 is independently -N(CH 3 )COCBr 3 . In embodiments, R 1 is independently -N(CH 3 )COCI 3 . In embodiments, R 1 is independently - N(CH 3 )COCHF 2 .
  • R 1 is independently -N(CH 3 )COCH 2 F. In embodiments, R 1 is independently -N(CH 3 )C0CHC1 2 . In embodiments, R 1 is independently -N(CH 3 )C0CH 2 C1. In embodiments, R 1 is independently -N(CH 3 )COCHBr 2 . In embodiments, R 1 is independently -N(CH 3 )COCH 2 Br. In embodiments, R 1 is independently -N(CH 3 )COCHI 2 . In embodiments, R 1 is independently -N(CH 3 )COCH 2 I. [0135] In embodiments of the compounds of formulae I-X, X 1 is -F. In embodiments, X 1 is - CI. In embodiments, X 1 is -Br. In embodiments, X 1 is -I. In embodiments, X 30 is -F. In embodiments, X 1 is -F. In embodiments, X 1 is -N(CH 3 )COCH
  • X is -CI. In embodiments, X is -Br. In embodiments, X is -I. In embodiments, z is 0. In embodiments, z is 1. In embodiments, z is 2. In embodiments, Ring A is an unsubstituted cyclopropyl. In embodiments, Ring A is an unsubstituted cyclobutyl. In embodiments, Ring A is an unsubstituted cyclopentyl.
  • X 1 is F.
  • R 30 is independently oxo
  • R 31 30 30 aryl, or R -substituted or unsubstituted heteroaryl.
  • X is halogen. In embodiments, X is F. [0138] R 31 is independently oxo,
  • NHC (0)H, -NHC(0)-OH, -NHOH, -OCX 2 3 , -OCHX 2 2 , R 33 -substituted or unsubstituted alkyl,
  • R -substituted or unsubstituted heteroalkyl R -substituted or unsubstituted cycloalkyl, R - substituted or unsubstituted heterocycloalkyl, R 33 -substituted or unsubstituted aryl, or R 33 - substituted or unsubstituted heteroaryl.
  • X 2 is halogen. In embodiments, X 2 is F. [0140] R 33 is independently oxo,
  • R 34 33 33 aryl, or R -substituted or unsubstituted heteroaryl.
  • X is halogen. In embodiments, X is F. [0141] R 34 is independently oxo,
  • R 37 is independently oxo
  • NHC (0)H, -NHC(0)-OH, -NHOH, -OCX 37 3 , -OCHX 37 2 , R 38 -substituted or unsubstituted alkyl,
  • R -substituted or unsubstituted heteroalkyl R -substituted or unsubstituted cycloalkyl, R - substituted or unsubstituted heterocycloalkyl, R 38 -substituted or unsubstituted aryl, or R 38 - substituted or unsubstituted heteroaryl.
  • X 37 is halogen. In embodiments, X 37 is F.
  • R -substituted or unsubstituted heteroalkyl R -substituted or unsubstituted cycloalkyl, R - substituted or unsubstituted heterocycloalkyl, R 39 -substituted or unsubstituted aryl, or R 39 - substituted or unsubstituted heteroaryl.
  • X 4 is halogen. In embodiments, X 4 is F.
  • R 39 is independently oxo
  • R 40 is independently oxo
  • X 40 is halogen. In embodiments, X 40 is F.
  • X 5 is halogen. In embodiments, X 5 is F.
  • R 42 is independently oxo
  • R 43 is independently oxo
  • R 32 , R 35 , R 38 , R 41 , and R 44 are independently hydrogen, oxo,
  • X 44 is halogen. In embodiments, X
  • the compound is any one of the compounds described herein (e.g., in an aspect, embodiment, claim, figure, table, or example).
  • a compound as described herein may include multiple instances of R 1 , R 2 , R 3 , R 4 , R 5 , and/or other variables. In such
  • each variable may optional be different and be appropriately labeled to distinguish each group for greater clarity.
  • R 1 , R 2 , R 3 , R 4 , and/or R 5 may be referred to, for example, as R 11 , R 1'2 , R 1'3 , R 1'4 , R 1'5 , R 21 , R 2'2 , R 2'3 , R 2'4 , R 2'5 , R 2'6 ,
  • R 3 is assumed by R 31 , R 3'2 , R 3'3 , R 3'4 , R 3'5 , R 3'6 , R 3'7 , R 3'8 , R 3'9 , R 310 , r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r
  • R 5 is assumed by R 5'1 , R 5'2 , R 5'3 , on r r r r
  • the compound is a compound described herein (e.g., in an aspect, embodiment, example, claim, table, scheme, drawaing, or figure).
  • the compound is described herein, including in an aspect, embodiment, example, figure, table, or claim.
  • the compound has the fomula:
  • R 1'1 , R 1'2 , and R 1'3 may have any value of R 1 as described herein.
  • one of R 1'1 , R 1'2 , and R 1'3 is hydrogen and two are not hydrogen.
  • R 1'1 , R 1'2 , and R 1'3 are each optionally different.
  • R 1'1 , R 1'2 , and R 1 3 are each different.
  • R 1 1 is H, -N0 2 , -OCH 3 , - COOCH 3 , -CH2CH 3 , -SCH 3 , -OCH2CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 1 1 is H. In embodiments, R 1 1 is -NO2. In embodiments, R 1 1 is -OCH 3 . In embodiments, R 1 1 is -COOCH 3 . In embodiments, R 1 1 is -CH 2 CH 3 . In embodiments, R 1 1 is - SCH 3 . In embodiments, R 1 1 is -OCH 2 CH 3 . In embodiments, R 1 1 is -CF 3 . In embodiments, R 1 1 is -F. In embodiments, R 1 1 is -CI. In embodiments, R 1 1 is -Br. In embodiments, R 1 1 is -I. In embodiments, R 1 1 is -SCH 2 CH 3 .
  • R 1 2 is H, -N0 2 , -OCH 3 , - COOCH 3 , -CH2CH 3 , -SCH 3 , -OCH2CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 1 2 is H. In embodiments, R 1 2 is -NO2. In embodiments, R 1 2 is -OCH 3 . In embodiments, R 1 2 is -COOCH 3 . In embodiments, R 1 2 is -CH 2 CH 3 . In embodiments, R 1 2 is - SCH 3 . In embodiments, R 1 2 is -OCH 2 CH 3 . In embodiments, R 1 2 is -CF 3 . In embodiments,
  • R 1 2 is -F. In embodiments, R 1 2 is -CI. In embodiments, R 1 2 is -Br. In embodiments, R 1 2 is -I. In embodiments, R 1 2 is -SCH 2 CH 3 . In embodiments, R 1 2 is not -CF 3 .
  • R 1 3 is H, -N0 2 , -OCH 3 , - COOCH 3 , -CH 2 CH 3 , -SCH 3 , -OCH 2 CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 1 3 is H. In embodiments, R 1 3 is -N0 2 . In embodiments, R 1 3 is -OCH 3 . In embodiments, R 1 3 is -COOCH 3 . In embodiments, R 1 3 is -CH 2 CH 3 . In embodiments, R 1 3 is - SCH 3 . In embodiments, R 1 3 is -OCH 2 CH 3 . In embodiments, R 1 3 is -CF 3 . In embodiments, R is -F. In embodiments, R is -CI. In embodiments, R is -Br. In embodiments, R is -I. In embodiments, R 1'3 is -SCH 2 CH 3 . In embodiments, R 1'3 is not -CF 3 .
  • R 1 1 is H;
  • R 1 2 is H, -N0 2 , -OCH 3 , -COOCH 3 , -CH 2 CH 3 , -SCH 3 , -OCH 2 CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 ;
  • R 1 3 is H, -NO2, -OCH3, -COOCH3, -CH 2 CH 3 , -SCH3, -OCH 2 CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 1 3 is H;
  • R 1 2 is H, -N0 2 , -OCH 3 , -COOCH 3 , -CH 2 CH 3 , -SCH 3 , -OCH 2 CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 ; and
  • R 1 1 is H, -N0 2 , -OCH 3 , -COOCH 3 , - CH 2 CH 3 , -SCH 3 , -OCH 2 CH 3 , -CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 1 2 is H;
  • R 1 1 is H, -NO2, -OCH3, -COOCH3, -CH2CH3, -SCH3, -OCH2CH3, -CF 3 , -F, -CI, -Br, -I, or - SCH2CH3;
  • R 1 3 is H, -N0 2 , -OCH3, -COOCH3, -CH 2 CH 3 , -SCH 3 , -OCH 2 CH 3 , - CF 3 , -F, -CI, -Br, -I, or -SCH 2 CH 3 .
  • R 2 is independently oxo,
  • R 3 , R 4 , and R 5 are independently hydrogen, R 2 -substituted or unsubstituted C1-C5 alkyl, or R 2 -substituted or unsubstituted 2 to 5 membered heteroalkyl.
  • the symbol X is independently halogen.
  • the compounds having a formula XI may include an embodiment described in a paragraph of this PARA- SUBSTITUTED COMPOSITIONS section or may include any combination of an embodiment of any paragraph in this PARA- SUBSTITUTED
  • R 1 is
  • R 1 is
  • R 1 may be
  • halogen -OR 5 , -SR 5 , -C3 ⁇ 4, -OCX 3 , -OCHX 2 , -OCH 2 X, -SCX 3 , -SCHX 2 , -SCH 2 X, R 2 -substituted or unsubstituted C1-C3 alkyl, or R 2 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 1 may be halogen, -OR 5 , -SR 5 , -CX 3 , -OCX 3 , -OCHX 2 , -OCH 2 X, -SCX 3 , -SCHX 2 , -SCH 2 X, unsubstituted Ci-C 2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 1 is halogen, -SR 5 , or unsubstituted Ci-C 2 alkyl.
  • R 3 and R 4 are independently hydrogen, R 2 -substituted or unsubstituted C1-C3 alkyl, or R 2 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 3 and R 4 are independently hydrogen, R 2 -substituted or unsubstituted Ci-C 2 alkyl, or R 2 -substituted or unsubstituted 2 membered heteroalkyl.
  • R 3 and R 4 are independently hydrogen, unsubstituted Ci-C 2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 3 and R 4 are independently hydrogen or
  • R 3 and R 4 are independently hydrogen or unsubstituted methyl. In embodiments, R 3 and R 4 are independently hydrogen.
  • R 5 is hydrogen, R 2 -substituted or unsubstituted C1-C3 alkyl, or R 2 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 5 is hydrogen, R 2 -substituted or unsubstituted Ci-C 2 alkyl, or R 2 -substituted or unsubstituted 2 membered heteroalkyl.
  • R 5 is hydrogen, unsubstituted Ci-C 2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 5 is hydrogen or
  • R 5 is unsubstituted Ci-C 2 alkyl.
  • R 5 is unsubstituted methyl.
  • R 5 is hydrogen or unsubstituted methyl.
  • R 5 is hydrogen.
  • R 2 is
  • R 2 is halogen, -NH 2 , -OH, or -SH. In embodiments, R 2 is oxo.
  • X is -F. In embodiments, X is -CI. In embodiments, X is -Br. In embodiments, X is -I.
  • R 1 is halogen. In embodiments, R 1 is -NR 3 R 4 . In embodiments, R 1 is -OR 5 . In embodiments, R 1 is -COOR 5 . In embodiments, R 1 is -SR 5 . In embodiments, R 1 is -COR 5 . In embodiments, R 1 is -CX 3 . In embodiments, R 1 is -ONR 3 R 4 . In embodiments, R 1 is -C(0)NR 3 R 4 . In embodiments, R 1 is -NR 3 C(0)R 5 . In embodiments, R 1 is -OCX 3 . In embodiments, R 1 is -OCHX2.
  • R 1 is -OCH2X. In embodiments, R 1 is -SCX 3 . In embodiments, R 1 is -SCHX 2 . In embodiments, R 1 is -SCH 2 X. In embodiments, R 1 is R 2 -substituted or unsubstituted C1-C3 alkyl. In embodiments, R 1 is R 2 - substituted or unsubstituted 2 to 3 membered heteroalkyl. In embodiments, R 1 is -F. In embodiments, R 1 is -CI. In embodiments, R 1 is -Br. In embodiments, R 1 is -I. In
  • R 1 is -CH 3 . In embodiments, R 1 is -CH 2 CH 3 . In embodiments, R 1 is -OCH 3 . In embodiments, R 1 is -OCH 2 CH 3 . In embodiments, R 1 is -SCH 3 . In embodiments, R 1 is -SCH 2 CH 3 . In embodiments, R 1 is -CH(CH 3 ) 2 . In embodiments, R 1 is -C(CH 3 ) 3 . [0169] In embodiments of the compounds of formula XI, R 1 is -SCH 2 F. In embodiments, R 1 is -SCHF 2 . In embodiments, R 1 is -SCF 3 . In embodiments, R 1 is -OCH 2 F.
  • R 1 is -OCHF 2 . In embodiments, R 1 is -OCF 3 . In embodiments, R 1 is -SCH 2 C1. In embodiments, R 1 is -SCHC1 2 . In embodiments, R 1 is -SCCI 3 . In embodiments, R 1 is -OCH 2 Cl. In embodiments, R 1 is -OCHCl 2 . In embodiments, R 1 is -OCCI 3 . In embodiments, R 1 is -SCH 2 Br. In embodiments, R 1 is -SCHBr 2 . In embodiments, R 1 is -SCBr 3 . In embodiments, R 1 is -
  • R 1 is -OCHBr 2 . In embodiments, R 1 is -OCBr 3 . In embodiments, R 1 is -SCH 2 I. In embodiments, R 1 is -SCHI 2 . In embodiments, R 1 is -SCI 3 . In embodiments, R 1 is -OCH 2 I. In embodiments, R 1 is -OCHI 2 . In embodiments, R 1 is -OCI 3 . In embodiments, R 1 is -SCC1 2 F. In embodiments, R 1 is -SCC1F 2 . In embodiments, R 1 is -OCCl 2 F. In embodiments, R 1 is -OCClF 2 . In embodiments, R 1 is -SCBr 2 F. In embodiments, R 1 is -
  • R 1 is -OCBr 2 F. In embodiments, R 1 is -OCBrF 2 . In embodiments, R 1 is -SCI 2 F. In embodiments, R 1 is -SCIF 2 . In embodiments, R 1 is -OCI 2 F. In embodiments, R 1 is -OCIF 2 . In embodiments, R 1 is -SCBr 2 Cl. In embodiments, R 1 is -SCBrCl 2 . In embodiments, R 1 is -OCBr 2 Cl. In embodiments, R 1 is -OCBrCl 2 . In embodiments, R 1 is - SCI 2 C1. In embodiments, R 1 is -SCIC1 2 .
  • R 1 is -OCI 2 Cl. In embodiments, R 1 is -OCICl 2 . In embodiments, R 1 is -SCBr 2 I. In embodiments, R 1 is -SCBrI 2 . In embodiments, R 1 is -OCBr 2 I. In embodiments, R 1 is -OCBrI 2 .
  • R 1 is -NHCH 3 .
  • R 1 is -NH 2 .
  • R 1 is -N(CH 3 ) 2 .
  • R 1 is -NHCH 2 CH 3 .
  • R 1 is -N(CH 3 )(CH 2 CH 3 ). In embodiments, R 1 is -N(CH 2 CH 3 ) 2 . In embodiments, R 1 is -COOH. In embodiments, R 1 is -COOCH 3 . In embodiments, R 1 is -COOCH 2 CH 3 . In embodiments, R 1 is -COOCF 3 . In embodiments, R 1 is -COOCCI 3 . In embodiments, R 1 is - COOCBr 3 . In embodiments, R 1 is -COOCI 3 . In embodiments, R 1 is -COOCHF 2 . In embodiments, R 1 is -COOCH 2 F. In embodiments, R 1 is -COOCHCI 2 .
  • R 1 is - COOCH 2 CI. In embodiments, R 1 is -COOCHBr 2 . In embodiments, R 1 is -COOCH 2 Br. In embodiments, R 1 is -COOCHI 2 . In embodiments, R 1 is -COOCH 2 I. [0171] In embodiments of the compounds of formula XI, R 1 is -COH. In embodiments, R 1 is -COCH 3 . In embodiments, R 1 is -COCH 2 CH 3 . In embodiments, R 1 is -COCF 3 ). In embodiments of the compounds of formula XI, R 1 is -COH. In embodiments, R 1 is -COCH 3 . In embodiments, R 1 is -COCH 2 CH 3 . In embodiments, R 1 is -COCF 3 ). In
  • R 1 is -COCCI 3 . In embodiments, R 1 is -COCBr 3 . In embodiments, R 1 is - COCI 3 . In embodiments, R 1 is -COCHF 2 . In embodiments, R 1 is -COCH 2 F. In embodiments, R 1 is -COCHCl 2 . In embodiments, R 1 is -COCH 2 Cl. In embodiments, R 1 is -COCHBr 2 . In embodiments, R 1 is -COCH 2 Br. In embodiments, R 1 is -COCHI 2 . In embodiments, R 1 is -
  • R 1 is -ONHCH 3 . In embodiments, R 1 is -ONH 2 . In embodiments, R 1 is -ON(CH 3 ) 2 . In embodiments, R 1 is -ONHCH 2 CH 3 . In embodiments, R 1 is -ON(CH 3 )(CH 2 CH 3 ). In embodiments, R 1 is -ON(CH 2 CH 3 ) 2 . [0173] In embodiments of the compounds of formula XI, R 1 is -C(0)NHCH 3 . In embodiments of the compounds of formula XI, R 1 is -C(0)NHCH 3 . In embodiments of the compounds of formula XI, R 1 is -C(0)NHCH 3 . In
  • R 1 is -C(0)NH 2 . In embodiments, R 1 is -C(0)N(CH 3 ) 2 . In embodiments, R 1 is -C(0)NHCH 2 CH 3 . In embodiments, R 1 is -C(0)N(CH 3 )(CH 2 CH 3 ). In embodiments, R 1 is -C(0)N(CH 2 CH 3 ) 2 .
  • R 1 is -NHCOH. In embodiments, R 1 is -NHCOCH 3 . In embodiments, R 1 is -NHCOCH 2 CH 3 . In embodiments, R 1 is -NHCOCF 3 . In embodiments, R 1 is -NHCOCCI 3 . In embodiments, R 1 is -NHCOCBr 3 . In embodiments, R 1 is -NHCOCI 3 . In embodiments, R 1 is -NHCOCHF 2 . In embodiments, R 1 is -NHCOCH 2 F. In embodiments, R 1 is -NHCOCHCl 2 . In embodiments, R 1 is -NHCOCH 2 Cl. In embodiments, R 1 is -NHCOCHBr 2 . In embodiments, R 1 is -NHCOCH 2 Br. In embodiments, R 1 is -NHCOCHI 2 . In embodiments, R 1 is -NHCOCH 2 I.
  • R 1 is -N(CH 3 )COH.
  • R 1 is -N(CH 3 )COCH 3 . In embodiments, R 1 is -N(CH 3 )COCH 2 CH 3 . In embodiments, R 1 is -N(CH 3 )COCF 3 . In embodiments, R 1 is -N(CH 3 )C0CC1 3 . In embodiments, R 1 is -N(CH 3 )COCBr 3 . In embodiments, R 1 is -N(CH 3 )COCI 3 . In embodiments, R 1 is - N(CH 3 )COCHF 2 . In embodiments, R 1 is -N(CH 3 )COCH 2 F. In embodiments, R 1 is - N(CH 3 )C0CHC1 2 .
  • R 1 is -N(CH 3 )C0CH 2 C1. In embodiments, R 1 is - N(CH 3 )COCHBr 2 . In embodiments, R 1 is -N(CH 3 )COCH 2 Br. In embodiments, R 1 is - N(CH 3 )COCHI 2 . In embodiments, R 1 is -N(CH 3 )COCH 2 I.
  • the compound is any one of the compounds described herein (e.g., in an aspect, embodiment, claim, figure, table, or example).
  • the compound is described herein, including in an aspect, embodiment, example, figure, table, or claim.
  • composition including a
  • pharmaceutically acceptable salt thereof is included in a therapeutically effective amount.
  • the pharmaceutical composition includes a second agent (e.g. therapeutic agent).
  • the pharmaceutical composition includes a second agent (e.g. therapeutic agent) in a therapeutically effective amount.
  • the second agent is an Irel inhibitor (e.g., AD60).
  • the second agent is a PERK inhibitor (e.g., GSK, GSK2606414, GSK2656157).
  • the second agent is a proteasome inhibitor (e.g., bortezomib).
  • the second agent is thalidomide.
  • the second agent is lenalidomide.
  • the second agent is a treatment for a neurodegenerative disease (e.g., rivastigmine, galantamine, memantine, donepezil; levodopa, carbidopa, apomorphine, bromocriptine, rotigotine, pramipexole, ropinirole, benzotropine, trihexyphenidyl, selegiline, rasagiline, entacapone, tolcapone, amantadine, droxidopa; tetrabenazine, haloperidol, chlorpromazine, risperidone, quetiapine, levetiracetam, clonazepam; riluzole).
  • a neurodegenerative disease e.g., rivastigmine, galantamine, memantine, donepezil; levodopa, carbidopa, apomorphine, bromocriptine, rotigotine, pr
  • a method of treating a disease caused by abnormal levels of Irel activity in a subject in need of such treatment may be caused by an amount of Irel activity that is too low or too high.
  • the disease may be caused by a deficiency in Irel activity or by abnormally high Irel activity (e.g., hyperactivity of Irel).
  • the method may include administering to the subject a therapeutically effective amount of an Irel modulator as described above (e.g., compound described herein).
  • a compound as described herein for use as a medicament is provided.
  • the medicament is useful for treating a disease caused by abnormal levels of Irel activity in a subject in need of such treatment.
  • the disease may be caused by an amount of Irel activity that is too low or too high.
  • the disease may be caused by a deficiency in Irel activity or by abnormally high Irel activity (e.g., hyperactivity of Irel).
  • the use may include administering to the subject a therapeutically effective amount of an Irel modulator as described above (e.g., compound described herein).
  • a compound as described herein for use in the treatment of a disease caused by abnormal levels of Irel activity in a subject in need of such treatment.
  • the disease may be caused by an amount of Irel activity that is too low or too high.
  • the disease may be caused by a deficiency in Irel activity or by abnormally high Irel activity (e.g., hyperactivity of Irel).
  • the use may include administering to the subject a therapeutically effective amount of an Irel modulator as described above (e.g., compound described herein).
  • Irel deficiency is a decreased amount of Irel activity compared to normal levels of Irel activity in a particular subject or a population of healthy subjects. The decreased amount of Irel activity may result in excessive amounts of misfolded protein accumulation thereby causing the disease state.
  • Irel hyperactivity is an increased amount of Irel activity compared to normal levels of Irel activity in a particular subject or a population of healthy subjects. The increased amount of Irel activity may result in, for example, excessive amounts of cell proliferation thereby causing the disease state.
  • the disease is associated with Irel activity deficiency.
  • diseases may include, but are not limited to, cystic fibrosis, retinitis pigmentosa, diabetes, or a neurodegenerative disease.
  • the neurodegenerative disease may include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HlV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson'
  • the disease is associated with abnormally high Irel activity.
  • diseases may include, but are not limited, to cancers, inflammatory diseases, or autoimmune diseases.
  • Exemplary cancers include, but are not limited to, breast cancer (e.g., triple negative breast cancer) or multiple myeloma.
  • the disease is multiple myeloma.
  • Exemplary inflammatory diseases include, but are not limited to, asthma, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease; reperfusion injury, rheumatoid arthritis, transplant rejection, and vasculitis.
  • Exemplary autoimmune diseases include, but are not limited to, XBP1- linked Crohn's disease, Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sj5gren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis.
  • the disease is XBP1 -linked Crohn's disease.
  • the subject of treatment for the disease is typically a mammal.
  • the mammals treated with the compound may be humans, nonhuman primates, and/or non- human mammals (e.g., rodents, canines).
  • a method of treating an Irel activity-associated disease in a subject in need of such treatment including administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • a compound as described herein for use as a medicament may be useful for treating an Irel activity-associated disease in a subject in need of such treatment.
  • the use may include administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • a compound for use in the treatment of an Irel activity- associated disease in a subject in need of such treatment may include administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • Also provided herein is a method of treating a disease caused by abnormal levels of PERK activity in a subject in need of such treatment.
  • the disease may be caused by an amount of PERK activity that is too low or too high.
  • the disease may be caused by a deficiency in PERK activity or by abnormally high PERK activity (e.g., hyperactivity of PERK).
  • the method may include administering to the subject a therapeutically effective amount of a PERK modulator as described above (e.g., compound described herein).
  • a compound as described herein for use as a medicament is provided.
  • the medicament is useful for treating a disease caused by abnormal levels of PERK activity in a subject in need of such treatment.
  • the disease may be caused by an amount of PERK activity that is too low or too high.
  • the disease may be caused by a deficiency in PERK activity or by abnormally high PERK activity (e.g., hyperactivity of PERK).
  • the use may include administering to the subject a therapeutically effective amount of a PERK modulator as described above (e.g., compound described herein).
  • a compound as described herein for use in the treatment of a disease caused by abnormal levels of PERK activity in a subject in need of such treatment.
  • the disease may be caused by an amount of PERK activity that is too low or too high.
  • the disease may be caused by a deficiency in PERK activity or by abnormally high PERK activity (e.g., hyperactivity of PERK).
  • the use may include administering to the subject a therapeutically effective amount of a PERK modulator as described above (e.g., compound described herein).
  • PERK deficiency is a decreased amount of PERK activity compared to normal levels of PERK activity in a particular subject or a population of healthy subjects. The decreased amount of PERK activity may result in excessive amounts of misfolded protein accumulation thereby causing the disease state.
  • PERK hyperactivity is an increased amount of PERK activity compared to normal levels of PERK activity in a particular subject or a population of healthy subjects.
  • the increased amount of PERK activity may result in, for example, excessive amounts of cell proliferation thereby causing the disease state.
  • the disease is associated with PERK activity deficiency.
  • diseases may include, but are not limited to, cystic fibrosis, retinitis pigmentosa, diabetes, or a neurodegenerative disease.
  • the neurodegenerative disease may include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HlV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson'
  • the disease is associated with abnormally high PERK activity.
  • diseases may include, but are not limited, to cancers, inflammatory diseases, or autoimmune diseases.
  • Exemplary cancers include, but are not limited to, breast cancer (e.g., triple negative breast cancer) or multiple myeloma.
  • the disease is multiple myeloma.
  • Exemplary inflammatory diseases include, but are not limited to, asthma, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease; reperfusion injury, rheumatoid arthritis, transplant rejection, and vasculitis.
  • Exemplary autoimmune diseases include, but are not limited to, XBP1- linked Crohn's disease, Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sj5gren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis.
  • the disease is XBP1 -linked Crohn's disease.
  • the subject of treatment for the disease is typically a mammal.
  • the mammals treated with the compound may be humans, nonhuman primates, and/or non- human mammals (e.g., rodents, canines).
  • a method of treating a PERK activity-associated disease in a subject in need of such treatment including administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • a compound as described herein for use as a medicament is provided.
  • the medicament may be useful for treating a PERK activity-associated disease in a subject in need of such treatment.
  • the use may include administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • a compound for use in the treatment of a PERK activity- associated disease in a subject in need of such treatment may include administering a compound, or a pharmaceutically acceptable salt thereof, as described herein, including embodiments (e.g. an example, table, figure, or claim).
  • the compound, or pharmaceutically acceptable salt thereof is included in an effective amount. In embodiments of the method or use, the compound, or pharmaceutically acceptable salt thereof, is included in a therapeutically effective amount. In embodiments of the method or use, the compound, or pharmaceutically acceptable salt thereof, is included in a prophylactically effective amount.
  • the method includes administering a second agent (e.g. therapeutic agent). In embodiments, the method includes administering a second agent (e.g. therapeutic agent) in a therapeutically effective amount. In embodiments, the use includes a second agent (e.g. therapeutic agent). In embodiments, the use includes a second agent (e.g. therapeutic agent) in a therapeutically effective amount. In embodiments, the second agent is a PERK inhibitor (e.g., GSK). In embodiments, the PERK inhibitor is a protein (e.g., antibody, antibody fragment), nucleic acid (e.g., antisense nucleic acid, siRNA, miRNA), or small molecule (e.g., GSK).
  • PERK inhibitor is a protein (e.g., antibody, antibody fragment), nucleic acid (e.g., antisense nucleic acid, siRNA, miRNA), or small molecule (e.g., GSK).
  • the second agent is an Irel inhibitor (e.g., AD60).
  • the Irel inhibitor is a protein (e.g., antibody, antibody fragment), nucleic acid (e.g., antisense nucleic acid, siRNA, miRNA), or small molecule (e.g., AD60).
  • the second agent is a PERK inhibitor (e.g., GSK, GSK2606414, GSK2656157).
  • the second agent is a proteasome inhibitor (e.g., bortezomib).
  • the second agent is thalidomide.
  • the second agent is lenalidomide.
  • the second agent is a treatment for a neurodegenerative disease (e.g., rivastigmine, galantamine, memantine, donepezil;
  • levodopa carbidopa, apomorphine, bromocriptine, rotigotine, pramipexole, ropinirole, benzotropine, trihexyphenidyl, selegiline, rasagiline, entacapone, tolcapone, amantadine, droxidopa; tetrabenazine, haloperidol, chlorpromazine, risperidone, quetiapine, levetiracetam, clonazepam; riluzole).
  • the treatment is prevention.
  • the compounds set forth herein are provided as pharmaceutical compositions including the compound and a
  • the disease is described herein, including in an aspect, embodiment, example, figure, table, definition (e.g., an example of a metabolic disease, cancer,
  • neurodegenerative disease inflammatory disease, disease associated with Irel activity, or disease associated with PERK activity), or claim.
  • a method of treating a disease including administering an effective amount of a compound as described herein.
  • a compound as described herein for use as a medicament e.g., for treatment of a disease.
  • a compound as describe herein for use in the treatment of a disease e.g., including administering an effective amount of a compound as described herein.
  • the disease is arrhythmia (e.g., associated with diabetic cardiomyopathy) (Liu et al. Cardiovasc Res. 2014 Jul 15; 103 Suppl 1 :S116), diabetic nephropathy (Zhuang et al, J Endocrinol. 2014 Jun 30.
  • cystic fibrosis cystic fibrosis, retinitis pigmentosa, diabetes, a neurodegenerative disease, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado-Joseph disease
  • Schizophrenia Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, cancer, an inflammatory disease, an autoimmune disease, breast cancer (e.g., triple negative breast cancer), multiple myeloma, asthma, chronic inflammation, chronic prostatitis, glomerulonephritis,
  • hypersensitivities inflammatory bowel diseases, pelvic inflammatory disease; reperfusion injury, rheumatoid arthritis, transplant rejection, vasculitis, XBP1 -linked Crohn's disease, Coeliac disease, Celiac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sj5gren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, or rheumatoid arthritis.
  • IDDM diabetes mellitus type 1
  • SLE systemic lupus erythematosus
  • Sj5gren's syndrome Churg-Strauss Syndrome
  • Hashimoto's thyroiditis Graves' disease, idiopathic thrombocytopenic purpura, or rheumatoid arthritis.
  • a method of modulating Irel activity in a subject in need thereof including administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.
  • the method includes activating Irel activity.
  • the method includes inhibiting Irel activity.
  • the cell is a mammalian cell, such as a human cell.
  • the cell may be isolated in vitro, form part of a tissue in vitro, or may form part of an organism.
  • Modulating Irel activity includes directly or indirectly affecting one or more functions of Irel and/or one or more downstream effects of Irel. In other words, the function or effect of Irel is altered compared to the function or effect of Irel when the modulator is not present.
  • the Irel modulator (e.g., compound as described herein, including in embodiments) is an Irel inhibitor that decreases one or more of: activation of Irel by misfolded proteins, initiation of UPR, trans-autophosphorylation of Irel, co-factor binding by Irel, translation of Hacl, translation of Xbpl, Irel oligomerization, Irel ribonuclease activity, or correct protein folding.
  • an effective amount of Irel inhibitor is an amount that decreases Xbpl expression relative to the expression of Xbpl in the absence of Irel inhibitor.
  • an effective amount of Irel inhibitor is an amount sufficient to decrease Irel activity in the cell to reduce UPR relative to the amount of UPR in the absence of Irel inhibitor.
  • an effective amount of Irel modulator e.g., inhibitor
  • the Irel modulator (e.g., compound as described herein, including in embodiments) is an Irel activator that increases one or more of: activation of Irel by misfolded proteins, initiation of UPR, trans-autophosphorylation, co-factor binding, translation of Hacl, translation of Xbpl, Irel oligomerization, Irel ribonuclease activity, or corrected protein folding.
  • an effective amount of Irel activator is an amount sufficient to increase Irel activity in the cell to reduce misfolded protein accumulation relative to the amount of misfolded protein accumulation in the absence of Irel activator.
  • an effective amount of Irel modulator is an amount sufficient to decrease apoptosis.
  • modulating Irel activity comprises direct binding of the Irel modulator (e.g., a compound described herein) to Irel. In another embodiment, modulating Irel activity is accomplished indirectly.
  • a method of modulating PERK activity in a subject in need thereof including administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.
  • the method includes inhibiting PERK activity.
  • the method includes increasing PERK activity.
  • the cell is a mammalian cell, such as a human cell.
  • the cell may be isolated in vitro, form part of a tissue in vitro, or may form part of an organism.
  • Modulating PERK activity includes directly or indirectly affecting one or more functions of PERK and/or one or more downstream effects of PERK. In other words, the function or effect of PERK is altered compared to the function or effect of PERK when the modulator is not present.
  • the PERK modulator e.g., compound as described herein, including in embodiments
  • the PERK modulator is a PERK inhibitor that decreases one or more of: activation of
  • an effective amount of PERK inhibitor is an amount sufficient to decrease PERK activity in the cell to reduce UPR relative to the amount of UPR in the absence of the PERK inhibitor.
  • an effective amount of PERK modulator e.g., inhibitor
  • an effective amount of PERK inhibitor is an amount sufficient to decrease apoptosis.
  • an effective amount of PERK inhibitor is an amount sufficient to decrease translation attenuation (e.g., relative to control).
  • the PERK modulator (e.g., compound as described herein, including in embodiments) is a PERK activator that increases one or more of: activation of PERK by misfolded proteins, initiation of UPR, co-factor binding, translation of Hacl, translation of Xbpl, phosphorylation of PERK, phosphorylation of eIF2a, or corrected protein folding.
  • an effective amount of PERK activator is an amount sufficient to increase PERK activity in the cell to reduce misfolded protein accumulation relative to the amount of misfolded protein accumulation in the absence of PERK activator.
  • an effective amount of PERK modulator (e.g., activator) is an amount sufficient to increase apoptosis. In another embodiment, an effective amount of PERK activator is an amount sufficient to increase translation attenuation (e.g., relative to control).
  • modulating PERK activity comprises direct binding of the PERK modulator (e.g., a compound described herein) to PERK. In another embodiment, modulating PERK activity is accomplished indirectly.
  • PERK modulator e.g., a compound described herein
  • a method of modulating Ire 1 activity and PERK activity in a subject in need thereof including administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.
  • the method includes co-administering a PERK inhibitor (e.g., GSK).
  • the method includes co-administering an Irel inhibitor (e.g., AD60).
  • pect is provided a compound having the formula:
  • R 3 , R 4 , and R 5 are independently hydrogen, R 2 -substituted or unsubstituted C1-C5 alkyl, or R 2 -substituted or unsubstituted 2 to 5 membered heteroalkyl;
  • X is independently halogen.
  • halogen -OR 5 , -SR 5 , -CX 3 , -OCX3, -OCHX 2 , -OCH 2 X, -SCX 3 , -SCHX 2 , -SCH 2 X, unsubstituted C1-C2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • R 2 -substituted or unsubstituted C1-C 3 alkyl independently hydrogen, R 2 -substituted or unsubstituted C1-C 3 alkyl, or R 2 -substituted or unsubstituted 2 to 3 membered heteroalkyl.
  • R 2 -substituted or unsubstituted Ci-C 2 alkyl independently hydrogen, R 2 -substituted or unsubstituted Ci-C 2 alkyl, or R 2 -substituted or unsubstituted 2 membered heteroalkyl.
  • halogen -CN, -NO, -N0 2 , - H 2 , -OH, -SH, -CX 3 , -NHNH 2 , -ONH 2 , -NHOH, -OCX3, -OCHX 2 , unsubstituted Ci-C 2 alkyl, or unsubstituted 2 membered heteroalkyl.
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of one of embodiments 1 to 24, or a pharmaceutically acceptable salt thereof.
  • Molecules made contain a urea-linked and variably m- and ⁇ -substituted phenyl group.
  • IREla-KR43 comprises the soluble cytoplasmic portion of IREla, composed of the kinase and RNase domains plus 43 amino acids of the linker that bridges the kinase to its transmembrane domain.
  • IREla prefers a substituent with polarizable character at the ara-position of the terminal phenyl ring, perhaps indicating that the ring occupies a hydrophobic region in IREla's active site. Increasing bulkiness at the ara-position resulted in decreased activities (cmp8, cmp9, and cmplO), imply that the size of this pocket must be limited.
  • cmp6 we decided to further characterize compound cmp6, based on its robust activation properties. Henceforth, we refer to cmp6 as IPA (for IREl/PERK Activator). To validate that binding of IPA to IREla's kinase domain is critical for IREla RNase activation, we generated a control compound, IP Ax. In IP Ax, the pyrazole ring bears an additional l -methyl group that is predicted to sterically interfere with binding to the gatekeeper pocket in IREla (FIG. 1A).
  • IP Ax was inactive showing an indistinguishable effect from the DMSO control (FIG. IB).
  • ATP mimetics such as APY29
  • IREla-KR43 oligomerization induces the RNase activity 14 ' 15 .
  • RNase activity was measured as a function of enzyme concentration in the presence of saturating compound concentrations (20 ⁇ ; FIG. 1C and FIG. 6).
  • apo-IREla-KP43 RNase activity measured as k 0 b s under single-turnover conditions, increased sharply with increasing enzyme concentration, reaching 1 ⁇ 2 k maX;0 b s at 8 ⁇ (FIG. 1C, diagonal crossed circles, far right curve).
  • addition of IP Ax did not enhance IREla activation, showing activation kinetics that were in all respects indistinguishable from the no-compound control (FIG.
  • HEK293T, T-REx293 and wild type, Irel ⁇ and Perk ⁇ cells MEF cells were maintained at 37°C, 5% C0 2 in Dulbecco's Modified Eagle Medium (DMEM)
  • FBS Fetal Bovine Serum
  • Peptomycin 10 units/ml penicillin
  • 10 ⁇ g/ml streptomycin 10 units/ml
  • Tunicamycin was obtained from Sigma.
  • Transient and stable transfections were performed using the Lipofectamine 2000 (Invitrogen) and FuGene6 reagent (Roche). Stable cell lines expressing IRE1-3F6HGFP T-REx293 were described previously 5 .
  • RNA Isolation and RT-PCR Cells were lysed and total RNA was collected (Total R A Kit 1, EZ-R A, EZNA (USA)). PolyA + mRNA was reverse transcribed with M-MLV (Invitrogen) and the resulting cDNA was used as template for PCR amplification across the fragment oiXBPl cDNA containing the intron. Primers used to amplify human XBP1: 5'- TTACGAGAGAAAACTCATGGC - 3 ' (SEQ ID NO:3) and 5'-
  • AGGCAACAGTGTCGAGTCC - 3' (SEQ ID NO:6).
  • PCR conditions were as follows: 95°C for 5 min, 95°C for 1 min, 58°C for 30 s, 72°C for 30 s, and 72°C for 5 min, with 35 cycles of amplification.
  • PCR products were resolved on a 2.5% agarose tris-acetate EDTA (TAE) gel.
  • TAE agarose tris-acetate EDTA
  • PCR conditions were: 95°C x 5 min, 95°C x 1 min, 56°C x 30 s, 72°C for 30 s for 30 cycles, 72°C for 5 min at 4°C. PCR products were resolved on a 1% agarose TAE gel.
  • the 6xHis was cleaved as described 5 and the resulting protein was then loaded onto a HisTrap HP, 5 x 5 ml column to remove the uncleaved protein.
  • the cleaved protein was then loaded on a Mono-S 5/50 GL column (GE Healthcare), and the eluate was then concentrated to 5 mg/ml and loaded on a Superdex 200 HR 10/300 (GE Healthare) column in buffer containing 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 10% Glycerol and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP).
  • TCEP tris(2-carboxyethyl)phosphine
  • the murine PERK kinase domain (580-1077aa) was cloned into a pGEX4Tl vector to create a fusion protein containing N- terminal GST.
  • Cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgC12, 3 mM ⁇ - mercaptoethanol, 2.5% Glycerol) and lysed by sonication. After centrifugation, the supernatant was applied to a GSH-Sepharose column and washed with buffer A and buffer A containing 500 mM KC1 and 1 mM ATP. Sample was eluted with buffer A containing 20 mM glutathione.
  • buffer A 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgC12, 3 mM ⁇ - mercaptoethanol, 2.5% Glycerol
  • GST- PERK kinase domain was then concentrated and further purified on a Superdex 200 10/300 gel filtration column equilibrated in buffer B (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , 3 mM DTT, 1% Glycerol). Fractions containing GST-PERK kinase domain were concentrated and flash-frozen in liquid nitrogen and stored at -80°C.
  • anti-P-actin 1 10,000 (catalog number A5441, Sigma), anti-total PERK at 1 : 1000 (catalog number C33E10, Cell Signaling Technology), anti-phospho eIF2a at 1 : 1,000 (catalog number 3597S, Cell Signaling Technology), anti-total eIF2a at 1 : 1,000 (L57A5, Cell Signaling Technology).
  • membranes were washed in PBS with 0.05% Tween and incubated in HRP-coupled secondary antibody anti-rabbit (catalog number 611-1302, Rockland) or anti-mouse (catalog number 610-1302, Rockland) diluted at 1 :5,000 in wash buffer.
  • IRE1 endoribonuclease activity was detected employing two in vitro assays. k 0bs and Hill coefficients were determined from the cleavage kinetics of [ 32 P]-labeled RNA substrates as previously described 4 . The assay was started by adding 1 ⁇ of [ 32 P]-labeled RNA to 9 ⁇ of premixture containing 20 mM HEPES pH 7.4, 70 mM NaCl, 2 mM MgCl 2 , 4 mM DTT, 5% glycerol, 1 ⁇ of 20 ⁇ compound in DMSO.
  • Reactions were performed at 30°C. Reactions were conducted under single turnover conditions. They contained ⁇ 1 pM [ 32 P]-labeled RNA and increasing concentrations of purified IREla- KR43. Reactions were quenched at time intervals with 6 ⁇ stop solution (10 M urea, 0.1% SDS, 0.1 mM EDTA, 0.05% xylene cyanol, and 0.05% bromophenol blue). Samples were analyzed in 10-20% Urea-PAGE gels. Gels were scanned using a Typhoon variable mode imager (GE)
  • EC5 0 values were determined using a FRET-based cleavage assay.
  • a FRET probe FRET IDT l 7 56- FAM/rCrArCrCrUrCrUrGrCrArGrCrArGrGrUrG/IABlk_FQ) (SEQ ID NO: 13) was purchased from IDT (RNase free HPLC purification) and dissolved in RNase-free H2O. Excitation: 485 (480-490) and emission: 517 (512-520).
  • RNA FRET probe 100 nM
  • RNA FRET probe 100 nM
  • RNA FRET probe 100 nM
  • a premixture containing 20 mM HEPES, pH 7.4, 70 mM NaCl, 2 mM MgCl 2 , 4 mM DTT, 5% glycerol, and compound in DMSO not to exceed 1%.
  • Reactions were performed at 30°C and contained 150 nM enzyme under single turnover conditions. Reactions were quenched at time intervals with equal volumes of formamide.
  • Samples were analyzed using a SpectraMax M5 plate reader equipped with SoftMax®Pro 5 and data acquisition software (Molecular Devices). The data were plotted using SigmaPlot software package (Systat Software). Experiments were repeated 3-4 times and mean values were computed.
  • IRE1 Kinase Assay In vitro IRE1 Kinase Assay. Kinase assays were performed using 0.1 ⁇ of purified IREla-KR43 in kinase buffer (20 mM HEPES, pH 7.0, 70 mM NaCl, 2 mM Mg(OAc) 2 , 5 mM DTT, and 5% glycerol) and varying concentrations of compounds. Kinase reactions were initiated by the addition of 100 ⁇ cold ATP supplemented with 5 ⁇ ⁇ -[ 32 ⁇ ] ATP and allowed to proceed at room temperature.
  • kinase buffer 20 mM HEPES, pH 7.0, 70 mM NaCl, 2 mM Mg(OAc) 2 , 5 mM DTT, and 5% glycerol
  • Titration data were fit to a sigmoidal dose response to derive IC5 0 values using the SigmaPlot software package (Systat software). Dose responses were based on a 12-point inhibitor titration, using a semi-logarithmic dilution series starting from 100 ⁇ . Experiments were repeated 2-4 times and mean values were computed.
  • AD60 IRE la inhibitor
  • XBPl mRNA-splicing reporter that produces an 5 7-luciferase fusion protein only after IRE 1 -mediated splicing of its mRNA
  • IC5 0 0.85 ⁇ for AD60 in HEK293T cells
  • AD60 inhibited Tm-induced XBPl mRNA splicing (FIG. 2C, lane 3).
  • AD60 fully ablated IPA-induced XBPl mRNA splicing (FIG. 2C, lanes 9-12), even at the highest IPA concentration tested. Taken together, these results confirm that IPA binds to the ATP -pocket of IRE la to induce XBP1 mRNA splicing in cells.
  • T-REx293 cell line containing a genome-integrated doxycycline- inducible IREla-GFP fusion gene, to monitor IRE la oligomerization in vivo 5 .
  • IREla-GFP was expressed and localized to the ER (FIG. 2D).
  • Tm rapidly induced the relocalization of IREla-GFP into discrete foci, indicative of IREla oligomerization and activation 5 .
  • the foci dissolved by eight hours of treatment as cells attenuated IREla signaling even in the presence of unmitigated ER stress (FIG. 2D, upper panels) 5 .
  • IPA caused the rapid relocalization of IREla-GFP into foci, consistent with sustained IRE1 activation (FIG. 2D, lower panels). While Tm- induced foci became larger and fewer over time as previously observed, IPA-induced foci remained small and numerous. Interestingly, IPA-induced foci remained stable even at late time points, consistent with the results obtained for XBP1 mRNA splicing (FIG. 2A). These results suggest that ⁇ - binding to IREla stabilizes IRE la's oligomeric state, interfering with its attenuation. [0280] We next tested the effect of IPA on cell viability. As shown in FIG. 2E, IPA killed cells with an LD 50 of 0.82 ⁇ .
  • T-REx293 cells were split two days before imaging onto glass- bottom micro-well dishes (MatTek) at 5 x 10 4 cells/dish.
  • Doxycyclin-containing medium (10 nM doxycycline) was added for 24 h, withdrawn before imaging and replaced with imaging media (Hank's Balanced Salt Solution (GIBCO), 2% FBS, and 5 mM HEPES pH 7.0). Images were acquired on a spinning-disk confocal microscope as described 5
  • HEK293T cells were grown in DMEM (Sigma) complete media containing 10% FBS, 10 units/ml penicillin (Invitrogen) and 10 ⁇ g/ml streptomycin (Invitrogen). Cells were plated at a density of 30,000 cells per well in 96-well black plates with clear flat bottoms (Corning) 24 h before the experiment. A 12-point semi-logarithmic dilution series of compounds was made starting at 30 ⁇ not to exceed 0.1% DMSO after final dilution into the growth media. Viability assays were conducted over the course of 24 h. Cell viability was determined using CellTiter-Blue (Promega) following the manufacturer's instructions.
  • the coding sequence of the reporter was amplified by PCR using primers with engineered BamHI and EcoRI sites, and was subsequently cloned into the cognate sites of the retroviral expression vector pBABE.puro (Addgene) to generate construct DAA-A171.
  • DAA-A171 was used to generate recombinant retroviral particles using standard methods and the resulting retroviral supernatant was used to transduce HEK293T cells, which were then subsequently selected with puromycin to create a stable reporter cell line.
  • PERK activation by IPA could be due to direct binding of IPA to PERK, or it could be caused by indirect activation of PERK by IRE1. If the latter were true, IPA should not trigger PERK activation in the absence of IRE 1.
  • MEFs mouse embryonic fibroblasts
  • the membrane was washed in 1% phosphoric acid and the sheets were washed five times in buffer, dried, and transferred radioactivity was measured using a Typhoon variable mode imager (GE Healthcare) and quantified using the ImageQuant (Molecular Devices). Titration data were fit to a sigmoidal dose response to derive IC5 0 values using the SigmaPlot software package (Systat software). Dose responses were based on a 12-point inhibitor titration, using a semi-logarithmic dilution series starting from 30 ⁇ . Experiments were completed 2-4 times and mean values were computed. E. IPA activates PERK at low concentrations but inhibits it at higher concentrations
  • FIGS. 3c and 4a The observation of IPA-induced PERK inhibition (shown in FIGS. 3c and 4a) poses the conundrum of why we observed IPA-induced activation in the low micromolar IPA range, producing an unusual, bell-shaped dose response (FIG. 3C).
  • This behavior could be explained by a model posing that at lower IPA concentrations, IPA occupies the ATP -binding sites of a subset of PERK molecules causing activation the kinase activity of neighboring, unoccupied PERK molecules, as previously shown for other kinases 21"23 . At higher IPA concentrations, IPA would saturate all PERK molecules, thereby inhibiting the pathway.
  • PERK inhibitors block IPA-induced PERK activation and restore translation
  • GSK2606414 24 abbreviated as "GSK”
  • GSK selective PERK inhibitor
  • GSK added at 1 ⁇ protected cells from IPA-induced cell death, shifting the LD 50 from 0.8 ⁇ to 6.2 ⁇ (FIG. 5D).
  • PI small molecules activators
  • IPA a strong activator of IREla signaling in vitro, by trapping IREla's kinase domain in its active (DFG-in) conformation, which promotes IRE1 oligomerization.
  • IREl 's RNase becomes activated 4 , offering the unique opportunity to read-out the conformational status of a kinase domain in the absence of phospho-transfer.
  • a compound, AD60 that traps IREl 's kinase domain in its inactive (DFG-out) conformation acts as an inhibitor of IRE 1 signaling.
  • IREl 's kinase domain acts as a conformational switch, in which ligand binding to the ATP binding pocket— rather than enzymatic phospho-transfer— controls activity and down-stream signal transduction events 14 .
  • IPA exerts its IRE1 activating activity by causing general ER stress, we explored its effects on the two other branches of the UPR that signal through ATF6 and PERK. Unexpectedly, we discovered that IPA also activated PERK.
  • PERK activation displayed a bell-shaped dose response: PERK was activated at a low IPA concentrations while being inhibited at a higher ones. Strikingly, this mode of PERK activation by IPA mirrors RAF activation by PLX4720 22 ' 23 , a well-documented phenomenon governing the modulation of this kinase by ATP-mimetics.
  • the PERK/IPA system resembles the RAF/PLX4720 system and broadens the number of still exotic examples where an ATP-competitive binder would activate rather than inhibit a kinase.
  • this mode of kinase regulation is now documented for two different protein kinases, it could be a common feature of multiple protein kinases. If so, it could be important to consider this feature during therapeutic uses of kinase inhibitors.
  • IPA-mediated activation of IRE 1 and PERK occurred in the absence of ER stress. This property contrasts with that of the previously described activator 1NM-PP1, which binds in the ATP binding pocket to a cognate, analog-sensitized IRE1 allele (IREl-as). Activation of IREl-as by 1NM-PP1 requires the additional induction of ER stress 15 , or overproduction of IREl-as driving IREl-as oligomerization by mass action 16 .
  • IPA therefore presents a unique pharmacological tool with which activation of wild-type IREl can be studied in living cells directly in the absence of these complications.
  • the select application of combinations of UPR modulators has allowed us to begin dissecting the individual contributions of the signaling branches of the UPR.
  • the lethality that IPA displays at high doses can be partially overcome if the PERK pathway is inactivated using a selective PERK inhibitor, GSK, increasing the EC5 0 with which IPA drives cells into apoptosis by about an order of magnitude. This observation is consistent with the proposed roles of both the IREl and PERK branches, providing
  • cytoprotective and pro-apoptotic outputs respectively 20 .
  • AD60 as a DFG-out kinase inhibitor, reversed the effects of IPA on IREl.
  • IPA, AD60, and other compounds developed to date therefore provide a stepping stone towards developing novel methodologies for the selective pharmacological tuning of the UPR.
  • Diseases such as multiple myeloma, a cancer of highly secretory plasma cells where the UPR is thought to play a major cytoprotective role 26 ' 27 , or triple-negative breast cancer 28 , in which high XBP1 activity has been correlated with poor patient prognosis, have exposed the potential significance of targeting the UPR in cancers.
  • the chemical biology tools developed during this work make a step toward UPR-based therapies, as well as offer fundamental mechanistic insights into a key mechanism that keeps the healthy balance of protein folding in the ER.

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Abstract

L'invention concerne, entre autres, des compositions et des méthodes pour moduler la réponse aux protéines mal repliées.
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WO2018102751A1 (fr) 2016-12-02 2018-06-07 Quentis Therapeutics, Inc. Inhibiteurs de petites molécules ire1
WO2020117634A1 (fr) * 2018-12-03 2020-06-11 Cornell University Inhibiteurs à petites molécules d'ire1
CN111566089A (zh) * 2017-11-10 2020-08-21 康奈尔大学 Ire1小分子抑制剂
CN113493436A (zh) * 2020-04-03 2021-10-12 中国医学科学院药物研究所 胺基取代吡啶衍生物及其制法和药物组合物与用途

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US20110319436A1 (en) * 2008-09-15 2011-12-29 The Regents Of The University Of California Methods and Compositions for Modulating IRE1, SRC, and ABL Activity

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10934275B2 (en) 2016-12-02 2021-03-02 Cornell University IRE1 small molecule inhibitors
EP3548476A4 (fr) * 2016-12-02 2020-05-20 Quentis Therapeutics, Inc. Inhibiteurs de petites molécules ire1
WO2018102751A1 (fr) 2016-12-02 2018-06-07 Quentis Therapeutics, Inc. Inhibiteurs de petites molécules ire1
US11634403B2 (en) 2017-11-10 2023-04-25 Cornell University IRE1 small molecule inhibitors
CN111566089A (zh) * 2017-11-10 2020-08-21 康奈尔大学 Ire1小分子抑制剂
CN111566089B (zh) * 2017-11-10 2023-11-17 康奈尔大学 Ire1小分子抑制剂
US12071424B2 (en) 2017-11-10 2024-08-27 Cornell University IRE1 small molecule inhibitors
WO2020117634A1 (fr) * 2018-12-03 2020-06-11 Cornell University Inhibiteurs à petites molécules d'ire1
EP4201924A1 (fr) * 2018-12-03 2023-06-28 Cornell University Inhibiteurs à petites molécules d'ire1
US11945784B2 (en) 2018-12-03 2024-04-02 Cornell University IRE1 small molecule inhibitors
US12404248B2 (en) 2018-12-03 2025-09-02 Cornell University IRE1 small molecule inhibitors
CN113493436A (zh) * 2020-04-03 2021-10-12 中国医学科学院药物研究所 胺基取代吡啶衍生物及其制法和药物组合物与用途
CN113493436B (zh) * 2020-04-03 2023-10-20 中国医学科学院药物研究所 胺基取代吡啶衍生物及其制法和药物组合物与用途

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