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WO2024243015A2 - Composés pour traitement de paraplégie spastique héréditaire - Google Patents

Composés pour traitement de paraplégie spastique héréditaire Download PDF

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Publication number
WO2024243015A2
WO2024243015A2 PCT/US2024/029856 US2024029856W WO2024243015A2 WO 2024243015 A2 WO2024243015 A2 WO 2024243015A2 US 2024029856 W US2024029856 W US 2024029856W WO 2024243015 A2 WO2024243015 A2 WO 2024243015A2
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substituted
compound
unsubstituted
alkyl
certain embodiments
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WO2024243015A3 (fr
Inventor
Darius EBRAHIMI-FAKHARI
Mustafa SAHIN
Jed L. Hubbs
Masato Satoh
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Boston Childrens Hospital
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Boston Childrens Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • C07D405/02—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 two hetero rings
    • C07D405/12—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 two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Adaptor protein complex 4 (AP-4)-related hereditary spastic paraplegia (AP-4-HSP, which includes AP4B1-associated SPG47 (OMIM #614066), AP4M1-associated SPG50 (OMIM #612936), AP4E1-associated SPG51 (OMIM #613744) and AP4S1-associated SPG52 (OMIM #614067), is a rare but prototypical form of childhood-onset complex hereditary spastic paraplegia (HSP) and an important genetic mimic of cerebral palsy.
  • HSP hereditary spastic paraplegia
  • AP-4-HSP Children with AP-4-HSP present with features of both a neurodevelopmental disorder (e.g., early-onset global developmental delay and seizures, microcephaly, and developmental brain malformations) and a neurodegenerative disease (e.g., progressive spasticity and weakness, loss of ambulation, and extrapyramidal movement disorders).
  • AP-4-HSP is caused by bi- allelic loss-of-function variants in any of the four AP-4 subunits ( ⁇ , ⁇ 4, ⁇ 4, ⁇ 4), leading to impaired AP-4 assembly and function.
  • AP-4 is an obligate heterotetrameric protein complex that mediates transport from the trans-Golgi network (TGN) to the cell periphery, including sites of autophagosome biogenesis.
  • TGN trans-Golgi network
  • the core autophagy protein and lipid scramblase ATG9A has been identified as a major cargo of AP-4, linking loss of AP-4 function to defective autophagy.
  • AP-4 deficiency in non-neuronal and neuronal cells leads to an accumulation of ATG9A in the TGN, including in iPSC-derived neurons from AP-4-HSP patients.
  • AP-4 is required for trafficking of ATG9A from the TGN;
  • loss-of-function variants in AP-4 subunits lead to a loss of AP-4 function;
  • ATG9A accumulates in the TGN leading to a reduction of axonal delivery of ATG9A;
  • lack of ATG9A at the distal axon impairs autophagy leading to axonal degeneration.
  • AP-4 cargo proteins identified to date include the poorly characterized transmembrane proteins SERINC1 and SERINC3, and the endocannabinoid producing enzyme DAG lipase beta (DAGLB).
  • AP-4 cargo proteins identified to date include the poorly characterized transmembrane proteins SERINC1 and SERINC3, and the endocannabinoid producing enzyme DAG lipase beta (DAGLB).
  • AP-4 cargo proteins identified to date include the poorly characterized transmembrane proteins SERINC1 and SERINC3, and the endocannabinoid producing enzyme DAG lipase beta (DAGLB).
  • AP-4 cargo proteins identified to date include the poorly characterized transmembrane proteins SERINC1 and SERINC3, and the endocannabinoid producing enzyme DAG lipase beta (DAGLB).
  • AP-4 cargo proteins identified to date include the poorly characterized transmembrane proteins SERINC1 and SERINC3,
  • compounds of the disclosure restore ATG9A pathology in multiple disease models, including patient-derived fibroblasts and iPSC-derived neurons.
  • multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches identify putative molecular targets of the disclosed compounds and their mechanisms of action.
  • Molecular regulators of intracellular ATG9A trafficking are also identified.
  • each occurrence of R 1 is, independently, halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, -OR A , -N(R A ) 2 , -SR A , -CN, -S
  • compositions comprising a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, and a pharmaceutically acceptable excipient.
  • kits comprising a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition of the disclosure, and instructions for administering the compound or pharmaceutical composition to a subject in need thereof.
  • methods of treating a neurological disease or disorder comprising administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition of the disclosure, to a subject in need thereof.
  • ATG9A Autophagy Related 9A trafficking in or from a cell
  • the methods comprising contacting an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition of the disclosure, with the cell.
  • methods of modulating intracellular vesicle trafficking and increasing autophagic flux in a cell comprising contacting an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition of the disclosure, with the cell.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition of the disclosure, with the cell.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19 F with 18 F, or the replacement of 12 C with 13 C or 14 C are within the scope of the disclosure.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • a range of values is listed, it is intended to encompass each value and sub-range within the range.
  • C 1-6 alkyl is intended to encompass, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
  • aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups.
  • heteroaliphatic refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C 1-10 alkyl”). In certain embodiments, an alkyl group has 1 to 9 carbon atoms (“C 1-9 alkyl”). In certain embodiments, an alkyl group has 1 to 8 carbon atoms (“C 1-8 alkyl”). In certain embodiments, an alkyl group has 1 to 7 carbon atoms (“C 1-7 alkyl”). In certain embodiments, an alkyl group has 1 to 6 carbon atoms (“C 1-6 alkyl”). In certain embodiments, an alkyl group has 1 to 5 carbon atoms (“C 1-5 alkyl”).
  • an alkyl group has 1 to 4 carbon atoms (“C 1-4 alkyl”). In certain embodiments, an alkyl group has 1 to 3 carbon atoms (“C 1-3 alkyl”). In certain embodiments, an alkyl group has 1 to 2 carbon atoms (“C 1-2 alkyl”). In certain embodiments, an alkyl group has 1 carbon atom (“C 1 alkyl”). In certain embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2-6 alkyl”).
  • C 1-6 alkyl groups include methyl (C 1 ), ethyl (C 2 ), propyl (C 3 ) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C 5 ) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C 6 ) (e.g., n-hexyl).
  • alkyl groups include n-heptyl (C 7 ), n- octyl (C 8 ), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F).
  • substituents e.g., halogen, such as F
  • the alkyl group is an unsubstituted C 1-10 alkyl (such as unsubstituted C 1-6 alkyl, e.g., ⁇ CH 3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)).
  • unsubstituted C 1-6 alkyl such as unsubstituted C 1-6 alkyl, e.g., ⁇ CH 3 (Me),
  • the alkyl group is a substituted C 1-10 alkyl (such as substituted C 1-6 alkyl, e.g., ⁇ CF 3 , Bn).
  • haloalkyl is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.
  • the haloalkyl moiety has 1 to 8 carbon atoms (“C 1-8 haloalkyl”).
  • the haloalkyl moiety has 1 to 6 carbon atoms (“C 1-6 haloalkyl”).
  • the haloalkyl moiety has 1 to 4 carbon atoms (“C 1-4 haloalkyl”). In certain embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C 1-3 haloalkyl”). In certain embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C 1-2 haloalkyl”). Examples of haloalkyl groups include ⁇ CF 3 , ⁇ CF 2 CF 3 , ⁇ CF 2 CF 2 CF 3 , ⁇ CCl 3 , ⁇ CFCl 2 , ⁇ CF 2 Cl, and the like.
  • heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-10 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-9 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-8 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-7 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-6 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC 1-5 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC 1-4 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-3 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-2 alkyl”). In certain embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC 1 alkyl”).
  • a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC 2-6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC 1-10 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC 1-10 alkyl.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds).
  • an alkenyl group has 2 to 9 carbon atoms (“C 2-9 alkenyl”).
  • an alkenyl group has 2 to 8 carbon atoms (“C 2-8 alkenyl”).
  • an alkenyl group has 2 to 7 carbon atoms (“C 2-7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C 2-6 alkenyl”).
  • an alkenyl group has 2 to 5 carbon atoms (“C 2-5 alkenyl”). In certain embodiments, an alkenyl group has 2 to 4 carbon atoms (“C 2-4 alkenyl”). In certain embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2-3 alkenyl”). In certain embodiments, an alkenyl group has 2 carbon atoms (“C 2 alkenyl”).
  • the one or more carbon- carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • Examples of C 2-4 alkenyl groups include ethenyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1- butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkenyl groups as well as pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like.
  • Additional examples of alkenyl include heptenyl (C 7 ), octenyl (C 8 ), octatrienyl (C 8 ), and the like.
  • each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents.
  • the alkenyl group is an unsubstituted C 2-10 alkenyl.
  • the alkenyl group is a substituted C 2-10 alkenyl.
  • heteroalkenyl refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-10 alkenyl”).
  • a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-9 alkenyl”). In certain embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-8 alkenyl”). In certain embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-7 alkenyl”).
  • a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-6 alkenyl”). In certain embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-5 alkenyl”). In certain embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1or 2 heteroatoms within the parent chain (“heteroC 2-4 alkenyl”).
  • a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC 2-3 alkenyl”). In certain embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC 2-10 alkenyl.
  • the heteroalkenyl group is a substituted heteroC 2-10 alkenyl.
  • alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C 2-10 alkynyl”).
  • an alkynyl group has 2 to 9 carbon atoms (“C 2-9 alkynyl”).
  • an alkynyl group has 2 to 8 carbon atoms (“C 2-8 alkynyl”).
  • an alkynyl group has 2 to 7 carbon atoms (“C 2-7 alkynyl”).
  • an alkynyl group has 2 to 6 carbon atoms (“C 2-6 alkynyl”). In certain embodiments, an alkynyl group has 2 to 5 carbon atoms (“C 2-5 alkynyl”). In certain embodiments, an alkynyl group has 2 to 4 carbon atoms (“C 2-4 alkynyl”). In certain embodiments, an alkynyl group has 2 to 3 carbon atoms (“C 2-3 alkynyl”). In certain embodiments, an alkynyl group has 2 carbon atoms (“C 2 alkynyl”). The one or more carbon- carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • Examples of C 2-4 alkynyl groups include, without limitation, ethynyl (C 2 ), 1-propynyl (C 3 ), 2- propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkynyl groups as well as pentynyl (C 5 ), hexynyl (C 6 ), and the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents.
  • the alkynyl group is an unsubstituted C 2-10 alkynyl.
  • the alkynyl group is a substituted C 2-10 alkynyl.
  • heteroalkynyl refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-10 alkynyl”).
  • a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-9 alkynyl”). In certain embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-8 alkynyl”). In certain embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2- 7 alkynyl”).
  • a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-6 alkynyl”). In certain embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-5 alkynyl”). In certain embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1or 2 heteroatoms within the parent chain (“heteroC 2-4 alkynyl”).
  • a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC 2-3 alkynyl”). In certain embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents.
  • the heteroalkynyl group is an unsubstituted heteroC 2-10 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC 2-10 alkynyl.
  • the term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C 3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In certain embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C 3-10 carbocyclyl”).
  • a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3-8 carbocyclyl”). In certain embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3-7 carbocyclyl”). In certain embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3-6 carbocyclyl”). In certain embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C 4-6 carbocyclyl”). In certain embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C 5-6 carbocyclyl”).
  • a carbocyclyl group has 5 to 10 ring carbon atoms (“C 5-10 carbocyclyl”).
  • Exemplary C 3-6 carbocyclyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
  • Exemplary C 3-8 carbocyclyl groups include, without limitation, the aforementioned C 3-6 carbocyclyl groups as well as cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like.
  • Exemplary C 3-10 carbocyclyl groups include, without limitation, the aforementioned C 3-8 carbocyclyl groups as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C 3-14 carbocyclyl.
  • the carbocyclyl group is a substituted C 3-14 carbocyclyl.
  • “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C 3-14 cycloalkyl”).
  • a cycloalkyl group has 3 to 10 ring carbon atoms (“C 3-10 cycloalkyl”).
  • a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3-8 cycloalkyl”).
  • a cycloalkyl group has 3 to 6 ring carbon atoms (“C 3-6 cycloalkyl”).
  • a cycloalkyl group has 4 to 6 ring carbon atoms (“C 4-6 cycloalkyl”). In certain embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5-6 cycloalkyl”). In certain embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C 5-10 cycloalkyl”). Examples of C 5-6 cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ).
  • C 3-6 cycloalkyl groups include the aforementioned C 5-6 cycloalkyl groups as well as cyclopropyl (C 3 ) and cyclobutyl (C 4 ).
  • Examples of C 3-8 cycloalkyl groups include the aforementioned C 3-6 cycloalkyl groups as well as cycloheptyl (C 7 ) and cyclooctyl (C 8 ).
  • each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • the cycloalkyl group is an unsubstituted C 3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C 3-14 cycloalkyl.
  • “Carbocyclylalkyl” is a subset of “alkyl” and refers to an alkyl group substituted by a carbocyclyl group, wherein the point of attachment is on the alkyl moiety.
  • heterocyclyl refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”).
  • heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon- carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.
  • the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”).
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”).
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”).
  • the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl.
  • Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione.
  • Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8- naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole,
  • Heterocyclylalkyl is a subset of “alkyl” and refers to an alkyl group substituted by an heterocyclyl group, wherein the point of attachment is on the alkyl moiety.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C 6-14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C 6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C 10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C 14 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an unsubstituted C 6- 14 aryl.
  • the aryl group is a substituted C 6-14 aryl.
  • “Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
  • heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
  • the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6- bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
  • Heteroaralkyl is a subset of “alkyl” and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety. Affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.
  • alkylene
  • a group is optionally substituted unless expressly provided otherwise.
  • the term “optionally substituted” refers to being substituted or unsubstituted.
  • alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group).
  • substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound.
  • the present invention contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • the invention is not intended to be limited in any manner by the exemplary substituents described herein.
  • halo or “halogen” refers to fluorine (fluoro, ⁇ F), chlorine (chloro, ⁇ Cl), bromine (bromo, ⁇ Br), or iodine (iodo, ⁇ I).
  • hydroxyl or “hydroxy” refers to the group ⁇ OH.
  • amino refers to the group ⁇ NH 2 .
  • substituted amino by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino. In certain embodiments, the “substituted amino” is a monosubstituted amino or a disubstituted amino group.
  • trisubstituted amino refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups, and includes groups selected from ⁇ N(R bb ) 3 and ⁇ N(R bb ) 3 + X ⁇ , wherein R bb and X ⁇ are as defined herein.
  • acyl groups include aldehydes ( ⁇ CHO), carboxylic acids ( ⁇ CO 2 H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas.
  • Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyl
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms.
  • the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an “amino protecting group”).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Nitrogen protecting groups such as carbamate groups include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t- butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1- methylethyl carbamante, 9-fluorenylmethyl carbamate (Fmo
  • Nitrogen protecting groups such as sulfonamide groups include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide
  • Ts p-toluenesulfonamide
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)- acyl derivative, N’-p-toluenesulfonylaminoacyl derivative, N’-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3- oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5- triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyr
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”).
  • Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-meth
  • the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”).
  • Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • salt refers to any and all salts, and encompasses pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • suitable inorganic and organic acids and bases include those derived from suitable inorganic and organic acids and bases.
  • pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (C 1-4 alkyl) 4 ⁇ salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • solvate refers to forms of the compound, or a salt thereof, that are associated with a solvent, usually by a solvolysis reaction.
  • This physical association may include hydrogen bonding.
  • Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • the compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates.
  • solvates include hydrates, ethanolates, and methanolates.
  • hydrate refers to a compound that is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R ⁇ x H 2 O, wherein R is the compound, and x is a number greater than 0.
  • Tautomerizations i.e., the reaction providing a tautomeric pair
  • exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.
  • isomers compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”.
  • stereoisomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.
  • stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”.
  • enantiomers When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or ( ⁇ )-isomers respectively).
  • a chiral compound can exist as either individual enantiomer or as a mixture thereof.
  • a mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
  • the term “polymorph” refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All polymorphs have the same elemental composition.
  • prodrugs refers to compounds that have cleavable groups and become by solvolysis or under physiological conditions the compounds described herein, which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
  • Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups pendant on the compounds described herein are particular prodrugs.
  • double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
  • C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, aryl, C 7-12 substituted aryl, and C 7 -C 12 arylalkyl esters of the compounds described herein may be preferred.
  • a “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal. “Disease,” “disorder,” and “condition” are used interchangeably herein.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from the specified infectious disease or inflammatory condition, which reduces the severity of the infectious disease or inflammatory condition, or retards or slows the progression of the infectious disease or inflammatory condition (“therapeutic treatment”), and also contemplates an action that occurs before a subject begins to suffer from the specified infectious disease or inflammatory condition (“prophylactic treatment”).
  • the “effective amount” of a compound refers to an amount sufficient to elicit the desired biological response.
  • the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject.
  • An effective amount encompasses therapeutic and prophylactic treatment.
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of an infectious disease or inflammatory condition, or to delay or minimize one or more symptoms associated with the infectious disease or inflammatory condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the infectious disease or inflammatory condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of infectious disease or inflammatory condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • a “prophylactically effective amount” of a compound is an amount sufficient to prevent an infectious disease or inflammatory condition, or one or more symptoms associated with the infectious disease or inflammatory condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the infectious disease or inflammatory condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • FIGs.1A-1M show the establishment of a cell-based phenotypic small molecule screening platform using ATG9A translocation as a surrogate for AP-4 function and primary screening of 28,864 novel small molecule compounds.
  • FIG.1A shows an overview of the primary screening of 28,864 novel small molecule compounds in fibroblasts from a patient with AP-4-HSP due to biallelic loss-of-function variants in AP4B1.
  • FIG.1B shows an illustration of the automated image analysis pipeline.
  • FIG.1C shows an overview of the high-throughput platform and workflow. The assay was miniaturized to 96- or 384-well microplates.
  • FIGs.1D-1F show the distribution of ATG9A fluorescence intensities inside (FIG.1D) and outside (FIG.1E) the TGN, as well as the ATG9A ratio (FIG.1F) are shown on a per cell basis.99,927 WT/LoF and 119,522 LoF/LoF cells were quantified.
  • FIG.1G shows that cell counts were measured for each experimental well.1312 wells were analyzed per condition.
  • FIGs.1H and 1I show that replicate plots were generated by random sampling of the 82 plates from the primary screen in two groups. Similar positions on the assay plates were plotted against each other with respect to the ATG9A fluorescence intensity inside the TGN (FIG.1H) and the ATG9A ratio (FIG.1I). Replicate correlations for both analysis strategies were assessed by averaging the Pearson correlation coefficients of 100 random sampling tests.
  • the ATG9A ratio shows a mean Pearson correlation coefficient (r) of 0.9, while the ATG9A fluorescence inside the TGN shows an average r of 0.82.
  • FIG.1J demonstrates the discriminative power of the ATG9A ratio in separating positive and negative controls.
  • Statistical testing was done using the T- Test. Quantification was done using per well means.1312 wells per condition were included. Positive and negative controls showed a robust separation (p ⁇ 0.0001).
  • FIG.1K shows that in order to test the robustness of separation of the ATG9A ratio between positive (WT/LoF) and negative controls (LoF/LoF), a dataset containing measurement for 99,927 WT/LoF and 119,522 LoF/LoF cells was partitioned into a training set (70% of data) and a test set (30%). A generalized linear model was trained using the training set.
  • FIG.1K The performance of the prediction model using the test set is shown in (FIG.1K).
  • the AUC was 0.96.
  • FIG.1L shows the impact of 28,864 compounds applied for 24 hours at a concentration 10 ⁇ M.
  • Z-scores for the primary metric, the ATG9A ratio are shown. All data points represent per well means.
  • the mean of the positive control (WT/LoF) is shown as a grey dotted line, indicated at “A”.
  • the grey shaded areas at “A” represent ⁇ 1 SD.
  • Active compounds were a priori defined as those reducing the ATG9A ratio by at least 3 SD compared to negative controls.
  • Toxicity was defined as a reduction of cell count of at least 2SD compared to the negative control.501 compounds show activity by reducing the ATG9A ratio by more than 3 SD.
  • FIG.1M shows the distribution of Z-scores of all non-toxic 27,412 compounds. Active compounds are highlighted in dark grey.
  • FIGs.2A-2C show that the counter-screen in fibroblasts from AP-4-HSP patients confirms 16 compounds that lead to dose-dependent redistribution of ATG9A.
  • FIG.2A shows an overview of the counter-screen of the 503 active compounds identified in the primary screen.
  • FIG.2C shows dose-response curves that were fitted using a four-parameter logistic regression model and then ED50 concentrations were calculated. All concentrations were tested in biologic duplicates. Most ED50 were in the low micromolar range (median: 4.66 ⁇ M, IQR: 8.63). Black dashed lines represent the a priori defined thresholds of +/- 3SD compared to the negative control (LoF/LoF).
  • Triangles represent toxic concentrations based on the a priori defined threshold of a reduction of cell counts of at least 2 SD compared to the negative control.
  • the “B” dashed line represents the mean of negative controls, while the “A” dashed line depicts the mean of the positive controls (WT/LoF).
  • Representative images of the ED50 are shown for each active compound. Representative images show a merge of the 4 channels: Phalloidin, DAPI, TGN and ATG9A, as well as the TGN and ATG9A channels in greyscale.
  • FIGs.3A-3O show that the orthogonal assays in AP4B1 KO SH-SY5Y cells confirm 5 active compounds.
  • FIG.3A shows an overview of the orthogonal screen of 16 active compounds in differentiated AP4B1 KO SH-SY5Y cells, a neuronal model of AP-4 deficiency.
  • Active compounds were a priori defined as those reducing the ATG9A ratio by at least 3 SD compared to negative controls. Toxicity was defined as a reduction of cell count of at least 2 SD compared to the negative control.
  • FIG.3B shows the baseline differences in ATG9A ratios of AP4B1 WT vs. AP4B1 KO SH-SY5Y cells which were quantified from 160 AB4B1 WT and 158 AB4B1 KO wells from 5 assay plates. Statistical testing was performed using a T-Test.
  • FIGs.3C-3G show dose-response curves for ATG9A ratios in AB4B1 KO cells treated with different compounds. Data points represent per-well means from 3 different assay plates. Dashed lines show mean Z-scores for positive (“A”) and negative (“B”) controls. Shaded areas represent ⁇ 1 SD.
  • FIG. 3H shows representative images of the intracellular ATG9A distribution for individual compounds. The merged image shows beta-3 tubulin, DAPI, the TGN and ATG9A. The TGN and ATG9A channels are further separately depicted in greyscale. Scale bar: 10 ⁇ m.
  • FIG.3I shows the baseline differences of DAGLB ratios in AP4B1 WT vs. AP4B1 KO cells were quantified from 192 AB4B1 WT and 192 AB4B1 KO wells from 4 assay plates. Statistical testing was done using a T-Test. Positive and negative controls showed a robust separation (p ⁇ 0.0001).
  • FIGs.3J-3N show the dose-response curves for DAGLB ratios in AB4B1 KO cells treated with different compounds. All data points represent per-well means from 4 different assay plates. Dashed lines show mean Z-scores for positive (“A”) and negative (“B”) controls. Shaded areas represent ⁇ 1 SD.
  • FIG.3O shows representative images of the intracellular DAGLB distribution for individual compounds.
  • the merge shows beta-3 tubulin, DAPI, the TGN and DAGLB.
  • the TGN and DAGLB channels are further separately depicted in greyscale. Scale bar: 10 ⁇ m.
  • FIGs.4A-4G show multiparametric profiling of 5 active compounds in AP4B1 KO SH- SY5Y cells.
  • FIG.4A shows multiparametric profiling of images of 5373 cells acquired using 4 fluorescent channels. Scale: 10 ⁇ m.
  • a total of 90 measurements per cell were generated for the cytoskeleton (beta-3 tubulin), the nucleus (DAPI), the TGN (TNG46) and ATG9A vesicles (ATG9A).
  • FIG.4B demonstrates that PCA shows different clusters of cells based on 85 phenotypic features.
  • the first two principal components (PC1 and PC2) explain 43.2% of the observed variance.
  • FIG.4C shows a bar plot summarizing the variance explained by the first 10 principal components (PCs). Most of the variance is explained by PC1 and to a lesser degree PC2.
  • FIG.4D shows the correlation analysis of PC1 with all 85 features using the Pearson correlation coefficient. Grey lines, labeled “A”, represent cut-offs for correlations >0.75.
  • FIG.4E shows a zoom-in on selected features of interest showing a correlation with PC1 >0.75.
  • FIG.4F shows a measurements of TGN intensity and descriptors of TGN shape and network complexity for the individual hit compounds as line graphs and FIG.4G is summarized using heatmap visualization.
  • FIGs.5A-5I show that compound C-01 restores ATG9A and DAGLB trafficking in iPSC-derived neurons from AP-4-HSP patients.
  • FIG.5A shows an overview of the testing of 5 active compounds in iPSC-derived cortical neurons from a patient with AP4M1-associated SPG50 compared to heterozygous controls (same-sex parent). Active compounds were defined as those reducing the ATG9A ratio by at least 3 SD compared to negative controls (patient-derived iPSC-neurons treated with vehicle).
  • FIG.5B shows baseline differences of ATG9A ratios in controls vs. patient-derived iPSC-neurons were quantified using per well means of 60 wells per condition from 5 plates. Statistical testing was done using a T-Test. Positive and negative controls showed a robust separation (p ⁇ 0.0001).
  • FIG. 5C shows representative images of iPSC-neurons from a patient with SPG50 treated with individual compounds at 5 ⁇ M for 24 hours ( ⁇ ED50 in prior experiments). The merge shows beta-3 tubulin, DAPI, the Golgi and ATG9A. The Golgi and ATG9A channels are further separately depicted in greyscale.
  • FIGs.5D-5F show dose-response curves for ATG9A ratios in iPSC-neurons from a patient with SPG50 treated with individual compounds for 24 hours, along with their morphological profiles depicted as heatmaps. All data points represent per-well means of 3-4 independent differentiations. Dashed lines show mean Z-scores for positive (“A”) and negative (“B”) controls. Shaded areas represent ⁇ 1SD.
  • FIG.5G shows the chemical synthesis and structure of compound C-01.
  • FIGs.5H-5I show dose-response curves for ATG9A and DAGLB ratios in iPSC-neurons from a patient with SPG50 (FIG.5H) and an additional patient with SPG47 (FIG.5I) after prolonged treatment with C-01 for 72 hours, along with the morphologic profile depicting changes in cellular ATG9A and DAGLB distribution, TGN intensity and morphology and cell count. All data points represent per-well means of 2 independent differentiations. Dashed lines show mean Z-scores for positive (“A”) and negative (“B”) controls. Shaded areas represent ⁇ 1 SD.
  • FIGs.6A-6F show target deconvolution using bulk RNA sequencing and weighted gene co-expression network analysis in AP4B1 KO SH-SY5Y cells treated with C-01.
  • FIG.6A shows hierarchical clustering of 12 samples using average linkage showed two main clusters based on treatment with vehicle vs. C-01, irrespective of cell line.
  • FIG.6B shows a cluster dendrogram of 18,506 expressed genes based on topological overlap. Clusters of co- expressed genes (“modules”) were isolated using hierarchical clustering and adaptive branch pruning.
  • FIG.6C shows a heatmap visualization of the correlation of gene expression profiles (“module eigengene”, ME) of each module with measured traits. Pearson correlation coefficients are shown for each cell of the heatmap.
  • FIG.6D shows intramodular analysis of module membership (MM) and gene significance (GS) for highly correlated modules, allowing identification of genes that have high significance with treatment as well as high connectivity to their modules.
  • FIG.6E shows ME expression profiles for the top 5 co- expressed modules.
  • FIG.6F shows that gene ontology enrichment analysis showed enriched pathways in 3/5 modules. Pathways were considered differentially expressed with an FDR ⁇ 0.05.
  • FIGs.7A-7D show target deconvolution using unbiased quantitative proteomics in AP4B1 KO SH-SY5Y cells and AP-4-HSP patient-derived iPSC-neurons treated with C-01.
  • FIGs.7A-7C show differential protein enrichment analysis. Statistical testing was done using protein-wise linear models and empirical Bayes statistics.
  • FIG. 7A shows SH-SY5Y cells: 8141 unique proteins were analyzed. PCA of the top 500 variable proteins shows robust separation between experimental conditions.
  • the volcano plot summarizes differential protein enrichment for AP4B1 WT and AP4B1 KO cells pooled into two groups, vehicle vs. C-01 treated. Differentially enriched proteins are depicted in black. Proteins with the most consistent enrichment profiles across all experimental conditions (see FIGs.14A-14D) are labeled and have an adjacent arrow.
  • FIG.7B shows iPSC-derived neurons: 7386 unique proteins were analyzed.
  • FIG.7C shows the integrated analysis of SH-SY5Y cells and iPSC-derived neurons: 5357 unique proteins were analyzed.
  • the volcano plot summarizes differential protein enrichment for control and AP-4-deficient cells pooled into two groups, vehicle vs. C-01.
  • FIG.7D shows the RAB protein family members RAB1B, RAB3C and RAB12 showed the most consistent profiles in response to C-01 treatment and were selected for further analysis.
  • LFQ intensities in SH- SY5Y cells (AP4B1 WT and AP4B1 KO pooled) and neurons (control and patient pooled) are shown.
  • Statistical testing was done using pairwise T-tests. P-values were adjusted for multiple testing using the Benjamini-Hochberg procedure.
  • FIGs.8A-8I show that RAB3C and RAB12 are involved in C-01-mediated vesicle trafficking and enhancement of autophagic flux.
  • FIG.8B shows AP4B1 KO SH-SY5Y cells were transfected for 72 hours with RNPs targeting RAB3C, RAB12 or both compared to NLRP5 as a non-essential control.
  • Vehicle vs. C-01 treatment at a concentration of 5 ⁇ M was administered for 24 hours.
  • Each experimental condition was tested in 8-12 wells from 2-3 independent plates.
  • the dashed line represents a reduction of the ATG9A ratio of -2 SD compared to the negative control (AP4B1 KO + sgNLRP5).
  • Knockout of RAB12 did not significantly alter the ATG9A ratio, while RAB3C knockout led to a reduction of -2 SD. Combining the knockout of RAB3C and RAB12 did not result in an additive effect.
  • FIGs.8C-8F show representative western blots of whole cell lysates. Cells were treated with vehicle vs. C- 01 at a concentration of 5 ⁇ M for 72 hours. All experiments were performed in four biological replicates. AP4E1 levels were reduced in AP4B1 KO cells, indicating reduced AP-4 complex formation. ATG9A ratios were significantly increased in AP4B1 KO cells and were not altered by C-01 treatment.
  • FIGs. 8G-8I show western blots of whole cell lysates of AP4B1 KO SH-SY5Y cells transfected for 72 hours with RNPs against RAB3C, RAB12 or both, compared to NLRP5. Vehicle vs. C-01 treatment was administered for 48 hours.
  • FIGs.9A-9B show quality metrics of the ATG9A translocation assay in the primary screen and counter-screen. Assay performance was monitored in the (FIG.9A) primary screen and (FIG.9B) counter-screen using criteria proposed by Zhang et al. and included a Z’ robust ⁇ 0.3, a strictly standardized median difference (SSMD) ⁇ 3 and an inter-assay coefficient of variation ⁇ 10%. All metrics were calculated with respect to the positive and negative controls of the same assay plate to avoid bias by inter-plate variability. Predefined thresholds (“A” lines) were met by all assay plates.
  • FIGs.10A-10B show a summary of the counter-screen in AP-4-HSP patient-derived fibroblasts.
  • ED50 are indicated where possible.17 compounds demonstrated a clear and reproducible dose-response relationship and raised no suspicion for autofluorescence on automated and manual review.34 compounds were active but showed autofluorescence or resulted in imaging artifacts. Dose-response curves for all 503 compounds tested in the secondary screen.
  • FIG.11 shows that the orthogonal screen identified 11 compounds that showed no activity in AP4B1 KO SH-SY5Y cells. Eleven of 16 compounds were excluded due to either lacking activity (D-01, E-01, L-01, M-01, N-01, O-01, P-01), suspicion for artefacts or autofluorescence (I-01, J-01, K-01), or obvious changes in cellular morphology (A-01).
  • FIGs.12A-12F show multiparametric profiling of 5 active compounds in AP4B1 KO SH-SY5Y cells. PCA analysis of 85 extracted features of the nucleus, cytoskeleton/global cell morphology, TGN and ATG9A vesicles is shown.
  • FIG.12A shows baseline analysis of AP4B1 WT and AP4B1 KO cells. Cell lines clustered closely together and were only separated by the ATG9A signal.
  • FIGs.12B-12F show spatial clustering of the 5 active compounds in relation to the positive and negative controls. Compound concentrations are depicted by the legend.
  • FIGs.13A-13C show that bulk RNA sequencing in AP4B1 KO SH-SY5Y cells treated with C-01 shows a small number of differentially expressed genes, mainly involved in ER stress response.
  • FIG.13A shows volcano plots depicting the results of bulk RNA Sequencing in different experimental conditions in SH-SY5Y cells (AP4B1 WT vs.
  • AP4B1 KO treated with vehicle AP4B1 WT treated with vehicle vs. AP4B1 WT treated with C-01
  • AP4B1 WT and AP4B1 KO cells pooled in two groups vehicle vs. C-01).
  • Differential expression analysis was done following the TREAT approach developed by McCarthy and Smyth (2009). Dots labeled with “A” represent differentially expressed genes with a log 2 fold change >0.3 and an FDR ⁇ 0.05.
  • FIG.13B shows that gene ontology analysis shows enriched pathways of the pooled analysis. Pathways were considered differentially expressed with an FDR ⁇ 0.05.
  • FIG.13C portrays a Gene-Concept Network showing differentially expressed genes and their pathway membership.
  • FIGs.14A-14L show unbiased quantitative proteomics in AP4B1 KO SH-SY5Y cells and AP-4-HSP patient-derived iPSC-neurons treated with C-01.
  • FIGs.14A-14D show SH- SY5Y cells: 8141 unique proteins were analyzed. Volcano plots summarize differential protein enrichment for different experimental conditions: FIG.14A shows AP4B1 WT vs. AP4B1 KO treated with vehicle, FIG.14B shows AP4B1 WT treated with vehicle vs. AP4B1 WT treated with C-01, FIG.14C shows AP4B1 KO treated with vehicle vs.
  • FIG.14E-14H show iPSC-derived neurons: 7386 unique proteins were analyzed. Volcano plots summarize differential protein enrichment for different experimental conditions.
  • FIG.14E shows controls vs. patient- derived neurons treated with vehicle
  • FIG.14F shows controls treated with vehicle vs. controls treated with C-01
  • FIG.14G shows patient-derived neurons treated with vehicle vs. patient-derived neurons treated with C-01
  • FIG.14H shows controls and patient-derived neurons pooled in two groups, treated with vehicle vs. C-01.
  • FIGs.14I-14L show integrated analysis of SH-SY5Y cells and iPSC- derived neurons: 5357 unique proteins were analyzed. Volcano plots summarize differential protein enrichment for different experimental conditions.
  • FIG.14I shows controls vs. AP-4- deficient cells treated with vehicle
  • FIG.14J shows controls treated with vehicle vs. controls treated with C-01
  • FIG.14K shows AP-4-deficient cells treated with vehicle vs. AP-4- deficient cells treated with C-01
  • FIG.14L shows controls and AP-4-deficient cells pooled into two groups, vehicle vs. C-01.
  • Differentially enriched proteins are depicted in black.
  • FIGs.15A-15E show mRNA transcript expression and correlation analysis of RAB3C and RAB12.
  • FIGs.16A-16D show original western blots.
  • FIG.16A shows uncropped, original blots corresponding to FIG.8C.
  • FIG.16B shows an uncropped, original blot corresponding to FIG.8G.
  • FIG.16C shows an uncropped, original blot corresponding to FIG.8H.
  • FIG.16D shows an uncropped, original blot corresponding to FIG.8I.
  • FIGs.17A-B show a dose response curve and cell count number for exemplary Compound 10 in an assay testing for its effectiveness in the treatment of AP-4 deficiency.
  • FIGs.18A-B show a dose response curve and cell count number for exemplary Compound 17 in an assay testing for its effectiveness in the treatment of AP-4 deficiency.
  • the present disclosure describes the use of intracellular ATG9A mislocalization as a cellular readout for AP-4 deficiency to develop a large-scale, automated, multi-parametric, unbiased phenotypic small molecule screen for modulators of ATG9A trafficking in patient- derived cellular models.
  • a diverse library of novel small molecules were screened in AP-4- deficient patient fibroblasts to identify compounds that redistribute ATG9A from the TGN to the cytoplasm.
  • neuronal cells including differentiated AP4B1KO SH-SY5Y cells and iPSC-derived neurons from patients.
  • compounds that restore neuronal phenotypes of AP-4-deficiency were discovered. Accordingly, described herein are compounds of Formula (I). The compounds restore neuronal phenotypes of AP-4-deficiency, modulate intracellular vesicle trafficking, and increase autophagic flux.
  • each occurrence of R 1 is, independently, hydrogen, halogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, -OR A , -N(R A ) 2 , -SR A , -CN,
  • the compound is not of the formula of any one or more of the following: R 1
  • each occurrence of R 1 is, independently, unsubstituted C 1-4 alkyl. In certain embodiments, each occurrence of R 1 is, independently, unsubstituted C 1-3 alkyl. In certain embodiments, each occurrence of R 1 is, independently, unsubstituted C 1-2 alkyl. In certain embodiments, each occurrence of R 1 is, independently, -CH 3 . In certain embodiments, each occurrence of R 1 is, independently, -OR A . In certain embodiments, each occurrence of R 1 is, independently, -OR A , wherein each occurrence of R A is, independently, hydrogen or substituted or unsubstituted alkyl.
  • each occurrence of R 1 is, independently, -OR A , wherein each occurrence of R A is, independently, hydrogen or unsubstituted C 1-4 alkyl. In certain embodiments, each occurrence of R 1 is, independently, -OR A , wherein each occurrence of R A is, independently, hydrogen or unsubstituted C 1-4 alkyl. In certain embodiments, each occurrence of R 1 is, independently, - OR A , wherein each occurrence of R A is, independently, hydrogen or unsubstituted C 1-3 alkyl. In certain embodiments, each occurrence of R 1 is, independently, -OR A , wherein each occurrence of R A is, independently, hydrogen or unsubstituted C 1-2 alkyl.
  • each occurrence of R 1 is, independently, -OH or -OCH 3 . In certain embodiments, each occurrence of R 1 is, independently, -OH. In certain embodiments, each occurrence of R 1 is, independently, -OCH 3 . In certain embodiments, each occurrence of R 1 is, independently, hydrogen, -OH or - CH 3 . In certain embodiments, each occurrence of R 1 is, independently, hydrogen. In certain embodiments, each occurrence of R 1 is, independently, -OH or -CH 3 . As described herein, t is 0 or a positive integer. In certain embodiments, t is an integer from 0-10. In certain embodiments, t is an integer from 0-8.
  • t is an integer from 0-6. In certain embodiments, t is an integer from 0-5. In certain embodiments, t is an integer from 0-4. In certain embodiments, t is an integer from 0-3. In certain embodiments, t is an integer from 0-2. In certain embodiments, t is 0 or 1. In certain embodiments, t is 1. In certain embodiments, t is 0.
  • the compound of Formula (I) is a compound of Formula (I- a): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 1 and t are as defined herein.
  • the compound of Formula (I) is a compound of Formula (I- b): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 1 and t are as defined herein.
  • the compound of Formula (I) is a compound of Formula (I- c): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 1 and t are as defined herein.
  • the compound of Formula (I) is a compound of Formula (I- d): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 1 is as defined herein.
  • the compound of Formula (I) is a compound of the formula:
  • the compound of Formula (I) is a compound of the formula: or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
  • the compound of Formula (I) is a compound of the formula: or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
  • the compound of Formula (I) is a compound of the formula: or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
  • the compound of Formula (I) is a compound of the formula: or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
  • compositions comprising a compound of the disclosure (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt, co- crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition described herein comprises a compound of the disclosure (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a compound of the disclosure e.g., a compound of Formula (I)
  • the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a neurological disease or disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a neurological disease or disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a neurological disease or disorder associated with aberrant protein trafficking. In certain embodiments, the effective amount is an amount effective for treating a neurological disease or disorder associated with aberrant protein trafficking in adaptor protein complex 4 (AP-4)- deficiency. In certain embodiments, the effective amount is an amount effective for treating a hereditary spastic paraplegia (HSP).
  • HSP hereditary spastic paraplegia
  • the effective amount is an amount effective for treating Adaptor protein complex 4 (AP-4)-related hereditary spastic paraplegia (AP-4-HSP) (e.g., AP4B1-associated SPG47 (OMIM #614066), AP4M1- associated SPG50 (OMIM #612936), AP4E1-associated SPG51 (OMIM #613744), AP4S1- associated SPG52 (OMIM #614067)).
  • AP-4-HSP Adaptor protein complex 4
  • the effective amount is an amount effective for modulating Autophagy Related 9A (ATG9A) trafficking in or from a cell.
  • ATG9A Autophagy Related 9A
  • the effective amount is an amount effective for modulating intracellular vesicle trafficking and increasing autophagic flux in a cell.
  • Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the composition comprising a compound of the disclosure (e.g., a compound of Formula (I)) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.
  • the compound and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, bucal, sublingual
  • intratracheal instillation, bronchial instillation, and/or inhalation and/or as an oral spray, nasal spray
  • the compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, and/or in reducing the risk to develop a disease in a subject in need thereof), improve bioavailability, improve their ability to cross the bloodbrain barrier, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
  • activity e.g., potency and/or efficacy
  • improve their activity e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, and/or in reducing the risk to develop a disease in a subject in
  • a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent exhibit a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.
  • the compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.
  • Pharmaceutical agents include therapeutically active agents.
  • Pharmaceutical agents also include prophylactically active agents.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S.
  • CFR Code of Federal Regulations
  • peptides proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent.
  • the additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses.
  • the particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • the subject is an animal. The animal may be of either sex and may be at any stage of development.
  • the subject described herein is a human. In certain embodiments, the subject is a non-human animal.
  • the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal.
  • kits e.g., pharmaceutical packs.
  • the kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein.
  • the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.
  • kits including a first container comprising a compound or pharmaceutical composition described herein.
  • the kits are useful for treating a neurological disease or disorder (e.g., hereditary spastic paraplegia (HSP)) in a subject in need thereof.
  • the kits are useful for preventing a neurological disease or disorder (e.g., hereditary spastic paraplegia (HSP)) in a subject in need thereof.
  • the kits are useful for reducing the risk of developing a neurological disease or disorder (e.g., hereditary spastic paraplegia (HSP)) in a subject in need thereof.
  • kits are useful for for modulating Autophagy Related 9A (ATG9A) trafficking in or from a cell.
  • the kits are useful for modulating intracellular vesicle trafficking and increasing autophagic flux in a subject and/or a cell.
  • a kit described herein further includes instructions for using the kit.
  • a kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA).
  • the information included in the kits is prescribing information.
  • a kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
  • the present disclosure provides a method for treating a neurological disease or disorder. In certain embodiments, the present disclosure provides a method of treating a neurological disease or disorder associated with aberrant protein trafficking. In certain embodiments, the present disclosure provides a method of treating a neurological disease or disorder associated with aberrant protein trafficking in adaptor protein complex 4 (AP-4)-deficiency. In certain embodiments, the present disclosure provides a method of treating a hereditary spastic paraplegia (HSP). In certain embodiments, the present disclosure provides a method of treating Adaptor protein complex 4 (AP-4)-related hereditary spastic paraplegia. In certain embodiments, the present disclosure provides a method of treating AP4B1-associated SPG47 (OMIM #614066).
  • the present disclosure provides a method of treating AP4M1-associated SPG50 (OMIM #612936). In certain embodiments, the present disclosure provides a method of treating AP4E1-associated SPG51 (OMIM #613744). In certain embodiments, the present disclosure provides a method of treating AP4S1-associated SPG52 (OMIM #614067). In certain embodiments, the present disclosure provides a method of modulating Autophagy Related 9A (ATG9A) trafficking in or from a cell. In certain embodiments, the present disclosure provides a method of modulating intracellular vesicle trafficking and increasing autophagic flux in a subject and/or a cell. In certain embodiments, the cell is a mammalian cell.
  • the cell is a human cell. In certain embodiments, the cell is in a subject. In certain embodiments, the cell is in a mammal. In certain embodiments, the cell is in a human. In certain embodiments, the methods of the disclosure comprise administering to a subject an effective amount of a compound of the disclosure (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.
  • a compound of the disclosure e.g., a compound of Formula (I)
  • the effective amount
  • the subject being treated is an animal.
  • the animal may be of either sex and may be at any stage of development.
  • the subject is a mammal.
  • the subject being treated is a human.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal, such as a dog or cat.
  • the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal.
  • Certain methods described herein may comprise administering one or more additional pharmaceutical agent(s) in combination with the compounds described herein.
  • the additional pharmaceutical agent(s) may be administered at the same time as a compound of the disclosure (e.g., a compound of Formula (I)), or at different times than a compound of the disclosure (e.g., a compound of Formula (I)).
  • a compound of the disclosure e.g., a compound of Formula (I)
  • any additional pharmaceutical agent(s) may be on the same dosing schedule or different dosing schedules. All or some doses of a compound of the disclosure (e.g., a compound of Formula (I)) may be administered before all or some doses of an additional pharmaceutical agent, after all or some does an additional pharmaceutical agent, within a dosing schedule of an additional pharmaceutical agent, or a combination thereof.
  • the timing of administration of a compound of the disclosure (e.g., a compound of Formula (I)) and additional pharmaceutical agents may be different for different additional pharmaceutical agents.
  • the additional pharmaceutical agent comprises an agent useful in the treatment of a neurological disease or disorder.
  • the additional pharmaceutical agent is useful in the treatment of a neurological disease or disorder associated with aberrant protein trafficking. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of a neurological disease or disorder associated with aberrant protein trafficking in adaptor protein complex 4 (AP-4)-deficiency. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of a hereditary spastic paraplegia (HSP).
  • HSP hereditary spastic paraplegia
  • the additional pharmaceutical agent is useful in the treatment of Adaptor protein complex 4 (AP-4)-related hereditary spastic paraplegia (AP-4-HSP) (e.g., AP4B1-associated SPG47 (OMIM #614066), AP4M1- associated SPG50 (OMIM #612936), AP4E1-associated SPG51 (OMIM #613744) and AP4S1-associated SPG52 (OMIM #614067)).
  • AP-4-HSP Adaptor protein complex 4-related hereditary spastic paraplegia
  • the present disclosure provides methods for modulating Autophagy Related 9A (ATG9A) trafficking in or from a cell, the method comprising contacting the cell with a compound of the disclosure (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof.
  • a compound of the disclosure e.g., a compound of Formula (I)
  • the cell is in a subject.
  • the contacting is in a biological sample.
  • the contacting results in an increase in trafficking of ATG9A out of the trans- Golgi network (TGN).
  • the contacting results in a decrease of ATG9A in the trans-Golgi network (TGN). In certain embodiments, the contacting results in a decrease of ATG9A in the trans-Golgi network (TGN) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In certain embodiments, the contacting results in a decrease of the ratio of the concentration of ATG9A in the trans-Golgi network (TGN) to the concentration of ATG9A in the cytoplasm.
  • the ratio of the concentration of ATG9A in the trans-Golgi network (TGN) to the concentration of ATG9A in the cytoplasm is less than or equal to 1:1, less than or equal to 1.1:1, less than or equal to 1.2:1, less than or equal to 1.3:1, less than or equal to 1.4:1, less than or equal to 1.5:1, less than or equal to 1.6:1, less than or equal to 1.7:1, less than or equal to 1.8:1, less than or equal to 1.9:1, or less than or equal to 2:1 after contacting the the cell with a compound of the disclosure.
  • the ratio of the concentration of ATG9A in the trans-Golgi network (TGN) to the concentration of ATG9A in the cytoplasm is at least 1:1, at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, or at least 2:1 after contacting the the cell with a compound of the disclosure.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the present disclosure provides methods for modulating intracellular vesicle trafficking and increasing autophagic flux in a cell, the method comprising contacting the cell with a compound of the disclosure (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof.
  • a compound of the disclosure e.g., a compound of Formula (I)
  • the cell is in a subject.
  • the cell is in a biological sample.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • EXAMPLES In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope. Primary screening of 28,864 compounds in fibroblasts from AP-4-HSP patients identified 503 active compounds A diversity library of 28,864 novel small molecules was arrayed in 384-well microplates.
  • the primary screening was conducted in fibroblasts from a well-characterized patient with all core features of SPG47 and bi-allelic loss-of-function variants in AP4B1 (NM_001253852.3: c.1160_1161del (p.Thr387ArgfsTer30) / c.1345A>T (p.Arg449Ter)) (FIGs.1A-1B).
  • Fibroblasts from the sex-matched parent served as controls.
  • the assay was fully automated, miniaturized to 384-well microplates, and compounds were added for 24 hours at a single concentration of 10 ⁇ M (FIG.1C).
  • the ATG9A ratio (ATG9A fluorescence intensity inside the TGN vs. in the cytoplasm) was used as the primary assay metric.
  • the population distributions of the subcellular ATG9A signal inside and outside the TGN, at the level of single cells for negative (bi-allelic loss-of-function, LoF/LoF) and positive (heterozygous carriers, WT/LoF) controls are shown in FIG.1D and FIG.1E.
  • the ATG9A ratio demonstrated a normal distribution and robust separation of both groups (FIG.1F).
  • Cell counts were similar for positive and negative controls, excluding cell death or changes in proliferation rates as possible confounding factors (FIG.1G).
  • the ATG9A ratio as the primary outcome metric was further supported by a generalized linear model, which demonstrated high specificity and sensitivity (FIG.1K, AUC: 0.96).
  • assay performance was monitored using established quality control metrics for cell-based screens (Z’ robust ⁇ 0.3, strictly standardized median difference ⁇ 3, and an inter-assay coefficient of variation ⁇ 10%). All assay metrics were calculated for positive and negative controls of the same assay plate to avoid bias by inter-plate variability. Predefined thresholds were met by all assay plates (FIG.9A).
  • FIG.1L and FIG.1M The results of the primary screen are summarized in FIG.1L and FIG.1M.
  • 28864 compounds 26 were excluded due to non-quantifiable ATG9A signal, based on low cell counts or imaging artifacts.
  • the remaining 28,838 compounds were subsequently evaluated for changes in cell count and the ATG9A ratio.
  • Activity in the secondary screen was defined as the ability to reduce the ATG9A ratio by at least 3 SD in both replicates and at least 2 different concentrations, without exerting toxicity.51 compounds (10.1%) met these a priori defined criteria (FIGs.10A-10B). After manually verifying image quality and validating dose-response relationships, compounds were triaged (FIG.2A and FIGs.10A- 10B). Seventeen compounds demonstrated a clear and reproducible dose-response relationship, without evidence of image artifacts or autofluorescence.
  • Orthogonal assays in neuronal models of AP-4-deficiency confirmed 5 active compounds
  • the ATG9A assay was optimized for neuroblastoma-derived SH- SY5Y cells following a 5-day neuronal differentiation protocol with retinoic acid (FIG.3A).
  • SH-SY5Y cells with a stable AP4B1-knockout (AP4B1 KO ) served as negative controls while AP4B1-wildtype (AP4B1 WT ) cells were used as positive controls. All 16 active compounds were tested in 8-point dilutions (range: 50nM to 30 ⁇ M) with a treatment duration of 24 hours.
  • F- 01, G-01 and H-01 resulted in normalization of the intracellular DAGLB distribution, while B-01 and C-01 led to a moderate reduction of DAGLB ratios at higher concentrations (FIGs.3J-3K, FIG.3O).
  • B-01 and C-01 led to a moderate reduction of DAGLB ratios at higher concentrations (FIGs.3J-3K, FIG.3O).
  • a multiparametric morphological profiling approach was employed. Eighty-five measurements of the nucleus, cytoskeleton, global cell morphology, the TGN and ATG9A vesicles were automatically computed for each image, serving as a rich and unbiased source for interrogating biological perturbations induced by compound treatment.
  • FIG.4A and FIG.12 Principal component analysis was used to reduce dimensionality and cluster images based on their properties. Positive and negative controls clustered closely together and were separated only by the ATG9A signal (FIG.4B and FIG.12A). B-01, C-01 and G-01 showed properties comparable to positive and negative controls, suggesting little off-target effects (FIG.4B, FIGs.12B-12C, FIG.12E). F-01 and H-01, however, changed cellular morphology in a dose-dependent manner (FIG.4B and FIG.12D, FIG.12F), with changes mainly driven by the first principal component, accounting for 31.1% of the observed variance (FIG.4C).
  • TGN fluorescence intensity and morphological measures such as TGN area and elongation, as well as compactness and roughness, as indicators of the complexity of the TGN, were quantified for cells treated with all five active compounds (FIG. 4F-4G). While C-01 showed stable TGN signal and morphology across all assessed measurements, all other compounds depicted some degree of change. Again, F-01 and H-01 seemed to result in TGN changes in a dose-dependent manner while B-01 and G-01 led to only moderate alterations (FIG.4F-4G). Of note, these changes to TGN morphology were undetectable by visual inspection but only delineated through an automated analysis of ⁇ 600 images per group, s featuring the power of the automated, unbiased, high-throughput platform.
  • iPSCs from patients with AP-4-HSP due to biallelic loss-of-function variants in AP4M1 (NM_004722.4: c.916C>T (p.Arg306Ter) / c.694dupG (p.Glu232GlyfsTer21)) and AP4B1 (NM_001253852.3: c.1160_1161del (p.Thr387ArgfsTer30) / c.1345A>T (p.Arg449Ter)) were generated and differentiated into glutamatergic cortical neurons using established protocols.
  • iPSC-derived neurons from sex-matched parents served as controls (FIG.5A).
  • FIGs.7A- 7B Differential enrichment analyses for both cell lines are shown in FIGs.7A- 7B.
  • Baseline quantification of differentially expressed proteins in AP4B1 KO SH-SY5Y cells showed downregulation of AP-4 subunits, AP4B1, AP4E1 and AP4M1, and increased ATG9A levels (FIG.14A).
  • PCA analysis of SH-SY5Y cells demonstrated 4 distinct clusters separated by C-01 treatment (explaining 12.3% of variance) and genotype (explaining 8.7% of variance) (FIG.7A). Testing of vehicle vs.
  • C-01 treated cells showed broadly similar groups of dysregulated proteins in AP4B1 WT and AP4B1 KO SH-SY5Y cells (FIGs.14B-14D), suggesting a conserved mechanism of action independent of genotype, which allowed the pooling of cell lines to increase the power of the analysis (FIG.7A). Similar observations were made for iPSC-derived neurons (FIG.7B and FIG.14E-14H). Here cell lines were a stronger discriminator, likely due to heterogeneity of the positive and negative controls. Again, differentially enriched proteins following C-01 treatment in iPSC-neurons showed a high degree of similarity between patient and control lines (FIGs.14F-14H), allowing pooling of cell lines (FIG.7B).
  • RAB proteins involved in vesicle transport emerged as a consistent theme across cell types and genotypes, with the strongest evidence for the upregulation of RAB1B and downregulation of RAB3C and RAB12. While C-01 led to a significant change in protein levels of all three RAB protein family members in SH-SY5Y cells, only RAB3C and RAB12 reached significance in neurons (FIG.7D). This overall pattern of RAB protein modulation was further supported by upregulation of the RAB protein geranylgeranyltransferase components A1 (CHM) in SH- SY5Y cells and A2 (CHML) in both SH-SY5Y cells and neurons, which play a vital role for tethering RAB proteins to intracellular membranes.
  • CHM geranylgeranyltransferase components
  • FIG.7C upregulation of transferrin receptor protein 1 (TFRC) was observed FIG.7C), consistent with prior observations showing that reduction of RAB12 associates with increased protein levels of TFRC 38 .
  • TFRC transferrin receptor protein 1
  • mRNA levels of RAB3C and RAB12 in response to C-01 treatment were analyzed in AP4B1 WT and AP4B1 KO SH-SY5Y cells (FIGs.15A-15E). While no significant differences were detected, there was a trend toward a reduction of RAB3C (FIGs.15A, FIG.15C) and elevation of RAB12 (FIG. 15B, FIG.15D) mRNA levels.
  • the platform allows the user to determine the subcellular localization of the AP-4 cargo protein ATG9A in several cellular models of AP-4-deficiency based on ATG9A mislocalization being a key mechanism in the pathogenesis of AP-4-HSP.
  • ATG9A is the only conserved transmembrane autophagy-related protein and in mammalian cells cycles between the TGN and ATG9A vesicles, which associate with endosomes and autophagosome formation sites.
  • ATG9A has 4 transmembrane domains and forms homotrimers that have lipid scramblase activity, postulated to equilibrate lipids in the double-membrane layer of nascent autophagosomes.
  • Basal levels of autophagy are essential for neuronal survival, and neuron-specific ablation of the autophagy pathway leads to axonal degeneration and cell death.
  • autophagosomes form in the distal axon and are subject to active transport, thus efficient vesicular trafficking and spatial distribution of ATG9A are essential for axonal function as demonstrated in CNS-specific Atg9a knockout mice.
  • RAB1B the Rab proteins RAB1B, RAB3C and RAB12 were identified, as well as the interacting Rab geranyl transferase subunits CHM and CHML.
  • RAB3C and RAB12 showed the strongest and most consistent association with C-01 treatment in both SH-SY5Y cells and iPSC-derived neurons, and the analyses suggested that these two proteins are involved in C-01-mediated redistribution of ATG9A vesicles and increase of autophagic flux.
  • Rab proteins comprise a large family of small guanosine triphosphate (GTP) binding proteins that act as key regulators of intracellular membrane trafficking in eukaryotic cells, at every stage including cytoplasmic cargo sorting, vesicle budding, docking, fusion and membrane organization.
  • GTPases function both as soluble and specifically localized, integral-membrane proteins, the latter being mediated by prenylation.
  • the roughly 70 known Rab proteins more than 20 are primarily associated with the TGN, where they regulate Golgi organization, coordinate vesicle trafficking and interact with various steps of the autophagy pathway.
  • RAB3C which is part of the RAB3 superfamily, is primarily expressed in brain and endocrine tissues, where it localizes to the Golgi and synaptic vesicles and is involved in exocytosis and modulation of neurotransmitter release.
  • RAB12 is known to regulate endosomal trafficking and lysosomal degradation and has been identified as a modulator of autophagy through negative regulation of mechanistic target of rapamycin complex 1 (mTORC1).
  • mTORC1 mechanistic target of rapamycin complex 1
  • RAB12 is mainly localized to recycling endosomes where its known cargo is the transferrin receptor (TfR).
  • TfR transferrin receptor
  • Knockdown of RAB12 in mouse embryonic fibroblasts increased TfR protein levels, while overexpression led to its reduction.
  • treatment with C-01 was found to reduce RAB12 protein levels while robustly elevating transferrin receptor protein 1 (TFRC).
  • the sex-matched parent carries the heterozygous c.1160_1161del; p.Thr387Argfs*30 variant.
  • Patient 2 was diagnosed with AP4M1-associated SPG50 and carries the following compound-heterozygous variants: NM_004722.4, c.916C>T (p.Arg306Ter) / c.694dupG (p.Glu232GlyfsTer21).
  • the sex- matched parent carries the heterozygous c.694dupG (p.Glu232GlyfsTer21) variant. Antibodies and reagents.
  • Bovine serum albumin (AmericanBIO, Cat# 9048-46-8), saponin (Sigma, #47036-50G-F), normal goat serum (Sigma-Aldrich, Cat# G9023-10ML), Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Cat# 14190-250), trypsin (Thermo Fisher Scientific, Cat#25200056), 4% paraformaldehyde (4%) (Boston BioProducts, Cat# BM-155), dimethyl-sulfoxide (DMSO) (American Bioanalytical, Cat# AB03091-00100), Bafilomycin A1 (Enzo Life Sciences, Cat# BML-CM110-0100), Molecular Probes Hoechst 33258 (Thermo Fisher Scientific, Cat# H3569) and ALEXA FLUORTM 647-labelled phalloidin (Thermo Fisher Scientific, Cat#A22287).
  • DPBS Dulbecco's
  • Anti-AP4E1 at 1:500 (BD Bioscience, Cat# 612019), anti-ATG9A at 1:500-1000 (Abcam, Cat# ab108338), anti- DAGLB at 1:500 (Abcam, Cat# 191159), anti-TGN46 at 1:800 (Bio-Rad, Cat# AHP500G), anti-Golgi 971:500 (Abcam, Cat# 169287), anti-beta-Tubulin III 1:1000 (Synaptic Systems, Cat# 302304), anti-beta-Actin 1:10,000 (Sigma, Cat# A1978-100UL), anti-LC3B 1:1000 (Novus, Cat#100-2220).
  • Fluorescently labelled secondary antibodies for immunocytochemistry were used at 1:2000 (Thermo Fisher Scientific, Cat# A11008, A11016, A21245), for western blotting at 1:5000 (LI-COR Biosciences, Cat# 926-68022, 926-68023, 926-32212, 926-32213).
  • Small molecule library A diversity small molecule library containing 28,864 compounds was provided by Astellas Pharma Inc.. Compounds were arrayed in 384-well microplates at a final concentration of 10mM (1000-fold the screening concentration) in DMSO. Assay plates were stored at -80 °C and thawed 30 minutes prior to cell plating.
  • Active compounds from the primary screen were re-screened in a secondary screen, using eleven-point concentrations (range: 0.04 ⁇ M, 0.08 ⁇ M, 0.16 ⁇ M, 0.31 ⁇ M, 0.63 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M) in two biological replicates.
  • Fibroblast cell culture Fibroblast lines were established from routine skin punch biopsies in both patients and their respective sex-matched heterozygous parents. Primary human skin fibroblasts were cultured and maintained.
  • fibroblasts were seeded onto 384-well plates (Greiner Bio-One, #781090) at a density of 2 ⁇ 10 3 per well using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, #11388-558). Media changes were done every 2-3 days and drugs administered 24 hours before fixation.
  • SH-SY5Y cell culture AP4B1 wild type (AP4B1 WT and AP4B1 knockout (AP4B1 KO )) SH-SY5Y cells were generated previously. Undifferentiated SH-SY5Y cells were maintained in DMEM/F12 (Gibco, Cat# 11320033) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Cat# 10438026), 100U/ml penicillin and 100 ⁇ g/ml streptomycin at 37°C under 5% CO 2 .
  • SH-SY5Y cells were passaged every 2-3 days and differentiated into a neuron-like state using a 5-day differentiation protocol with all-trans-retinoic acid (MedChemExpress, #HY-14649).
  • differentiated SH-SY5Y cells were plated in 96-well plates (Greiner Bio-One, Cat# 655090), at a density of 10,000 cells per well. Media changes were done every 2-3 days and drugs administered 24-72 hours before fixation.
  • the immunocytochemistry protocol was optimized for high- throughput staining by using automated pipettes and reagent dispensers (Thermo Fisher Scientific MULTIDROPTM Combi Reagent Dispenser, Integra VIAFLO 96/384 liquid handler, Integra VOYAGER pipette).
  • Fibroblasts and SH-SY5Y cells were fixed using 3% and 4% PFA, respectively, permeabilized with 0.1% saponin in PBS and blocked in 1% BSA/0.01% saponin (blocking solution) in PBS.
  • iPSC-derived neurons were fixed in 4% PFA, and permeabilized and blocked using 0.1% TRITONTM X-100/2% BSA/0.05% NGS in PBS.
  • High-throughput confocal imaging was performed on an ImageXpress Micro Confocal Screening System (Molecular Devices) using an experimental pipeline.
  • images were acquired using a 20x S Plan Fluor objective (NA 0.45 ⁇ M, WD 8.2-6.9 mm).
  • 4 fields were acquired in a 2x2 format (384-well plates).
  • 384-well plates For experiments in SH-SY5Y cells and iPSC neurons, up to 36 fields were acquired in a 6x6 format (96-well plate) using a 40x S Plan Fluor objective ((NA 0.60 ⁇ m, WB 3.6-2.8 mm).
  • the image analysis was performed using a customized image analysis pipeline in MetaXpress (Molecular Devices): Briefly, cells were identified based on the presence of DAPI signal inside a phalloidin (fibroblasts) or TUBB3 (SH-SY5Y cells and iPSC-neurons)-positive cell body. Sequential masks were generated for (1) the TGN by outlining the area covered by TGN marker TGN46 (TGN46- positive area, in fibroblasts and SH-SY5Y cells) or Golgi 97 (Golgi 97-positive area, in iPSC neurons) and (2) for the cell area outside the TGN (actin-positive area minus TGN46-positive area).
  • TGN46 TGN46- positive area, in fibroblasts and SH-SY5Y cells
  • Golgi 97 Golgi 97-positive area, in iPSC neurons
  • ATG9A fluorescence intensity was measured in both compartments in each cell and the ATG9A ratio was calculated by dividing the ATG9A fluorescence intensity the TGN by the ATG9A fluorescence intensity in the remaining cell body (FIG.1B).
  • Z’-factor robust values and strictly standardized median difference (SSMD) were calculated for each plate and only plates that met the predefined quality metrics of a Z’-factor robust ⁇ 0.3 and SSMD ⁇ 3 were included in subsequent analyses.
  • Western blotting Western blotting was done.
  • Equal amounts of protein were solubilized in LDS sample buffer (Thermo Fisher Scientific, Cat# NP0008) under reducing conditions, separated by gel electrophoresis, using 4–12% (Thermo Fisher Scientific, Cat# NW04125BOX) or 12% Bis-Tris gels (Thermo Fisher Scientific, Cat# NP0343BOX) and MOPS or MES buffer (Thermo Fisher Scientific, #NP0001 and #NP0002) and transferred to a PVDF or nitrocellulose membranes (EMD Millipore, #SLHVR33RS). Following blocking with blocking buffer (LI-COR Biosciences, #927-70001), membranes were incubated overnight with the respective primary antibodies.
  • RNA extraction Near-infrared fluorescent-labeled secondary antibodies (IR800CW, IR680LT; LI-COR Biosciences) were used and quantification was done using the Odyssey infrared imaging system and Image Studio Software (LI-COR Biosciences).
  • Sample preparation for RNA extraction SH-SY5Y cells were differentiated with retinoic acid as described above and subsequently treated with compounds of interest for 72 hours, prior to lysis using the Quiagen RTL-Buffer supplemented with 1% ß- mercaptoethanol. RNA extraction, library preparation and sequencing were conducted at Azenta Life Sciences (South Plainfield, NJ, USA). Total RNA was extracted from frozen cell pellet samples using Qiagen RNEASYTM mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany).
  • RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized.
  • cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles.
  • the sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
  • the sequencing libraries were clustered on 3 lanes of a flowcell. After clustering, the flowcell was loaded on the Illumina instrument (HiSeq 4000 or equivalent) according to manufacturer’s instructions.
  • the samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and de- multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. Downstream RNA sequencing analysis. Sequencing reads were mapped to the GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.7.9a. Differential expression analysis was done using the TREAT approach developed by McCarthy and Smyth (McCarthy DJ, Smyth GK. Bioinformatics 25, 765-771 (2009)), implemented in the edgeR package in R.
  • PE Paired End
  • Genes were then grouped based on topological overlap and clusters were isolated using hierarchical clustering and adaptive branch pruning of the hierarchical cluster dendrogram, giving rise to groups of co-expressed genes, so called modules.
  • Gene expression profiles within each module were summarized using the “module eigengene” (ME), defined as the first principal component of a module.
  • ME module eigengene
  • association of MEs with measured clinical traits was examined by correlation analysis. For these selected modules, module eigengene based connectivity was determined for every gene by calculating the absolute value of the Pearson correlation between the expression of the gene and the respective ME, producing a quantitative measure of module membership (MM).
  • MM quantitative measure of module membership
  • GS gene significance
  • peptide samples for analysis by mass spectrometry, 30-50 ⁇ g protein were precipitated by overnight incubation in 5 volumes of ice- cold acetone at ⁇ 20° C and pelleted by centrifugation at 10,000 ⁇ g for 5 minutes at 4° C. All subsequent steps were performed at room temperature. Precipitated protein pellets were air- dried, resuspended for denaturation and reduction in digestion buffer (50 mM Tris pH 8.3, 8M Urea, 1 mM dithiothreitol (DTT)) and incubated for 15 minutes. Proteins were alkylated by addition of 5 mM iodoacetamide for 20 minutes in the dark.
  • digestion buffer 50 mM Tris pH 8.3, 8M Urea, 1 mM dithiothreitol (DTT)
  • proteins were enzymatically digested by addition of LysC (1 ⁇ g per 50 ⁇ g of protein; Wako, Cat# 129-02541) for an overnight incubation. Samples were then diluted four- fold with 50 mM Tris pH 8.3 before addition of Trypsin (1 ⁇ g per 50 ⁇ g of protein; Sigma- Aldrich, Cat# T6567) for 3 hours. The digestion reaction was stopped by addition of 1% (v/v) trifluoroacetic acid (TFA) and samples were incubated on ice for 5 minutes to precipitate contaminants, which were pelleted by centrifugation at 10,000 ⁇ g for 5 minutes.
  • TFA trifluoroacetic acid
  • Acidified peptides were transferred to new tubes, before purification by solid-phase extraction using poly(styrenedivinylbenzene) reverse-phase sulfonate (SDB-RPS; Sigma-Aldrich, Cat# 66886-U) StageTips 76 .
  • SDB-RPS poly(styrenedivinylbenzene) reverse-phase sulfonate
  • StageTips with three SDB-RPS plugs were washed with 100% acetonitrile, equilibrated with StageTip equilibration buffer (30% [v/v] methanol, 1% [v/v] TFA), and washed with 0.2% (v/v) TFA.20 ⁇ g of peptides in 1% TFA were then loaded onto the activated StageTips, washed with 100% isopropanol, and then 0.2% (v/v) TFA.
  • Peptides were eluted in three consecutive fractions by applying a step gradient of increasing acetonitrile concentrations: 20 ⁇ L SDB-RPS-1 (100 mM ammonium formate, 40% [v/v] acetonitrile, 0.5% [v/v] formic acid), then 20 ⁇ L SDB-RPS-2 (150 mM ammonium formate, 60% [v/v] acetonitrile, 0.5% [v/v] formic acid), then 30 ⁇ L SDB-RPS-3 (5% [v/v] NH4OH, 80% [v/v] acetonitrile).
  • Eluted peptides were dried in a centrifugal vacuum concentrator, resuspended in Buffer A* (0.1% (v/v) TFA, 2% (v/v) acetonitrile), and stored at ⁇ 20° C until analysis by mass spectrometry.
  • Mass spectrometry Mass spectrometry was performed on an Exploris 480 mass spectrometer coupled online to an EASY ⁇ nLC 1200, via a nano-electrospray ion source (all Thermo Fisher Scientific). Per sample, 250 ng of peptides were loaded on a 50 cm by 75 ⁇ m inner diameter column, packed in-house with ReproSil-Pur C18-AQ 1.9 ⁇ m silica beads (Dr Maisch GmbH).
  • the column was operated at 50° C using an in-house manufactured oven.
  • Peptides were separated at a constant flow rate of 300nL/minute using a linear 110 minute gradient employing a binary buffer system consisting of Buffer A (0.1% [v/v] formic acid) and Buffer B (80% acetonitrile, 0.1% [v/v] formic acid).
  • the gradient ran from 5 to 30% B in 84 minutes, followed by an increase to 60% B in 8 minutes, a further increase to 95% B in 4 minutes, a constant phase at 95% B for 4 minutes, and then a washout decreasing to 5% B in 5 minutes, before re-equilibration at 5% B for 5 minutes.
  • the Exploris 480 was controlled by Xcalibur software (v.4.4, Thermo Fisher Scientific) and data were acquired using a data- dependent top-15 method with a full scan range of 300 - 1650 Th.
  • MS1 survey scans were acquired at 60,000 resolution with an automatic gain control (AGC) target of 3 ⁇ 10 6 charges and a maximum ion injection time of 25 milliseconds.
  • AGC automatic gain control
  • Selected precursor ions were isolated in a window of 1.4 Th and fragmented by higher-energy collisional dissociation (HCD) with normalized collision energies of 30.
  • MS2 fragment scans were performed at 15,000 resolution, with an AGC target of 1 ⁇ 10 5 charges, a maximum injection time of 28 milliseconds, and precursor dynamic exclusion for 30 seconds.
  • Mass spectrometry raw files were processed in MaxQuant Version 2.1.4.0, using the human SwissProt canonical and isoform protein database, retrieved from UniProt (2022_09_26; uniprot.org). Label-free quantification was performed using the MaxLFQ algorithm. Matching between runs was enabled to match between equivalent and adjacent peptide fractions, within replicates. LFQ minimum ratio count was set to 1 and default parameters were used for all other settings. All downstream analyses were performed on the ‘protein groups’ file output from MaxQuant. Proteomic downstream data analysis. Differential enrichment analysis of proteomics data was done using the DEP package in R.
  • Preprocessing and quality filtering was performed separately for SH-SY5Y cells and iPSC-derived neurons. Proteins that were only identified by a modification site, or matched the reversed part of the decoy database, as well as commonly occurring contaminants were removed. Duplicate proteins were removed based on the corresponding gene names by keeping those with the highest total MS/MS count across all samples. All following steps were done separately for each cell type (SH-SY5Y cells (FIG.7A and FIGs.14A-14D) and iPSC-derived neurons (FIG.7B and FIGs.14E-14H) and for the pooled dataset (FIG.7C and FIGs.14I-14L).
  • Proteins were considered as differentially enriched with a false discovery rate of ⁇ 0.05 and a log 2 fold change > 0.3.
  • the biological information contained in differentially enriched proteins was summarized using Reactome pathway annotation in clusterProfiler. Pathways were considered differentially expressed with an FDR ⁇ 0.05. Electroporation. sgRNAs against NLRP5, RAB3C and RAB12 were purchased as multi-guide knockout kits from Synthego, diluted to the desired stock concentrations and kept at -80°C. Electroporation was performed under RNAse free conditions on a Lonza 4D- Nucleofector according to the manufacturer’s protocol.
  • SH-SY5Y cells were harvested and resuspended in Nucleofector Solution at a concentration of 400x10 ⁇ 3 cells/ml.
  • sgRNAs were incubated with Cas9 protein to form ribonucleoprotein complexes (RNPs) according to the manufacturer's instructions.
  • the cell solution was then incubated with an amount of the respective RNPs and transferred into a nucleofection cuvette.
  • Cuvettes were placed in the 4D-Nucleofector System, and electroporation was done using the G-004 program. Following electroporation, pre-warmed medium was added, and cells were plated. Compound treatment was started 48 hours after electroporation.
  • sgRNAs Knockout efficiency of sgRNAs was assessed using the Synthego ICE Analysis online tool. For this, genomic DNA was extracted from nucleofected cells using a **kit** according to manufacturer’s instructions and sequenced using the following primers: NLRP5 forward: CTTGAGAATTTGCTGCAAGATCCT, NLRP5 reverse: CGATTCTTCCCTGTTCCCATGAG, RAB3C forward: CCACTCGCCTCCTGAGTGTCTG, RAB3C reverse: GAACAAGGCAGAAAGTTTCTCCC, RAB12 forward: CGAGTAGGGAGGAGTGAAAAGG, RAB12 reverse: GGCACGAAAACCTCTGCCAGGC. Statistical testing.
  • the active compounds identified in the counter-screen were re-screened in differentiated AP4B1 WT and AP4B1 KO SH-SY5Y cells using an 8-point titration, ranging from 50nM to 30 ⁇ M. Active compounds were a priori defined as those reducing the ATG9A ratio or DAGLB ratio by at least 3SD compared to negative controls, in more than one concentration. Toxicity was defined as a reduction of cell count of at least 2SD compared to the negative control.
  • Compound Synthesis Compounds of Formula (I) were prepared following the synthetic schemes and procedures described in detail below.
  • AP4B1 wildtype (AP4B1 WT ) and AP4B1 knockout (AP4B1 KO ) SH-SY5Y cells were generated previously (PMID: 38233389, PMID: 35217685). Undifferentiated SH-SY5Y cells were maintained in DMEM/F12 (Gibco, Cat# 11320033) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Cat# 10438026), 100 U/mL penicillin and 100 #g/mL streptomycin at 37 °C under 5% CO2.
  • SH- SY5Y cells were passaged every 2–3 days and differentiated into a neuron-like state using a 5-day differentiation protocol with all- trans-retinoic acid (MedChemExpress, #HY-14649).
  • differentiated SH-SY5Y cells were plated in 96- well plates (Greiner Bio-One, Cat# 655090) at a density of 1 ⁇ 10 4 cells per well. Media changes were done every 2–3 days and drugs were administered 24h before fixation. Immunocytochemistry.
  • the immunocytochemistry workflow was optimized for high- throughput using automated pipettes and reagent dispensers (Thermo Fisher Scientific Multidrop Combi Reagent Dispenser, Integra VIAFLO 96/384 liquid handler, Integra VOYAGER pipette).
  • SH-SY5Y cells were fixed using 4% PFA, permeabilized with 0.1% saponin in PBS and blocked in 1% BSA/0.01% saponin (blocking solution) in PBS.
  • Primary antibody diluted in blocking solution
  • High-content imaging and automated image analysis were performed on the ImageX-press Micro Confocal Screening System (Molecular Devices) using an experimental pipeline modified from the pipeline described in Behne et al. (PMID: 31915823). Up to 36 fields were acquired in a 6 ⁇ 6 format (96-well plate) using a 40x S Plan Fluor objective (NA 0.60 ⁇ m, WB 3.6–2.8 mm). Image analysis was performed using a customized image analysis pipeline in MetaXpress (Molecular Devices): Briefly, cells were identified based on the presence of DAPI signal inside a TUBB3-positive cell body.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features.

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Abstract

L'invention concerne des composés qui modulent le trafic lié à l'autophagie 9A (ATG9A) et/ou augmentent le flux autophagique. L'invention concerne également des compositions pharmaceutiques comprenant les composés, ainsi que des méthodes de traitement d'une maladie ou d'un trouble neurologique, tel qu'une paraplégie spastique héréditaire (PSH).
PCT/US2024/029856 2023-05-19 2024-05-17 Composés pour traitement de paraplégie spastique héréditaire Pending WO2024243015A2 (fr)

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