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WO2024155689A1 - Composés et méthodes d'inhibition du récepteur de type iii de tyrosines kinases - Google Patents

Composés et méthodes d'inhibition du récepteur de type iii de tyrosines kinases Download PDF

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Publication number
WO2024155689A1
WO2024155689A1 PCT/US2024/011805 US2024011805W WO2024155689A1 WO 2024155689 A1 WO2024155689 A1 WO 2024155689A1 US 2024011805 W US2024011805 W US 2024011805W WO 2024155689 A1 WO2024155689 A1 WO 2024155689A1
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Prior art keywords
kit
type
polypeptide
iii
iii rtk
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Joseph Schlessinger
Stefan Krimmer
Nicole BERTOLETTI
Irit Lax
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Yale University
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Yale University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)

Definitions

  • RTKs type-III receptor tyrosine kinases
  • KIT KIT
  • PDGFRa PDGFRa
  • PDGFRP PDGFRP
  • CSF1R CSF1R
  • FLT3 FLT3
  • KIT KIT
  • PDGFRa PDGFRa
  • PDGFRP PDGFRP
  • CSF1R CSF1R
  • FLT3 FLT3
  • novel methods of inhibiting type-III RTKs as well as novel methods of treating, ameliorating and/or preventing diseases or disorders related to the overactivation/overexpression of the type-III RTKs.
  • the present invention addresses these needs.
  • the present invention is directed to the following non-limiting embodiments:
  • the present invention is directed to a method of inhibiting a type-111 receptor tyrosine kinase (RTK).
  • RTK type-111 receptor tyrosine kinase
  • the method comprising contacting the type-III RTK with a compound that at least partially inhibits dimerization of the type-III RTK.
  • the compound binds to the (3G strand of the D5 domain of the type-III RTK. [008] In some embodiments, the compound binds to and at least partially blocks the PG strand of the D5 domain of the type-III RKT.
  • the compound is not able to cause the trans-phosphorylation of the type-III RKT.
  • the type-III RTK is proto-oncogene c-KIT (KIT) or a mutant thereof, and the compound binds to at least one KIT amino acid residue corresponding to A502, Y503.
  • the type-III RTK is platelet-derived grow th factor receptor a (PDGFRa) or a mutant thereof, and the compound binds to at least one PDGFRa amino acid residue corresponding to R512, E513, L514, K515, L516. V517 or A518 of the polypeptide as set forth in SEQ ID NO:2.
  • PDGFRa platelet-derived grow th factor receptor a
  • the type-III RTK is platelet-derived growth factor receptor (PDGFRP) or a mutant thereof, and the compound binds to at least one PDGFR p amino acid residue corresponding to Q519, E520, V521, 1522, V523, V524 or P525 of the polypeptide as set forth in SEQ ID NO: 3.
  • PDGFRP platelet-derived growth factor receptor
  • the type-III RTK is colony -stimulating factor- 1 receptor (CSF-1R) or a mutant thereof, and the compound binds to at least one CSF-1R amino acid residue corresponding to W496, A497, F498, 1499, P500, 1501 or S502 of the polypeptide as set forth in SEQ ID NO:4.
  • CSF-1R colony -stimulating factor- 1 receptor
  • the type-III RTK is fms-like tyrosine kinase 3 (FLT3) or a mutant thereof, and the compound binds to at least one FLT3 amino acid residue corresponding to E525, T526, 1527, L528, L529, N530 or S531 of the polypeptide as set forth in SEQ ID NO:5.
  • FLT3 fms-like tyrosine kinase 3
  • the compound is a polypeptide comprising the D5 domain of the type-III RTK or a nucleic acid encoding the polypeptide, and the polypeptide does not have tyrosine kinase activity.
  • the compound is a polypeptide comprising the D4-D5 section of the type-III RTK or a nucleic acid encoding the polypeptide.
  • the type-III RTK is KIT
  • the polypeptide comprises residues 408-508 (D5 domain) or residues 308-508 (D4-D5 segment) of SEQ ID NO: 1.
  • the KIT is a KIT with duplication of A502 and Y503 (KIT DupA502,Y503), and the polypeptide comprises duplication of A502 and Y503 in the sequence set forth in SEQ ID NO: 1.
  • the KIT is a KIT with T417I mutation and deletion of Y418 and D417 (KIT T417I. A418,419). and the polypeptide comprises T417I mutation and deletion of Y418 and D417 in the sequence set forth in SEQ ID NO: 1.
  • the compound comprises an antibody that binds to the (3G strand of the D5 domain of the t pe- 1 II RTK, or a nucleic acid encoding the antibody.
  • the antibody is a divalent antibody, and the antibody further binds to the residues in the D4 domain of the type-III RTK responsible for forming the salt bridge.
  • the type-III RTK is KIT
  • the antibody binds to at least one KIT residue corresponding to A502, Y503, F504, N505, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO: 1.
  • the type-III RTK is KIT
  • the antibody is divalent
  • the antibody further binds to at least one KIT residue corresponding to E386 or R381 of the polypeptide as set forth in SEQ ID NO: 1.
  • the type-III RTK is an isolated protein, or a protein in or on the surface of a cell.
  • the type-III RTK is in a subject. In some embodiments, the ty pe-III RTK is in a mammal. In some embodiments, the type-III RTK is in a human.
  • the type-III RTK is an overactivated mutant type-III RTK.
  • the present invention is directed to a method of treating, ameliorating, and/or preventing a disease or disorder caused by or involving an overactivation and/or an overexpression of a ty pe-III receptor ty rosine kinase in a subject in need thereof.
  • the method comprises: administering to the subject an effective amount of a compound inhibiting the dimerization of the type-III RTK.
  • the compound binds to the (3G strand of the D5 domain of the type-III RTK.
  • the compound binds to and at least partially blocks the J3G strand of the D5 domain of the type-III RKT, and the compound is not able to cause the transphosphorylation of the ty pe-III RKT.
  • the type-III RTK is proto-oncogene c-KIT (KIT) or a mutant thereof, and the compound binds to at least one KIT amino acid residue corresponding to A502, Y503, F504, N505, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO: 1
  • the type-III RTK is platelet-derived growth factor receptor a (PDGFRa) or a mutant thereof, and the compound binds to at least one PDGFRa amino acid residue corresponding to R512, E513, L514, K515, L516, V517 or A518 of the polypeptide as set forth in SEQ ID NO: 2.
  • PDGFRa platelet-derived growth factor receptor a
  • the type-III RTK is platelet-derived growth factor receptor [3 (PDGFRP) or a mutant thereof, and the compound binds to at least one PDGFR
  • PDGFRP platelet-derived growth factor receptor
  • the type-III RTK is colony-stimulating factor-1 receptor (CSF-1R) or a mutant thereof, and the compound binds to at least one CSF-1R amino acid residue corresponding to W496, A497, F498, 1499, P500, 1501 or S502 of the polypeptide as set forth in SEQ ID NO:4.
  • CSF-1R colony-stimulating factor-1 receptor
  • the type-III RTK is fms-like tyrosine kinase 3 (FLT3) or a mutant thereof, and the compound binds to at least one FLT3 amino acid residue corresponding to E525, T526, 1527, L528, L529, N530 or S531 of the polypeptide as set forth in SEQ ID NO:5.
  • FLT3 fms-like tyrosine kinase 3
  • the compound is a polypeptide comprising the D5 domain of the type-III RTK or a nucleic acid encoding the polypeptide, and the polypeptide does not have tyrosine kinase activity.
  • the compound is a polypeptide comprising the D4-D5 section of the type-III RTK or a nucleic acid encoding the polypeptide.
  • the type-III RTK is KIT
  • the polypeptide comprises residues 408-508 (D5 domain) or residues 308-508 (D4-D5 segment) of SEQ ID NO: 1.
  • the KIT is a KIT with duplication of A502 and Y503 (KIT DupA502,Y503), and the polypeptide comprises duplication of A502 and Y503 in the sequence set forth in SEQ ID NO: 1.
  • the KIT is a KIT with T417I mutation and deletion of Y418 and D417 (KIT T417I, A418,419), and the polypeptide comprises T417I mutation and deletion of Y418 and D417 in the sequence set forth in SEQ ID NO: 1.
  • the compound comprises an antibody that binds to the
  • the antibody is a divalent antibody, and the antibody further binds to one or more residues in the D4 domain of the type-III RTK responsible for forming the salt bridge.
  • the type-III RTK is KIT
  • the antibody binds to at least one KIT residue corresponding to A502, Y503, F504, N505, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO: 1.
  • the type-III RTK is KIT.
  • the antibody is divalent, and the antibody further binds to at least one KIT residue corresponding to E386 or R381 of the polypeptide as set forth in SEQ ID NO: 1.
  • the type-III RTK is KIT
  • the disease or disorder comprises a KIT-driven cancer, optionally a KIT-driven gastrointestinal stromal tumor (KIT- driven GIST), a KIT-driven core binding factor acute myeloid leukemia; or a mast cell diseases, optionally a systemic mastocytosis.
  • the type-III RTK is PDGFRa
  • the disease or disorder comprises a PDGFRa-driven cancer, optionally a PDGFRa-associated chronic eosinophilic leukemia or a PDGFRa-driven gastrointestinal stromal tumor (PDGFRa-driven GIST); or inflammatory fibroid polyps.
  • the type-III RTK is PDGFR0
  • the disease or disorder comprises a PDGFR0-driven cancer, optionally a PDGFR0-associated chronic eosinophilic leukemia; a primary familial brain calcification; an infantile myofibromatosis; a Kosaki overgrow th syndrome; or a premature aging syndrome, Penttinen type.
  • the type-III RTK is CSF1R, and the disease or disorder comprises a CSFIR-driven cancer, optionally a CSFIR-driven myeloid malignancy, a CSFIR-driven Hodgkin's lymphoma or a CSFIR-driven anaplastic large cell lymphoma.
  • the type-III RTK is FLT3, and the disease or disorder comprises a FLT3-driven cancer, optionally a FLT3-driven core binding factor acute myeloid leukemia or a FLT3-driven cytogenetically normal acute myeloid leukemia.
  • the disease or disorder is caused by or involves KIT overactivation, and the method further includes administering to the subject an effective amount of a stem cell factor (SCF) protein or a nucleic acid encoding the SCF.
  • SCF stem cell factor
  • the subject is a mammal. In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figs. 1A-1D Cryo-EM analysis of full-length wild-type KIT:SCF dimers reconstituted in an amphipol environment resolves the extracellular domain to high resolution, in accordance with some embodiments.
  • Fig. 1 A Representative negative staining EM 2D class averages. Regions corresponding to the extracellular domain (ECD) and the cytoplasmic domain (CD) are indicated.
  • Fig. IB Representative cryo-EM 2D class averages (front and side views only). The extracellular domain is sharply defined, whereas the cytoplasmic domain is blurred out.
  • Fig. 1C Cryo-EM 3D reconstruction after global refinement of the entire KIT:SCF complex.
  • cryo-EM map is displayed at low (left) and high (right) map contour levels.
  • Fig. ID Cryo-EM 3D reconstruction of the ECD of KIT:SCF dimers after local ECD refinement and post-processing (overall resolution: 3.45 A; KIT protomer A in beige, KIT protomer B in blue, SCF homodimer in green).
  • Figs. 2A-2D Cryo-EM structure of full-length wild-type KIT: SCF dimers reveals an asymmetric conformation of D5:D5' contacts, in accordance with some embodiments.
  • Fig. 2A Cryo-EM map (gray volume) of the ECD of KIT: SCF dimers. The structural model fitted into the cryo-EM map shows KIT protomer A, KIT protomer B, and SCF homodimer. The line in the middle represents the 2-fold rotation symmetry axis. NT. N-terminus; CT. C- terminus.
  • Fig. 2B Close-up view of the boxed asymmetric D5:D5' complex from Fig. 2A.
  • FIG. 2C Close-up view' of the boxed asymmetric 0G:
  • Backbone hydrogen bonds are shown as dotted lines (distances labeled in A).
  • Fig. 2D Schematic representation of the asymmetric PG: PG' backbone interactions. Relative to (3-strand PG. P- strand P is shifted by one residue toward the N-terminus, and rotated by 180°. Backbone hydrogen bonds are shown as dotted lines.
  • Figs. 2E-2G Side chain interactions at the D5:D5' interface.
  • Fig. 2E Top view of D5:D5' indicating side chain interactions at site-I (lower box) and site-II (upper box).
  • Figs. 2F- 2G Side chain interactions at site-I (Fig. 2FF) and site-II (Fig. 2G) of the D5:D5' interface. Hydrophobic packing interactions at the interface are delineated by black dotted lines. Side chains are shown as sticks, and their van der Waals radii are shown as semitransparent spheres to highlight shape complementarity. Backbone atoms are omitted for clarity. The side chain of F508' was omitted from the model due to poorly defined cryo-EM density. See also Figs. 8A-8G and 16.
  • Figs. 3A-3B Tyrosine autophosphorylation of wild-type and KIT mutants after stimulation with SCF, in accordance with some embodiments.
  • Fig. 3A Structure-based sequence alignments of P-strands G of human type-III RTK family members. Functionally conserved residues are highlighted.
  • Fig. 3B Lysates from SCF stimulated or unstimulated NIH 3T3 cells stably expressing wild-type KIT or KIT mutants were subjected to immunoprecipitation with anti-KIT antibodies followed by SDS-PAGE and immunoblotting with either anti-phospho-KIT(Y703) or anti-KIT antibodies.
  • Figs. 4A-4H Cryo-EM structure of full-length KIT (DupA502,Y503):SCF dimers reveals the ligand-sensitizing mechanism of oncogenic KIT mutant, in accordance with some embodiments.
  • Fig. 4A Superposition of D5:D5' of KIT(DupA502,Y503):SCF dimers (D5 and D5' colored differently) and D5:D5' of wild-type KIT:SCF dimers (D5 and D5' both in white).
  • the conformational change of P-sheet PA-PB-PE is indicated by black outlined arrows. Structures were superimposed on their ECDs excluding D5:D5' from the calculation of the superimposition.
  • Fig. 4B Enlarged view of boxed residues Y418, DupY503', and N505' from Fig. 4A. The shift in the location of N505' between wild-type and DupA502,Y503 mutant is indicated by the black outlined arrow.
  • Fig. 4C Same superposition as in Fig. 4A, rotated by 180°.
  • Fig. 4D Enlarged view of boxed residues Y418', DupY503, and N505 from Fig. 4C. The shift in the location of N505 between wild-ty pe and DupA502,Y503 mutant is indicated by the black outlined arrow.
  • Fig. 4B Enlarged view of boxed residues Y418, DupY503', and N505' from Fig. 4A. The shift in the location of N505' between wild-type and DupA502,Y503 mutant is indicated by the black outlined arrow.
  • Fig. 4C Same superposition
  • FIG. 4E Asymmetric D5:D5' interface formed by P-strands G and PG'. Dotted lines indicate backbone hydrogen bonds (distances labeled in A).
  • Figs. 4F-4H Side chain interactions at the D5:D5' interface.
  • Fig. 4F Top view of D5:D5' indicating side chain interactions at site-I (lower left box) and site-II (upper right box). Resides with side chains participating in interface interactions are shown as sticks.
  • Figs. 4G-4H Side chain interactions at site-I (Fig. 4G) and site-II (Fig. 4H) of the D5:D5' interface. Hydrophobic packing interactions at the interface are delineated by black dotted lines.
  • Figs. 5A-5C Cryo-EM map of full-length KIT(T417I,A418-419) dimers, in accordance with some embodiments.
  • Fig. 5A Cryo-EM map of a global refinement of the entire particle. The cryo-EM map is displayed at a low contour level (light gray) and a high contour level (dark gray).
  • Fig. 5B Close-up view of the boxed region from Fig. 5A. The crystal structure of the D4D5 fragment dimer of KIT(T417I,A418-419) (PDB ID 4PGZ) was rigid-body docked into the cryo-EM map.
  • Fig. 5C Cryo- EM map of a masked refinement of the ECD. Two copies of the crystal structure of monomeric KIT (PDB ID 2EC8) were docked into the map. See also Figs. 13A-13E and 16. [0059] Figs. 6A-6C: Cryo-EM structure of full-length KIT(T417I,A418-419):SCF dimers, in accordance with some embodiments. Fig.
  • FIG. 6A Cryo-EM map of the ECD of KIT(T417I,A418-419):SCF dimers.
  • KIT protomer A on the left, KIT protomer B on the right, SCF homodimer binds to both D2 of KIT promoter A and D2' of KIT promoter B.
  • the line in the middle represents the 2-fold rotation symmetry’ axis.
  • Fig. 6B Close-up view’ of the boxed D4:D4' interface from Fig. 6A, detailing the intact homotypic salt bridge interactions between R381 and E386.
  • Fig. 6C Close-up view of the boxed D5:D5' complex from Fig. 6A.
  • the cryo-EM map is displayed at a low contour level (light gray volume) and a high contour level (dark gray volume). See also Fig. 8A-8F and 16.
  • Fig. 7 Structural plasticity of the ligand-independent, constitutively active oncogenic KIT mutant, in accordance with some embodiments.
  • Binding of SCF to monomeric wild-ty pe KIT (1) results in the formation of KIT: SCF dimers (2).
  • KIT: SCF dimers the ECDs are held together by SCF binding to D1-D3, by D4:D4' homotypic interactions via symmetric contacts, and by D5:D5' interactions mediated by an asymmetric interface.
  • the ECDs of ligand-independent, constitutively active KIT(T417I,A418-419) dimers adopt a V-shaped conformation solely held together by D5:D5' contacts (3).
  • the D5:D5' complex of this constitutively active oncogenic KIT mutant adopts a strongly tilted, symmetric conformation with sufficient affinity to trigger KIT dimerization and activation in the absence of SCF binding. Binding of SCF to KIT(T417I,A418-419) dimers restores a wildtype-like conformation of the ECDs with an asymmetric D5:D5' interface (2).
  • CD cytoplasmic domain
  • TMD transmembrane domain.
  • Figs. 8A-8G Cryo-EM data processing and analysis of the structure of WT KIT: SCF dimers, in accordance with some embodiments.
  • Fig. 8A Representative cryo-EM micrograph and 2D class averages.
  • Fig. 8B Cryo-EM data processing flow-chart. The local resolution map of an ECD local refinement (overall resolution: 3.45 A) is displayed without sharpening (left) and after post-processing using deepEMhancer (right).
  • CS cryoSPARC.
  • Fig. 8C FSC curves of the ECD local refinement.
  • Fig. 8D Angular distribution plot of particles used for ECD local refinement.
  • Fig. 8A-8G Cryo-EM data processing and analysis of the structure of WT KIT: SCF dimers, in accordance with some embodiments.
  • Fig. 8A Representative cryo-EM micrograph and 2D class averages.
  • Fig. 8B Cryo-EM data processing flow-chart. The local
  • Fig. 8E Cryo-EM density of the homotypic D4:D4' salt bridge.
  • Fig. 8F Cryo-EM densities of residues of [3-strands (3A, (BG,
  • Fig. 8G Analysis of the ECD conformational flexibility using 3DVA of cryoSPARC. The two maps in show the two most distant conformations of the motion solved for the first eigenvector. Significant motion (indicated by black arrows) is observed for domains DI, DI', and for D5:D5'.
  • Figs. 9A-9E Structural analysis of the asymmetric D5:D5' conformation in the cryo-EM structure of WT KIT:SCF dimers, in accordance with some embodiments.
  • Fig. 9A Superposition of the ECDs of the cryo-EM structure (protomer A: left; protomer B: right; SCF homodimer: couples to D2 of promoter A and D2' of promoter B) and the crystal structure (PDB ID 2E9W; protomers A, B, and SCF in gray) of WT KIT:SCF dimers.
  • the line in the middle resembles the C2 symmetry axis of the crystal structure.
  • Fig. 9A Structural analysis of the asymmetric D5:D5' conformation in the cryo-EM structure of WT KIT:SCF dimers, in accordance with some embodiments.
  • Fig. 9A Superposition of the ECDs of the cryo-EM structure (protomer A: left; protomer B:
  • FIG. 9B Close-up view of boxed D5:D5' superposition from Fig. 9A. [3-strands [3D and [3D 1 were not included in the cryo-EM model due to poor local density in the cryo-EM map.
  • Fig. 9C Superposition of protomer A and protomer B. Resulting from the asymmetric conformation of D5:D5' in the cryo-EM structure, domains D5 and D5' do not superpose (left). In contrast, the conformations of D5 and D5' in crystal structure are symmetric, and thus D5 and D5' superpose well with each other (right).
  • Figs. 9D-9E Superpositions of D4-D5 and D4'-D5' of the cryo-EM structure.
  • Fig. 9D Superposition of D4 on D4'. Whereas D4 and D4' superpose well, D5 and D5' do not superpose well.
  • Fig. 9E Superposition of D5 on D5'. Whereas D5 and D5' superpose well, D4 and D4' do not superpose well.
  • Figs. 10A-10E D5:D5' contact formation requires conformational changes of interface residues, in accordance with some embodiments.
  • Fig. 10 A Close-up view of domain D5 (in white) of the crystal structure of the truncated ECD of monomeric KIT (PDB ID 2EC8). D5 side chain conformations in the cry stal structure of monomeric KIT are similar to D5 side chain conformations in the WT KIT:SCF crystal structure displayed in Fig. 10B.
  • Fig. 10B Close-up view of domains D5 (light gray) and D5’ (dark gray) of the crystal structure of the truncated ECD of WT KIT:SCF (PDB ID 2E9W).
  • Fig. 10C Close-up view of the D5:D5' complex of the cryo-EM structure of full-length WT KITSCF dimers. D5:D5' has an asymmetric quaternary conformation. D5 on the left, D5' on the right.
  • Fig. 10D Superposition of D5 of the cry stal structure from Fig. 10B and D5 of the cryo-EM structure from Fig. 10C. The significant conformational changes of Y418 and F504 upon D5:D5' complexation are indicated by arrows. Fig.
  • Figs. 11 A-l IB Tyrosine autophosphorylation of wild-ty pe and mutants of KIT and PDGFR/> in response to ligand stimulation, in accordance with some embodiments.
  • Fig. 11 A SCF stimulated or unstimulated NIH 3T3 cells stably expressing WT KIT or KIT mutants were lysed and subjected to immunoprecipitation with anti -KIT antibody followed by SDS- PAGE and immunoblotting with anti-KIT and anti-phospho-KIT (Y703) antibodies.
  • Full- length KIT migrates as two bands with an apparent MW of 145 kDa and 125 kDa.
  • Fig. 1 IB PDGF-BB stimulated or unstimulated MEF cells stably expressing WT PDGFR/> or PDGFR/> mutants were lysed and subjected to immunoprecipitation with anti -HA antibody followed by SDS-PAGE and immunoblotting with anti-PDGFR/i antibody or anti-phosphoty rosine antibody. Representative blots are shown from experiments performed in triplicate. Quantification of PDGFR/i phosphorylation is normalized relative to PDGFR/> expression levels. Quantification results show mean ⁇ s.d. of three independent experiments.
  • Figs. 12A-12F Cryo-EM data processing and analysis of the structure KIT(DupA502,Y503):SCF dimers, in accordance with some embodiments.
  • Fig. 12A Representative cryo-EM micrograph and 2D class averages.
  • Fig. 12B Cryo-EM data processing flow-chart. The unsharpened local resolution map of a ECD local refinement (overall resolution: 3.17 A) is displayed.
  • CS cryoSPARC.
  • Fig. 12C FSC curves of the ECD local refinement.
  • Fig. 12D Angular distribution plot of the particles used for the ECD local refinement.
  • Fig. 12E Cryo-EM density of the homotypic D4:D4' salt bridge.
  • Fig. 12F Cryo- EM densities of residues of P-strands A, PG. PA', and PG' forming the D5:D5' interface.
  • Figs. 13A-13E Cryo-EM data processing and analysis of the structure of KIT(T417I,A418-419) dimers, in accordance with some embodiments.
  • Fig. 13A Representative negative staining EM micrograph and 2D class averages.
  • Fig. 13B Representative cryo-EM micrograph and 2D class averages.
  • Fig. 13C Cryo-EM data processing flow-chart of a homogeneous refinement of the entire complex (8. 19 A).
  • CS cryoSPARC. Fig.
  • FIG. 13D Cr o-EM data processing flow-chart of a masked refinement of the ECD (13.1 A, mask-uncorrected FSC).
  • Fig. 13E Superposition of the cry stal structure of D4D5 fragment dimers of KIT(T417I.A418-419) (PDB ID 4PGZ; protomer A in beige, protomer B in blue) and the cryo-EM structure of full-length KIT(T417I,A418-419) dimers (in gray).
  • Figs. 14A-14F Comparison of the ECD structures of full-length WT KIT:SCF dimers and full-length KIT(T417I.A418-419) dimers, in accordance withs ome embodiments.
  • Fig. 14A Space-filling model of the cryo-EM structure of WT KIT:SCF dimers from Fig. 2A. Protomer A in beige, protomer B in blue, and SCF in green. The color-code is used throughout Figs. 14A-14F.
  • Fig. 14B Close-up view of the boxed D4D5:D4'D5' complex from Fig. 14A. CT, C-terminus.
  • Fig. 14A Close-up view of the boxed D4D5:D4'D5' complex from Fig. 14A. CT, C-terminus.
  • Fig. 14A Close-up view of the boxed D4D5:D4'D5' complex from Fig. 14A. CT, C-termin
  • FIG. 14C Close-up view of the boxed D5:D5' complex from Fig. 14B. Ca atoms of C-terminal residues A507 and F508' are shown as red spheres.
  • Fig. 14D Space-filling model of the cryo-EM structure of KIT(T417I,A418-419) dimers from Fig. 5C.
  • Fig. 14E Crystal structure of D4D5 fragment dimers of KIT(T417I,A418-419) (PDB ID 4PGZ) corresponding to the boxed region from Fig. 14D.
  • Fig. 14F Close-up view of the boxed D5:D5' complex from Fig. 14E. Ca atoms of C-terminal residues N505 and N505' are shown as red spheres.
  • Figs. 15A-15F Cryo-EM data processing and analy sis of KIT(T417I,A418- 419):SCF dimers, in accordance with some embodiments.
  • Figs. 15A Representative negative staining EM micrograph. Examples of single particles of KIT(T417EA418-419):SCF dimers showing the characteristic shape of the ligand-bound dimeric receptor are encircled in white.
  • Fig. 15B Representative cryo-EM micrograph and 2D class averages.
  • Fig. 15C Cryo-EM data processing flow-chart. The unsharpened local resolution map of an ECD local refinement (overall resolution: 3.96 A) is displayed.
  • CS cryoSPARC.
  • Fig. 15D FSC curves of the ECD local refinement.
  • Fig. 15E Angular distribution plot of the particles used for the ECD local refinement.
  • Fig. 15F Superposition of the cryo-EM structure of full-length KIT(T417I,A418-419):SCF dimers and the cry stal structure of D4D5 fragment dimer of KIT(T417I,A418-419). Due to steric pressure, binding of SCF to KIT(T417EA418-419) dimers converts the tilted conformation of D5:D5' to the WT-hke conformation of D5:D5' exhibiting parallel-oriented domains.
  • Fig. 16 Cryo-EM data collection, model refinement, and validation statistics in accordance withs some embodiments.
  • first and second features are formed in direct contact
  • additional features may be formed betw een the first and second features, such that the first and second features may not be in direct contact
  • present disclosure may repeat reference numerals and/or letters in the various examples.
  • the present study the cryo-EM structures of ligand-induced wild-type and ligand-sensitized oncogenic KIT mutants were solved. Additional biochemical and cellular analyses of the ligand-induced wild-type and ligand- sensitized oncogenic KIT mutants were also performed. The present study revealed a conserved site in the D5 domain of KIT which is involved in the dimerization of the KIT protein. In certain embodiments, this conserved site is a hotspot for activating somatic mutations in the KIT proteins.
  • the present study shows that the conserved site in the D5 domain can be therapeutically targeted for inhibition of wild type KIT, oncogenically activated KIT mutants, and/or other type-III receptor tyrosine kinases (RTKs). Furthermore, the disruption of the D4:D4' interaction in conjunction with the disruption of D5:D5' interaction potently antagonizes KIT activity. Sequence alignment indicates that the manner of by which KIT dimerizes.
  • a "disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • co-administered and “co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the disclosure along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein.
  • the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach.
  • the co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
  • composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient.
  • Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal. intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical. ophthalmic, pulmonary, and topical administration.
  • the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non- toxic, z.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function.
  • Each carrier must be “acceptable” in the sense of being compatible w ith the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient.
  • compositions that may sen e as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives.
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient.
  • pharmaceutically acceptable carrier may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
  • the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.
  • a “pharmaceutically effective amount,” “therapeutically effective amount,” or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • prevent means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.
  • the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • treatment is defined as the application or administration of a therapeutic agent, i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder and/or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder and/or the symptoms of the disease or disorder.
  • a therapeutic agent i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent
  • a therapeutic agent i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent
  • a therapeutic agent i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent
  • an isolated tissue or cell line from a patient (e.g., for diagnosis or ex
  • Non-limiting examples of such compounds include polypeptides including D5 domains of the tj pe-III RTKs, and monoclonal antibodies or other specific binders (such as (poly)peptides or non-(poly)peptides) that bind to D5 and block the dimerization interface of the D5 domain.
  • the instant specification is directed to a method of inhibiting a type-III receptor tyrosine kinase.
  • the type-III receptor tyrosine kinases include, but is not limited to, KIT, PDGFRa, PDGFR , CSF1R, and/or FLT3.
  • KIT refers to the receptor tyrosine kinase proteins encoded by the proto-oncogene C-Kit. and the genes encoding the same.
  • KIT refers to the tyrosine kinase protein having the amino acid sequence set forth in SEQ ID NO: 1, the gene that encodes the protein having the amino acid sequence set forth in SEQ ID NO: 1, genes that occupy the same allele as the gene encoding the protein having the amino acid sequence set forth in SEQ ID NO: 1 in the human genome, all protein products thereof, as well as the orthologs of the human proteins or genes in other species.
  • KIT refers to only the proteins described in this paragraph.
  • the definition as set forth in this paragraph includes isoforms of KIT, mutants of KIT (such as oncogenic mutants of KIT), such as those found in mammalian subjects such as humans.
  • the term “PDGFRa” refers to platelet-derived growth factor receptor a proteins, and genes encoding the same.
  • the term “PDGFRa” refers to the tyrosine kinase protein having the amino acid sequence set forth in SEQ ID NO:2. the gene that encodes the protein having the amino acid sequence set forth in SEQ ID NO:2, genes that occupy the same allele as the gene encoding the protein having the amino acid sequence set forth in SEQ ID NO:2 in the human genome, all protein products thereof, as well as the orthologs of the human proteins or genes in other species.
  • the term “PDGFRa” refers to only the proteins described in this paragraph.
  • the term “PDGFRP” refers to platelet-derived grow th factor receptor P proteins, and genes encoding the same.
  • the term “PDGFRP” refers to the tyrosine kinase protein having the amino acid sequence set forth in SEQ ID NO: 3, the gene that encodes the protein having the amino acid sequence set forth in SEQ ID NO: 3, genes that occupy the same allele as the gene encoding the protein having the amino acid sequence set forth in SEQ ID NO: 3 in the human genome, all protein products thereof, as well as the orthologs of the human proteins or genes in other species.
  • the term “PDGFRP” refers to only the proteins described in this paragraph.
  • CSF1R refers to colony stimulating factor 1 receptor proteins, and genes encoding the same.
  • CSF1R refers to the tyrosine kinase protein having the amino acid sequence set forth in SEQ ID NO:4, the gene that encodes the protein having the amino acid sequence set forth in SEQ ID NO: 4, genes that occupy the same allele as the gene encoding the protein having the amino acid sequence set forth in SEQ ID NO: 4 in the human genome, all protein products thereof, as well as the orthologs of the human proteins or genes in other species.
  • the term “CSF1R” refers to only the proteins described in this paragraph.
  • the term “FLT3” refers to fms-like tyrosine kinase 3 proteins, and genes encoding the same.
  • the term “FLT3” refers to the tyrosine kinase protein having the amino acid sequence set forth in SEQ ID NO:5, the gene that encodes the protein having the amino acid sequence set forth in SEQ ID NO:5, genes that occupy the same allele as the gene encoding the protein having the amino acid sequence set forth in SEQ ID NO:5 in the human genome, all protein products thereof, as well as the orthologs of the human proteins or genes in other species.
  • the term “FLT3” refers to only the proteins described in this paragraph.
  • the definition as set forth in this paragraph includes isoforms of FLT3, mutants of FLT3 (such as oncogenic mutants of FLT3), such as those found in mammalian subjects such as humans.
  • the method includes contacting the type- 1 II RTK with a compound inhibiting the dimerization of the pe-III RTK.
  • the compound binds to/occupies the [3G strand of the D5 domain of the type-III RTK.
  • the present study discovered that the
  • occupying the PG strand of the D5 domain can inhibit the dimerization of the type-III RTKs, thereby inhibiting the type-III receptor tyrosine kinases.
  • the compound binds to and fully or partially blocks the
  • the type-III RTK is proto-oncogene c-KIT (KIT) or a mutant thereof, and the compound binds to at least one KIT amino acid residue corresponding to A502, Y503, F504, N505, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO:1.
  • the type-III RTK is platelet-derived growth factor receptor a (PDGFRa) or a mutant thereof, and the compound binds to at least one PDGFRa amino acid residue corresponding to R512, E513, L514, K515, L516, V517 or A518 of the polypeptide as set forth in SEQ ID NO: 2.
  • PDGFRa platelet-derived growth factor receptor a
  • the type-III RTK is platelet-derived growth factor receptor (3 (PDGFR
  • 3 amino acid residue corresponding to Q519, E520, V521, 1522, V523, V524 or P525 of the polypeptide as set forth in SEQ ID NO:3.
  • the type-III RTK is colony-stimulating factor-1 receptor (CSF-1R) or a mutant thereof, and the compound binds to at least one amino acid residue in the CSF-1R corresponding to W496, A497, F498, 1499, P500, 1501 or S502 of the polypeptide as set forth in SEQ ID NO:4.
  • CSF-1R colony-stimulating factor-1 receptor
  • the type-III RTK is fms-like tyrosine kinase 3 (FLT3) or a mutant thereof, and the compound binds to at least one amino acid residue in the FLT3 corresponding to E525, T526, 1527, L528, L529, N530 or S531 of the polypeptide as set forth in SEQ ID NO: 5.
  • FLT3 fms-like tyrosine kinase 3
  • the compound is a polypeptide comprising the D5 domain of the type-III RTK or a nucleic acid encoding the polypeptide, and the polypeptide does not have tyrosine kinase activity.
  • the compound is a polypeptide comprising the D4-D5 section of the type-III RTK or a nucleic acid encoding the polypeptide.
  • the polypeptide does not have tyrosine kinase activity, such as does not include the kinase domain of the ty pe-III RTK.
  • the ty pe-III RTK is KIT
  • the polypeptide comprises residues 408-508 (D5 domain) or residues 308-508 (D4-D5 segment) of SEQ ID NO: 1.
  • the KIT is a KIT with duplication of A502 and Y503 (KIT DupA502,Y503), and the polypeptide comprises duplication of A502 and Y503 in the sequence set forth in SEQ ID NO: 1.
  • the KIT is a KIT with T417I mutation and deletion of Y418 and D417 (KIT T417I. A418,419). and the polypeptide comprises T417I mutation and deletion ofY418 and D417 in the sequence set forth in SEQ ID NO: 1.
  • the compound comprises an antibody that binds to the (3G strand of the D5 domain of the type-III RTK, or a nucleic acid encoding the antibody .
  • the antibody is a divalent antibody, and the antibody further binds to at least one residue in the D4 domain of the type-III RTK responsible for forming the salt bridge.
  • the type-III RTK is KIT
  • the antibody interacts with/binds to at least one KIT residue corresponding to A502, Y503, F504.
  • the ty pe-III RTK is KIT, wherein the antibody is divalent, and the antibody further interacts with/binds to at least one KIT residue corresponding to E386 or R381 of the polypeptide as set forth in SEQ ID NO: 1.
  • the type-III RTK is an isolated protein. In some embodiments, the type-III RTK is a protein within a cell and/or on the surface of a cell. In some embodiments, the cell is an isolated cell, such as a primary cell or a cell from a cell line. In some embodiments, the cell is in a subject, such as a mammal, such as a human.
  • the type-III RTK is in a subject, such as a mammal, such as a human.
  • the type-III RTK is an overactivated mutant type-III RTK.
  • tj pe-III RTK proteins Aberrantly activated or overexpressed tj pe-III RTK proteins are known to drive cancers or other diseases. As such, inhibiting the type-III RTK in subjects suffering from diseases or disorders caused by or involving overactive and/or overexpressed type-III RTK proteins can treat, ameliorate and/or prevent such diseases or disorders.
  • the instant specification is directed to a method of treating, ameliorating and/or preventing a disease or disorder caused by or involving overactive and/or overexpressed type-III RTK.
  • the method includes administering to the subject a compound capable of blocking dimerization of the type-III RTK.
  • the compound is the same as or similar to those as described elsewhere herein, such as the "Method of Inhibiting Type-III Receptor Tyrosine Kinases" section.
  • the type-III RTK is KIT
  • the disease or disorder includes KIT-driven cancers such as KIT-dnven gastrointestinal stromal tumors (KIT-dnven GISTs). KIT-driven core binding factor acute myeloid leukemia, or other KIT-driven cancers; mast cell diseases such as systemic mastocytosis (which is sometimes cancerous, as well); and urticaria and other skin related allergic and inflammatory ddiseases.
  • the type-III RTK is PDGFRa
  • the disease or disorder includes PDGFRa-driven cancers such as PDGFRa-associated chronic eosinophilic leukemia, PDGFRa-driven gastrointestinal stromal tumors (PDGFRa-driven GISTs), or other PDGFRa-driven cancers; and inflammatory fibroid polyps.
  • PDGFRa-driven cancers such as PDGFRa-associated chronic eosinophilic leukemia, PDGFRa-driven gastrointestinal stromal tumors (PDGFRa-driven GISTs), or other PDGFRa-driven cancers.
  • the type-III RTK is PDGFR0
  • the disease or disorder includes PDGFR(3-driven cancers such as PDGFR(3-associated chronic eosinophilic leukemia or other PDGFR
  • PDGFR(3-driven cancers such as PDGFR(3-associated chronic eosinophilic leukemia or other PDGFR
  • the type-III RTK is CSF1R
  • the disease or disorder includes CSFlR-driven cancers such as CSFIR-driven myeloid malignancy, CSFIR-driven Hodgkin's lymphoma, CSFIR-driven anaplastic large cell lymphoma, other types of CSFIR- driven cancers, or cancers drived by tumor associated machrophages.
  • the type-III RTK is FLT3, and the disease or disorder includes FLT3-driven cancers such as FLT3-driven core binding factor acute myeloid leukemia, FLT3-driven cytogenetically normal acute myeloid leukemia, or other FLT3- driven cancers.
  • FLT3-driven cancers such as FLT3-driven core binding factor acute myeloid leukemia, FLT3-driven cytogenetically normal acute myeloid leukemia, or other FLT3- driven cancers.
  • the disease or disorder is caused by or involves KIT overactivation, and the method further includes administering to the subject an effective amount of SCF.
  • the SCF is administered as a protein.
  • the SCF is administered as a nucleic acid encoding the SCF protein.
  • the compound for inhibiting type-III RTKs or the compounds for treating, ameliorating, and/or preventing diseases or disorders are in the form of nucleic acids.
  • Vectors can increase the stability of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells.
  • the compounds are incorporated into a vector.
  • the instant specification relates to a vector, including the nucleic acid sequence of the instant specification or the construct of the instant specification. The choice of the vector will depend on the host cell in which it is to be subsequently introduced.
  • the vector of the instant specification is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the instant specification to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available.
  • the vector is a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in virology 7 and molecular biology 7 manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6.326,193.
  • the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses.
  • AAV adeno-associated virus
  • the viral vector is a viral vector that can infect a human.
  • the desired nucleic acid sequence can be inserted between the inverted terminal repeats (ITRs) in the AAV.
  • the viral vector is an AAV2 or an AAV8.
  • the promoter can be a thyroxine binding globulin (TBG) promoter.
  • TBG thyroxine binding globulin
  • the promoter is a human promoter sequence that enables the desired nucleic acid expression in the desired site.
  • the AAV can be a recombinant AAV.
  • the capsid comes from one AAV serotype and the ITRs come from another AAV serotype.
  • the AAV capsid is selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid.
  • the ITR in the AAV is at least one ITR selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR.
  • the instant specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid.
  • the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein.
  • the AAV is an engineered AAVs for delivering nucleic acid across the blood brain barrier to the central and peripheral nervous systems, such as those as described by Chan et al., Nat Neurosci. 2017 Aug; 20(8): 1172— 1179. The entirety of this reference is incorporated herein by reference.
  • the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell.
  • vectors in which the nucleotide sequence of the instant specification or the gene construct of the instant specification can be inserted include a tet-on inducible vector for expression in eukaryote cells.
  • the vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.
  • the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the instant specification, described elsewhere herein.
  • a promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokary otic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory' regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology 7 , including PCRTM, in connection with the compositions disclosed herein (U.S. Patent 4,683,202. U.S. Patent 5,928,906).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • the recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells.
  • Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, (3-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
  • the selectable markers may be introduced on a separate vector from the nucleic acid of interest.
  • the method of treating, ameliorating, and/or preventing the diseases/disorders related to the type-III RTKs includes administering to the subject the effective amount of at least one compound contemplated within the disclosure.
  • the compound is formulated in a composition.
  • the subject is further administered at least one additional agent that treats, ameliorates, and/or prevents a disease and/or disorder contemplated herein.
  • the compound and the at least one additional agent are coadministered to the subject.
  • the compound and the at least one additional agent are co-formulated.
  • the compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds.
  • additional compounds may comprise compounds of the present disclosure and/or at least one additional agent for treating the ty pe-III RTK related conditions, and/or at least one additional agent that treats one or more diseases or disorders contemplated herein.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981 , Clin. Pharmacokinet.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and/or disorder contemplated herein.
  • dosages as well as staggered dosages may be administered daily 7 or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions contemplated within the disclosure to a patient may be carried out using know n procedures, at dosages and for periods of time effective to treat a disease and/or disorder contemplated herein in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and/or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5.000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g, physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease and/or disorder contemplated herein.
  • compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
  • the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two. days, every’ three days to once a week, and once every two weeks.
  • the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
  • the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
  • Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1.000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg. or less than about 500 mg. or less than about 200 mg. or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of type-III RTKs related conditions in a patient.
  • Formulations may be employed in admixtures with conventional excipients, z.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e. , lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers. coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g, other analgesic agents.
  • Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal. (trans )urethral. vaginal (e.g.. trans- and perivaginally), (intra)nasal and (trans)rectal).
  • transdermal transmucosal
  • transmucosal e.g., sublingual, lingual, (trans)buccal. (trans )urethral. vaginal (e.g.. trans- and perivaginally
  • vaginal e.g.. trans- and perivaginally
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry' powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g, cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g.. sodium starch gly collate); or wetting agents (e.g, sodium lauryl sulphate).
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa.
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g, lecithin or acacia); non-aqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g, lecithin or acacia
  • non-aqueous vehicles e.g, almond oil, oily esters or ethyl alcohol
  • preservatives e.g, methyl or propyl p-hydroxy benzoates or sorbic acid
  • the present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication.
  • a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5.972,389; 5.582,837; and 5.007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the disease/disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day. every 2 days, even’ 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (z.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days. 4 days. 5 days. 6 days. 7 days. 10 days, 12 days. 15 days, 20 days, 28 days. 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a longterm basis upon any recurrence of symptoms and/or infection.
  • the compounds for use in the method of the disclosure may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g.. about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50.
  • Capsid assembly modulators exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • Example 1 Cryo-EM analyses of wild-type and oncogenic KIT mutants reveal structural oncogenic plasticity and a novel "Achilles heel" for therapeutic intervention
  • the receptor tyrosine kinase KIT and its ligand SCF are required for the development of hematopoietic stem cells, germ cells and other cells.
  • a variety of human cancers such as acute myeloid leukemia and mast cell leukemia are driven by somatic gain- of-function KIT mutations.
  • the present study reports cryo-EM structural analyses of full-length wild-type and two oncogenic KIT mutants which show that the symmetric arrangement of ligand-occupied KIT dimers is converted into asymmetric D5 homotypic contacts juxtaposing the plasma membrane.
  • Mutational analysis of KIT reveals in D5 region a novel "Achilles-heel" for therapeutic intervention.
  • a ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation.
  • a constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. SCF binding to this mutant fully restores the conformation of wild-ty pe KIT dimers revealing an unexpected structural plasticity of oncogenic mutants that may offer new therapeutic modality.
  • RTK receptor tyrosine kinase
  • the first images of detergent-solubilized preparations of ligand-occupied full-length RTK dimers were obtained by analyzing negatively-stained EM samples of full-length EGF- induced EGF receptor (EGFR) dimers, SCF-induced KIT dimers, and PDGF-induced PDGF receptor (PDGFR) dimers. These studies revealed low-resolution structures of the ligand- occupied ECDs of these RTKs that are consistent and in accord with the high-resolution structures of corresponding ligand-occupied ECD complexes determined by X-ray crystallography.
  • EGFR EGF- induced EGF receptor
  • SCF-induced KIT dimers SCF-induced KIT dimers
  • PDGFR PDGF-induced PDGF receptor
  • KIT and its ligand SCF stem cell factor
  • SCF stem cell factor
  • KIT is a member of the type-III subfamily of RTKs, which also includes PDGFR-a, PDGFR-P, CsflR (colony stimulating factor 1 receptor or FMS), and Flt3R (FMS-like tyrosine kinase 3 receptor).
  • PDGFR-a PDGFR-a
  • PDGFR-P CsflR
  • Flt3R Flt3R
  • the present study presents cryo-EM analyses of the structures of full-length ligand- induced KIT dimers.
  • the cryo-EM structures of ligand-induced wild-type and ligand- sensitized oncogenic KIT mutants provide mechanistic insights into a new conserved "Achilles heel" in D5 of KIT, a hotspot for activating somatic mutations, that can be therapeutically targeted for inhibition of oncogenically activated KIT and other type-III RTKs.
  • the structural analyses demonstrate that the 2-fold symmetric arrangement of SCF bound to the KIT ligand-binding region (D1-D3) and of the salt bridges mediating homotypic D4:D4' contacts is converted into an asymmetric arrangement of D5:D5' contacts.
  • cryo-EM map of the dimeric cytoplasmic domains of wild-type KIT and its oncogenic mutants may arise from the small size (95 kDa) of the cytoplasmic domain and primarily from the dynamic nature of the cytoplasmic region that enables the tyrosine kinase domain of KIT to mediate efficient trans autophosphorylation within the context of a dimeric receptor complex.
  • Several recently published cryo-EM analyses of full-length RTKs similarly reported an inability to resolve structures of the cytoplasmic domain of ligand-activated full-length RTKs. This suggests that structural flexibility of the linker connecting the ECD with the transmembrane and cytoplasmic domains is a common feature of RTKs.
  • Example 2 The symmetric arrangement of ligand-occupied KIT dimers is converted into asymmetric D5:D5' contacts juxtaposing the plasma membrane
  • the present study determined the cryo-EM map of the ECD of full-length wild-type KIT:SCF at a global resolution of 3.45 A (Figs. 2A-2G and 8A-8G: local resolution map in Fig. 8B).
  • 3D variability analysis (Punjani and Fleet, 2021) of the cryo-EM map (Fig. 8G) reveals that the complex between neighboring domains D5 and D5' (D5:D5') is oscillating perpendicularly relative to the view of Fig. 2A, explaining the lower resolution of D5:D5' relative to rest of the ECD (Fig. 8B).
  • cryo-EM structure of SCF bound to the ligand-binding region of KIT (D1-D3) is virtually identical to the crystal structure of SCF in complex with the soluble ECD of KIT.
  • cryo-EM structures of domains D1-D4 and Dl'-D4' are very' similar to those of the crystal structure (Figs. 9A-9E).
  • the cryo-EM map also clearly reveals the salt bridge between E386 and R381 responsible for mediating the highly conserved D4:D4' interface (Fig. 8E).
  • the cryo-EM structure reveals a novel asymmetric interface formed between two neighboring domains D5 and D5' with a buried surface area of 292 A 2 (Fig. 2B).
  • D5:D5' interface is not present in the cry stal structure of the soluble ECD (Fig. 9B). It is clear that direct D5:D5' contact formation depends upon the integrity of the KIT receptor, requiring the presence of transmembrane and cytoplasmic domains in full-length KIT. D5 of KIT exhibits an Ig-like P-sandwich fold composed of antiparallel (3-sheets A- B- E-PD, and PC-PF-PG. The present study refrained from including P-strand PD, PD', and the loops proximal to the membrane into the models of D5:D5' (Fig. 9B) due to limited resolution of these regions in the cryo-EM map.
  • the backbone interactions of PG:PG' divide the side chain interactions at the interface into two separated sites, which the present study designated as site-I and site-II (Figs. 2E-2G). All side chains at the interface are hydrophobic, with the exception of the hydrophilic side chains of N505 and N505', which are solvent exposed. Because N505' is located at the center of site-I, the hydrophilic side chain of N505' largely prevents hydrophobic interactions at the interface, allowing only hydrophobic interactions between F506 and A507' (Fig. 2F, dotted line).
  • N505 in site-II is shifted by one residue toward the C-terminus due to the asymmetric interface, enabling hydrophobic interactions between Y503 and F504' (Fig. 2G). Furthermore, the shift of N505 enables Y418' in PA' to move closer toward the interface, enabling hydrophobic interactions between Y418' and Y503.
  • the single mutations F504A and F506A showed reduced ligand-induced KIT activation with the F506A mutant of KIT exhibiting approximately 50% reduced tyrosine autophosphorylation when the cells were stimulated with 1.5 nM of SCF, a saturating ligand concentration for wild-ty pe KIT expressed in these cells.
  • double mutation R381A,F506A exhibited a strong synergistic effect manifested by complete loss of SCF-induced tyrosine autophosphorylation of KIT.
  • the double mutation R381A.F504A showed less synergistic effect on SCF-induced tyrosine autophosphorylation of KIT.
  • Example 4 The ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5:D5' interface
  • the present stdy determined the cryo-EM map of the extracellular region of full-length KIT(DupA502,Y503):SCF dimers at an overall resolution of 3.17 A (Figs. 12A-12F; local resolution map in Fig. 12B).
  • the overall structure of domains D1-D4, D1 -D4', and bound SCF is virtually identical to the cryo-EM structure of wild- t pe KIT:SCF.
  • a more comprehensive asymmetric D5:D5' interface than in wild-type KIT was resolved in the structure of KIT DupA502,Y503 mutant (Figs.
  • DupY 503' now forms tight hydrophobic interactions with Y418 of 0-sheet 0A that is consistent with the compact conformation of the D5:D5' interface.
  • SCF dimers the polar side chain ofN505' repulses Y418 of 0-sheet 0A, resulting in less extensive D5:D5' contacts (Fig. 4B).
  • Fig. 4C the conformational change of 0-sheet 0A-0B-0E
  • SCF Fig. 4C
  • N505 is distant from and therefore does not make a direct contact with Y418' because of the asymmetric 0G:0G' interface conformation (Fig. 4D). Therefore, the insertion of DupA502 and DupY503 and the resulting shift of N505 by two residue positions to the C-terminus does not affect the conformation of 0-sheet 0A'.
  • the D5:D5' interface of wild-type KIT:SCF dimers Similar to the D5:D5' interface of wild-type KIT:SCF dimers, the D5:D5' interface of KIT(DupA502,Y503):SCF dimers has an asymmetric conformation (Fig. 4E).
  • N505 and N505' are C-terminally shifted by two residues, which prevents the disruption of hydrophobic interactions at the interface.
  • Example 5 The extracellular domain of a constitutively active oncogenic KIT mutant adopts a V-shaped conformation
  • the present study further used cryo-EM to determine the structure of full-length KIT(T417I,A418-419) — a constitutively active KIT mutant — in the absence of SCF.
  • Negative staining EM and cryo-EM 2D class averages reveal the formation of KIT(T417I,A418-419) dimers even in the absence of SCF binding (Figs. 13A-13B).
  • the dimeric complex of KIT(T417I,A418-419) has a high conformational flexibility which is due to lack of stabilization caused by SCF binding to the ligand-binding region.
  • the present study was unable to achieve a high-resolution 3D reconstruction of this cryo-EM dataset. Therefore, the present study refined two different cryo-EM maps at medium to low resolution.
  • the first cryo-EM map is a global refinement of the entire particle without the application of a mask (Fig. 13C). This map is relatively well resolved for D4-D5 and D4'-D5', revealing a V-shaped conformation for them (Figs. 5A-5B).
  • the cryo-EM map features two characteristic bulges at a map region corresponding to the D5:D5' complex.
  • the crystal structure of the D4D5 fragment dimer of KIT(T417I,A418-419) was docked into the map and achieved an overall good fit.
  • the angle between the two protomers in the crystal structure appears slightly larger than the one in the cryo-EM map, which can be attributed to the high degree of structural flexibility of this complex.
  • the docking pose of the D4D5 fragment dimer explains the two characteristic bulges in the map with the tilted orientation of the two domains of D5:D5'.
  • the ECDs D1-D5 and Dl'-D5' are fully resolved, exhibiting a characteristic V-shaped conformation (Fig. 5C).
  • the resolution of the map enables visualizing individual domains as separate density blobs at high contour levels.
  • Rigid-body docking of two copies of the previously reported cry stal structure of the monomeric ECD of KIT into the map followed by slight manual adjustment of D5 resulted in a good fit.
  • the resulting model shows the ECDs adopting a V-shaped conformation, with a similar angle as in the crystal structure of the D4D5 fragment dimer of KIT(T417I,A418-419) (Fig. 13E).
  • the only interactions between the ECDs are mediated via the D5:D5' contacts (the 'bottom' of the V), whereas Dl- D4 and DT-D4' are distant from each other (the 'arms' of the V).
  • cryo-EM maps of full-length KIT(T417I,A418-419) dimers confirm the V-shaped conformation of the crystal structure of D4D5 fragment dimers of KIT(T417I,A418-419). Furthermore, the cryo-EM results are fully consistent and support the molecular interactions at the D5:D5' interface as seen in the crystal structure of D4D5 fragment dimers of KIT(T417I,A418-419).
  • Example 6 The wild-type conformation of KIT is restored by ligand binding to the constitutively active oncogenic KIT mutant
  • cryo-EM map of D5:D5' is sufficient to unambiguously identify an asymmetric D5:D5' conformation similar to the conformation in wild-type KIT:SCF and interface formation via PG: PG' (Fig. 6C).
  • KIT molecules are expressed at the cell membrane as freely diffusing KIT monomers. Binding of SCF dimers to the ligand-binding region (D1-D3) of the extracellular domain of KIT brings two KIT receptors together in the cell membrane. The dramatic increase in local concentration caused by reduced dimensionality of two KIT molecules held together by SCF at the cell membrane combined with the flexibility 7 of inter-domain linkers play an essential role in KIT activation.
  • the flexible hinge regions connecting D3 to D4, D4 to D5, and the linker connecting D5 to the transmembrane region enable efficient formation of homotypic D4:D4' and D5:D5' contacts as well as additional interactions that may take place between the transmembrane and the cytoplasmic region with corresponding regions of neighboring KIT molecules.
  • This "zipper-like" mechanism enables formations of D4 and D5 homofypic contacts by weak binding affinities which are not strong enough to mediate dimerization and activation of unoccupied KIT molecules. However, these weak affinities are sufficiently effective due to the high local concentration of KIT at the cell membrane and because of the cooperative action of multiple homotypic contacts in each KIT molecule.
  • the homofypic interactions mediated by D4 and D5 at a region juxtaposing the transmembrane domain set the stage for efficient trans autophosphorylation of the tyrosine kinase domain, resulting in stimulation of tyrosine kinase activity followed by recruitment and activation of multiple cellular signaling pathways.
  • a similar "zipper-like" mechanism can be ascribed for the mechanism of activation of other type-III RTK family members stimulated by their specific ligands, and for other RTKs.
  • cryo-EM to determine the structures of full-length wild-type KIT and two full-length oncogenic KIT mutants.
  • the dynamic nature of the cytoplasmic region of KIT precludes resolving the structure of the cytoplasmic domain.
  • interesting and important new insights were revealed about the structure of the extracellular region in their native full-length state of SCF-occupied KIT dimers and the molecular mechanism underlying the action of two different oncogenic KIT mutants. Why does the D5.D5' interface adopt a distinctly asymmetric conformation?
  • cryo-EM structure of ligand-induced full-length wild-type KIT dimers clearly shows that D5:D5' homotypic contacts adopt an asymmetric conformation, which stands in stark contrast to the symmetric conformation of the remaining part of the ligand-occupied extracellular domain (domains D1-D4. DT-D4', and SCF; Figs. 9A and 9C).
  • the asymmetric conformation of the D5:D5' complex is the result of asymmetric interface interactions, which are mainly formed between the two neighboring p-strands
  • 3G and PG' run in the same direction but are rotated by about 180° along the long axis relative to each other. Therefore, to enable the formation of backbone hydrogen bonds, the two P-strands PG and PG' have to engage in asymmetric interactions shifted by one residue (Figs. 2C-2D).
  • an alternative, not mutually exclusive, interpretation is that the asymmetric conformation of D5:D5' reflects the most energetically stable interface.
  • This interpretation is consistent with the cryo-EM structure of the oncogenic ligand-sensitized KIT(DupA502,Y503) mutant in complex with SCF.
  • This oncogenic mutant exhibits a more comprehensive asymmetric D5:D5' interface, which significantly increases the buried surface areas to 479 A 2 compared to 292 A 2 of wild- t pe KIT.
  • the binding affinity- of the dimerization reaction of isolated D4D5 fragments of the oncogenic mutant is increased by 10 to 20-fold compared to the corresponding region of wild- type KIT.
  • the interpretation that the asymmetric conformation of D5:D5' reflects the most energetically stable interface was favored.
  • Double mutant R381 A,F506A which simultaneously disrupts both D4:D4' and D5:D5' interfaces, causes a complete loss of ty rosine autophosphorylation activity at any of the tested SCF concentrations (Fig. 3B).
  • Analysis of activities of similar mutations in D5 of PDGFR/> (Fig. 11B) suggest that asymmetric D5:D5' contacts may also take place during the course of ligand-induced activation of PDGFR/I and other members of type-III RTKs.
  • the oncogenic KIT(DupA502.Y503) mutant exhibits elevated basal tyrosine kinase activity and can be further activated by SCF binding.
  • the ty rosine kinase activity of this mutant relies entirely on the integrity of the salt bridge maintaining the D4:D4' contacts.
  • the increased dimerization affinity of this mutant shifts the monomer-dimer equilibrium toward dimer formation, thereby disturbing the delicately balanced ligand-mediated activation of wild-type KIT.
  • the elevated basal activity of KIT(DupA502.Y503) is caused by an increased population of active KIT mutant dimers even in the absence of SCF stimulation.
  • KIT(DupA502,Y503):SCF dimers overactivation upon stimulation with SCF is the result of an increase in the concentration of active KIT(DupA502,Y503):SCF dimers.
  • the cryo-EM structure of KIT(DupA502,Y503):SCF provides a satisfactory explanation for the biophysical properties of this oncogenic mutant.
  • Mutation DupA502,Y503 improves the shape complementarity at the D5:D5' interface, resulting in additional hydrogen bonds and hydrophobic interactions at the interface (Figs. 4E-4H). Increased D5:D5' interface interactions are responsible for increased affinity and stability of KIT(DupA502,Y503):SCF dimers.
  • mutation DupA502,Y503 stabilizes a wild-type-like conformation of D5:D5', which is compatible with D4:D4' contact formation and SCF binding. Indeed, introduction of mutation R381A into a background of KIT(DupA502.Y503) abolishes both the elevated basal activity and ligandstimulation of this oncogenic mutant.
  • the oncogenic mutation T417I,A418-419 located in D5 of KIT exhibits a constitutively active, ligand-independent tyrosine kinase activity.
  • the tyrosine kinase activity of this mutant is independent of the integrity of the salt bridge mediating D4:D4' contacts.
  • the binding affinity of isolated D4D5 fragments of KIT(T417I,A418-419) dimerization is 200-500 fold higher than the binding affinity 7 of D4D5 fragments of wild-ty pe KIT dimerization.
  • KIT(T417I,A418-419) molecules are localized intracellularly, and only a small population of this oncogenic mutant is located in the cell membrane.
  • the cryo-EM dataset of full-length KIT(T417I,A418-419) dimers reveals a V- shaped conformation of the extracellular region. Domains D1-D4 and DT-D4' are far apart from each other, whereas D5:D5' are tightly complexed (Figs. 5A-C). Ligand-independent dimerization of KIT(T417I,A418-419) mutant is entirely mediated by D5:D5 contacts.
  • the T417I, ⁇ 418-419 mutation of KIT triggers formation of a compact, strongly tilted interface of D5:D5' causing the V-shaped conformation of KIT(T417I,A418-419) dimers (Figs. 14D- 14F).
  • the tilted D5:D5' interface is formed by PG:(3A' and PG':PA, whereas the D5:D5' interface of wild-type KIT:SCF is only formed by PG:PG' (Figs. 14C and 14F).
  • the buried surface area of the D5:D5' interface increases from 292 A 2 to 1001 A 2 , and thus strongly increases the affinity of homotypic dimerization of KIT(T417I,A418-419) compared to wild-ty pe KIT.
  • SCF binding fully restores the hallmarks of the conformation of SCF-bound wild-type KIT including restoration of SCF interactions with the D1-D3 regions, restoration of the salt bridges responsible for mediating D4:D4' contacts and, importantly, restoration of the wild-type conformation of the D5:D5' interface (Fig. 7).
  • This is quite remarkable because of the large conformational change associated with a major tilt in D5:D5' contacts that increases the distance between C-terminal amino acids of the two D5 protomers of the oncogenic KIT mutant from 4.5 A to 15.0 A.
  • the D5:D5' contacts in SCF-bound KIT(T417I.A418-419) dimers are very similar, and likewise asymmetric as those seen in SCF-occupied wild-type KIT dimers.
  • Oncogenic KIT mutants that act inside the cell can be flushed out to the cell membrane by interfering with their ty rosine kinase activities using ty rosine kinase inhibitors such as imatinib and sunitinib. It is surmised that once relocated to the cell membrane (after imatinib or sunitinib treatment), an oncogenic mutant such as KIT(T417I,A418-419) that is treated with SCF could induce restoration of a wild-type-like KIT conformation to tame the harmful excessive activity of the oncogenic mutant.
  • a combination therapy involving a tyrosine kinase inhibitor together with ligand treatment can take advantage of the structural plasticity' of certain oncogenic mutations for therapeutic purposes.
  • SCF was expressed in E.coli BL21-CodonPlus (DE3)-RIPL, refolded from inclusion bodies, and purified.
  • K623A kinase-inactive KIT
  • Pl 0721-2 residues 32-976
  • a N-terminal FLAG-tag and a C- terminal 6xHis-tag preceded by a two-residues linker were subcloned into pFastBac 1 vector (Gibco).
  • ExpiSf9 cells were cultured in ExpiS® CD medium to a density of 5xl0 6 cells/mL, and infected with PO baculovirus stock. Infected cells were incubated for 48 h at 27 °C. Cells were harvested by centrifugation (4000xg, 10 min), resuspended in lysis buffer (10% glycerol, 20 mM HEPES pH 7.4.
  • KIT:SCF complexes For the formation of KIT:SCF complexes, SCF was added at a concentration of 2 pM. After incubation over night while rotating head-over-head at 4°C, 1% DDM (w/w) was added, followed by a further 1 h of incubation. The sample was ultracentrifuged (Beckman 45Ti, 40.000 rpm. 1 h), and the supernatant was applied to an anti-FLAG M2 affinity’ gel (Sigma). The resin was washed with resuspension buffer additionally containing 0.04% DDM, and the sample was eluted using 3xFLAG peptide (50 pM).
  • Protein-containing fractions were combined, and amphipol A8-35 (Anatrace) was added at a ratio of 1:4 (w/w), and the sample was incubated for 30 min while rocking at 4°C. Subsequently, Biobeads SM-2 resin (Bio-Rad) was added at a ratio of 1 :5 (w/v), and the sample w as incubated for 2 h while rocking at 4°C.
  • the sample was subjected to glycerol gradient ultracentrifugation in the presence of a chemical fixation reagent (5-20% glycerol, 0-0.2% glutaraldehyde, 20 mM HEPES pH 7.4, 200 mM NaCl). Gradients were prepared using a Gradient Master 108 (BioComp Instruments). Samples were ultracentrifuged using a Beckman SW41Ti rotor at 40,000 rpm for 18 h at 4°C. Samples w ere fractionated into 500 pL fractions from top to bottom, and glutaraldehyde was quenched by adding 100 mM Tris pH 7.4.
  • a chemical fixation reagent 5-20% glycerol, 0-0.2% glutaraldehyde, 20 mM HEPES pH 7.4, 200 mM NaCl.
  • Gradients were prepared using a Gradient Master 108 (BioComp Instruments). Samples were ultracentrifuged using a Beck
  • Negative staining EM sample preparation Prior to using a sample for cryo-EM. the quality of the sample was confirmed by negative staining EM. Protein samples (4 pL, 0.05 mg/mL) were applied on the carbon side of glow-discharged carbon-coated holey copper grids (EMS CF300-CU). After 1 min, excess protein solution was wicked away using filter paper. Particles were stained by floating the grid on a 50 pL drop of freshly prepared 1.5% (w/v) uranyl formate solution for 1 min, followed by wicking away excess liquid with filter paper. The procedure of staining and wicking away excess liquid was repeated once, followed by air-drying of the grid. Grids were screened on a FEI Tecnai T12 TEM operated at 120 kV, equipped with a Gatan UltraScan 4000 (4kx4k) CCD camera. 2D classification was performed using RELION 3.1.
  • Samples were used at concentrations of 5.5-6.0 mg/mL in sample buffer, to which fluorinated FC-8 w as added at a final concentration of 0.1% (v/v). All samples w ere prepared with glow-discharged holey carbon grids (C-flat 1.2/1.3, 300 mesh, gold support). Samples were vitrified using a Gatan CP3 (no wait time, room temperature, approximately 90% relative humidity, 3-4 s blotting time, blot force 0). and plunge-frozen in liquid ethane cooled by liquid nitrogen. Grids were screened on a Glacios Cryo-TEM (Thermo Fisher Scientific) operated at 200 keV, equipped w ith a K2 summit direct detection camera.
  • Glacios Cryo-TEM Thermo Fisher Scientific
  • KIT(T417I,A418-419):SCF was collected on a FEI Titan Krios G3i 300 keV TEM, equipped with a BioQuantum energy filter and a Gatan K3 direct electron detector, at the LBMS located at the Brookhaven National Laboratory. EPU (Thermo Fisher Scientific) was used for automatic data acquisition. All datasets were collected in super-resolution counting mode, operated in correlated-double sampling (CDS) imaging mode. Data collection parameters are detailed in Fig. 16.
  • Raw movies were corrected for beam-induced motion using patch motion correction (2 binmng).
  • CTF parameters were estimated using patch CTF estimation (default settings).
  • Micrographs of poor quality e.g., with large ice contaminants or cracked vitreous ice, were manually removed.
  • Initial particles were picked from a small fraction of micrographs using reference-free blob picking, and subjected to 2D classification.
  • Particle picks were extracted, and initially cleaned from incorrect picks (e.g., contaminants) by multiple rounds of 2D classification (default settings, 50-200 classes).
  • Particles were further cleaned by performing tw o iterative rounds of a six-classes ab-initio reconstruction followed by heterogeneous refinement, using the six ab-initio reconstructions as input models, followed by removal of 'junk' 3D classes. Subsequently, particles from clearly defined 3D classes were combined and used for the generation of a one-class ab-initio reconstruction, followed by re-centering of the particle by re-extraction using aligned shifts, CTF refinement, and non-uniform (NU) refinement.
  • NU non-uniform
  • NU refinement of all selected particles together resulted in a map with better resolution and more pronounced features compared to 3D classification using cryoSPARC or RELION and subsequent refinement of the individual classes or combinations thereof.
  • the map was postprocessed using deepEMhancer 0. 13 using the default tightTarget model.
  • the continuous flexibility of wild-type KIT:SCF (Fig. 8G) was analyzed using cryoSPARC's 3D variability 7 analysis (3DVA) algorithm (Punjani and Fleet, 2021).
  • Model quality was validated using MolProbity.
  • the models of KIT(DupA502,Y503):SCF and KIT(T417I,A418- 419):SCF were built using the final cryo-EM model of wild-type KITSCF as starting model. Model building and refinement was performed similarly as described for wild-type KITSCF. Adjustments of the models were performed using the sharpened and unsharpened maps. Loops of D5 and D5' proximal to the membrane, and [3-strands [3D and [3D' were omitted from both models. Due to the low resolution of D5:D5' in KIT(DupA502,Y503):SCF (Fig.
  • KIT DNA inserts containing the desired mutations were generated by PCR amplification using the Phusion High-Fidelity DNA Polymerase (New England Biolabs). The inserts were subcloned into either pFastBac 1 vector for structural studies or pBABE-puro vector for cell-based studies.
  • a cDNA encoding for full-length human PDGFR/> (NP_002600.1) was amplified by PCR and subcloned into lentiviral transfer plasmid pLenti CMV Hygro DEST. Using Agel and Mfel restriction sites.
  • PDGFR/> DNA inserts containing the desired mutations were generated by PCR amplification using the Phusion High-Fidelity 7 DNA Polymerase (New England Biolabs) and subcloned into pLenti CMV Hygro DEST for cellular studies. Recombinant plasmids were confirmed by restriction enzyme digestion and by 7 DNA sequencing (Keck DNA Sequencing Facility at Yale). Primers used for the generation of the inserts are listed in the Table below:
  • the retroviral pBABE-puro vector was used to generate NIH 3T3 cells stably expressing wild-type and mutants of KIT (1-972). MEFs deficient in endogenous PDGFRa and PDGFR/i were used to stably express wild-type and mutants of PDGFR/I (1-1106) with a C-terminal HA-tag. The generation of lentivirus for expressing the various PDGFR/> constructs was generated.
  • NIH 3T3 cells stably expressing wild-type and KIT mutants were culture at 37°C, 5% CO2 in DMEM (Gibco) supplemented with 5% FBS (Gibco), 5% BS (Gibco), 1% penicillin-streptomycin (Gibco), and 0.05% puromycin (Gibco).
  • Cells that reached 90% confluency were stimulated with SCF at increasing concentrations for 10 min at 37°C (Fig. 3B).
  • MEFs stably expressing w ild-type and PDGFR/I mutants of were culture at 37°C.
  • PDGFR/> was immunoprecipitated from the supernatant with 25 pL of monoclonal anti-HA antibody conjugated to Sepharose beads (Cell Signaling, no. C29F4). KIT and PDGFR/> immunocomplexes were washed three times with washing buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCh. 1 mM EGTA, 10% glycerol. 25 mM NaF. 1 mM NaiVO-i. 0.1% Triton-X 100, cOmplete protease inhibitor mixture (Roche)), and resuspended in 80 pL of reducing Laemmli buffer. Samples were separated on a 4-15% gradient SDS-PAGE gel, transferred to nitrocellulose membrane (Thermo Fisher Scientific), and immunoblot
  • anti-phospho KIT (Y703) antibody Cell Signaling Technology, no. D12E12
  • anli-PDGFR/> antibody Cell Signaling Technology 7 , no.3169S
  • anti-phosphoty rosine (pTyr) antibody Upstate Biotechnology, no. 4G10
  • All primary antibodies were used at a dilution of 1: 1000.
  • HRP-linked anti-rabbit IgG Cell Signaling Technology, no. 7074S
  • HRP-linked antimouse IgG Cell Signaling Technology, no. 7076S
  • the present invention is directed to the following non-limiting embodiments:
  • Embodiment 1 A method of inhibiting a type-III receptor tyrosine kinase (RTK). the method comprising contacting the ty pe- 111 RTK with a compound that at least partially' inhibits dimerization of the ty pe-III RTK.
  • RTK type-III receptor tyrosine kinase
  • Embodiment 2 The method of Embodiment 1, wherein the compound binds to the [3G strand of the D5 domain of the type-III RTK.
  • Embodiment 3 The method of any one of Embodiments 1-2, wherein the compound binds to and at least partially blocks the (3G strand of the D5 domain of the type-III RKT, and wherein the compound is not able to cause the trans-phosphorylation of the type-III RKT.
  • Embodiment 4 The method of any one of Embodiments 1-3, wherein at least one of the following applies:
  • the type-III RTK is proto-oncogene c-KIT (KIT) or a mutant thereof, and the compound binds to at least one KIT amino acid residue corresponding to A502, Y503.
  • the type-III RTK is platelet-derived growth factor receptor a (PDGFRa) or a mutant thereof, and the compound binds to at least one PDGFRa amino acid residue corresponding to R512, E513, L514. K515, L516. V517 or A518 of the polypeptide as set forth in SEQ ID NO:2;
  • the type-III RTK is platelet-derived grow th factor receptor (3 (PDGFR(3) or a mutant thereof, and the compound binds to at least one PDGFR (3 amino acid residue corresponding to Q519, E520, V521, 1522, V523, V524 or P525 of the polypeptide as set forth in SEQ ID NO:3;
  • the type-III RTK is colony-stimulating factor-1 receptor (CSF-1R) or a mutant thereof, and the compound binds to at least one CSF-1R amino acid residue corresponding to W496, A497, F498, 1499, P500, 1501 or S502 of the polypeptide as set forth in SEQ ID NO:4;
  • CSF-1R colony-stimulating factor-1 receptor
  • the type-III RTK is fms-like tyrosine kinase 3 (FLT3) or a mutant thereof, and the compound binds to at least one FLT3 amino acid residue corresponding to E525, T526, 1527, L528, L529, N530 or S531 of the polypeptide as set forth in SEQ ID NO:5.
  • FLT3 fms-like tyrosine kinase 3
  • Embodiment 5 The method of any one of Embodiments 1-4, wherein at least one of the following applies:
  • the compound is a polypeptide comprising the D5 domain of the type-III RTK or a nucleic acid encoding the polypeptide, and wherein the polypeptide does not have tyrosine kinase activity;
  • the compound is a polypeptide compnsing the D4-D5 section of the type-III RTK or a nucleic acid encoding the polypeptide.
  • Embodiment 6 The method of Embodiment 5, wherein the type-III RTK is KIT, and wherein the polypeptide comprises residues 408-508 (D5 domain) or residues 308-508 (D4-D5 segment) of SEQ ID NO: 1.
  • Embodiment 7 The method of Embodiment 6, wherein at least one of the following applies:
  • the KIT is a KIT with duplication of A502 and Y503 (KIT DupA502,Y503), and the polypeptide comprises duplication of A502 and Y503 in the sequence set forth in SEQ ID NOT;
  • the KIT is a KIT with T417I mutation and deletion of Y418 and D417 (KIT T417I, A418.419). and the polypeptide comprises T417I mutation and deletion of Y418 and D417 in the sequence set forth in SEQ ID NO: 1.
  • Embodiment 8 The method of any one of Embodiments 1-4, wherein the compound comprises an antibody that binds to the
  • Embodiment 9 The method of Embodiment 8, wherein the antibody is a divalent antibody, and wherein the antibody further binds to the residues in the D4 domain of the ty pe-III RTK responsible for forming the salt bridge.
  • Embodiment 10 The method of any one of Embodiments 8-9, wherein the type-III RTK is KIT. and wherein the antibody binds to at least one KIT residue corresponding to A502, Y503, F504, N 05, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO: I.
  • Embodiment 11 The method of any one of Embodiments 8-10, wherein the type-III RTK is KIT, wherein the antibody is divalent, and wherein the antibody further binds to at least one KIT residue corresponding to E386 or R381 of the polypeptide as set forth in SEQ ID NOT.
  • Embodiment 12 The method of any one of Embodiments 1-11, wherein the type-III RTK is an isolated protein, or a protein in or on the surface of a cell.
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the type-III RTK is in a subject, optionally a mammal such as a human.
  • Embodiment 14 The method of any one of Embodiments 1-13, wherein the ty pe-III RTK is an overactivated mutant type-III RTK.
  • Embodiment 15 A method of treating, ameliorating, and/or preventing a disease or disorder caused by or involving an overactivation and/or an overexpression of a type-III receptor tyrosine kinase in a subject in need thereof, the method comprising: administering to the subject an effective amount of a compound inhibiting the dimerization of the type-III RTK.
  • Embodiment 16 The method of Embodiment 15, wherein the compound binds to the [3G strand of the D5 domain of the ty pe-III RTK.
  • Embodiment 17 The method of any one of Embodiments 15-16, wherein the compound binds to and at least partially blocks the G strand of the D5 domain of the type- ill RKT, and wherein the compound is not able to cause the trans-phosphoiylation of the ty pe-III RKT.
  • Embodiment 18 The method of any one of Embodiments 15-17, wherein at least one of the following applies:
  • the type-III RTK is proto-oncogene c-KIT (KIT) or a mutant thereof, and the compound binds to at least one KIT amino acid residue corresponding to A502, Y503, F504, N505, F506, A507 or F508 of the polypeptide as set forth in SEQ ID NO: 1;
  • the type-III RTK is platelet-derived growth factor receptor a (PDGFRa) or a mutant thereof, and the compound binds to at least one PDGFRa amino acid residue corresponding to R512, E513, L514. K515, L516, V517 or A518 of the polypeptide as set forth in SEQ ID NO:2;
  • the type-III RTK is platelet-derived growth factor receptor P (PDGFRP) or a mutant thereof, and the compound binds to at least one PDGFR p amino acid residue corresponding to Q519, E520, V521, 1522, V523, V524 or P525 of the polypeptide as set forth in SEQ ID NO:3;
  • PDGFRP platelet-derived growth factor receptor P
  • the type-III RTK is colony-stimulating factor-1 receptor (CSF-1R) or a mutant thereof, and the compound binds to at least one CSF-1R amino acid residue corresponding to W496, A497, F498, 1499, P500, 1501 or S502 of the polypeptide as set forth in SEQ ID NO:4;
  • CSF-1R colony-stimulating factor-1 receptor
  • the type-III RTK is fins-like tyrosine kinase 3 (FLT3) or a mutant thereof, and the compound binds to at least one FLT3 amino acid residue corresponding to E525, T526, 1527, L528, L529, N530 or S531 of the polypeptide as set forth in SEQ ID NO:5.
  • Embodiment 19 The method of any one of Embodiments 15-18, wherein at least one of the following applies:
  • the compound is a polypeptide comprising the D5 domain of the type-III RTK or a nucleic acid encoding the polypeptide, and wherein the polypeptide does not have tyrosine kinase activity;
  • Embodiment 20 The method of Embodiment 19, wherein the type-III RTK is KIT, and wherein the polypeptide comprises residues 408-508 (D5 domain) or residues 308-508 (D4-D5 segment) of SEQ ID NO: 1.
  • Embodiment 21 The method of Embodiment 20, wherein at least one of the following applies:
  • the KIT is a KIT with duplication of A502 and Y503 (KIT DupA502,Y503), and the polypeptide comprises duplication of A502 and Y503 in the sequence set forth in SEQ ID NOT;
  • the KIT is a KIT with T417I mutation and deletion of Y418 and D417 (KIT T417I, A418,419), and the polypeptide comprises T417I mutation and deletion of Y418 and D417 in the sequence set forth in SEQ ID NO: 1.
  • Embodiment 22 The method of any one of Embodiments 15-19, wherein the compound comprises an antibody that binds to the
  • Embodiment 23 The method of Embodiment 22, wherein the antibody is a divalent antibody, and wherein the antibody further binds to one or more residues in the D4 domain of the type-III RTK responsible for forming the salt bridge.
  • Embodiment 24 The method of any one of Embodiments 22-23, wherein the type- III RTK is KIT, and wherein the antibody binds to at least one KIT residue corresponding to A502, Y503. F504, N505, F506. A507 or F508 of the polypeptide as set forth in SEQ ID NOT .
  • Embodiment 25 The method of any one of Embodiments 22-24, wherein the type- III RTK is KIT, wherein the antibody is divalent, and wherein the antibody further binds to at least one KIT residue corresponding to E386 or R381 of the polypeptide as set forth in SEQ ID NOT.
  • Embodiment 26 The method of any one of Embodiments 15-25, wherein at least one of the following applies:
  • the type-III RTK is KIT
  • the disease or disorder comprises a KIT-driven cancer, optionally a KIT-driven gastrointestinal stromal tumor (KIT-driven GIST), a KIT- driven core binding factor acute myeloid leukemia; or a mast cell diseases, optionally a systemic mastocytosis;
  • the type-III RTK is PDGFRa
  • the disease or disorder comprises a PDGFRa- driven cancer, optionally a PDGFRa-associated chronic eosinophilic leukemia or a PDGFRa- driven gastrointestinal stromal tumor (PDGFRa-driven GIST); or inflammatory' fibroid polyps;
  • the type-III RTK is PDGFR
  • the disease or disorder comprises a PDGFR0- driven cancer, optionally a PDGFRP-associated chronic eosinophilic leukemia; a primary familial brain calcification; an infantile myofibromatosis; a Kosaki overgrowth syndrome; or a premature aging syndrome, Penttinen type;
  • the type-III RTK is CSF1R
  • the disease or disorder comprises a CSFIR-driven cancer, optionally a CSFIR-driven myeloid malignancy, a CSFIR-driven Hodgkin's lymphoma or a CSFIR-driven anaplastic large cell lymphoma;
  • the type-III RTK is FLT3, and the disease or disorder comprises a FLT3-driven cancer, optionally a FLT3-driven core binding factor acute myeloid leukemia or a FLT3- driven cytogenetically normal acute myeloid leukemia.
  • Embodiment 27 The method of any one of Embodiments 15-26, wherein the disease or disorder is caused by or involves KIT overactivation, and the method further includes administering to the subject an effective amount of a stem cell factor (SCF) protein or a nucleic acid encoding the SCF.
  • SCF stem cell factor
  • Embodiment 28 The method of any one of Embodiments 15-27, wherein the subject is a mammal, optionally a human.

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Abstract

L'invention concerne une méthode d'inhibition du récepteur de type III de tyrosines kinases (RTK), telle que KIT, PDGFRα, PDGFRβ, CSF1R et/ou FLT3. La méthode comprend la mise en contact du RTK de type III avec un composé inhibant la dimérisation du RTK de type III. L'invention concerne également une méthode de traitement, d'atténuation et/ou de prévention d'une maladie ou d'un trouble provoqué par ou impliqué dans la suractivation et/ou la surexpression d'un RTK de type III chez un sujet en ayant besoin. La méthode comprend l'administration au sujet d'une quantité efficace d'un composé inhibant la dimérisation du RTK de type III.
PCT/US2024/011805 2023-01-17 2024-01-17 Composés et méthodes d'inhibition du récepteur de type iii de tyrosines kinases Ceased WO2024155689A1 (fr)

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Citations (2)

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US20110311538A1 (en) * 2007-06-05 2011-12-22 Yale University Inhibitors of receptor tyrosine kinases and methods of use thereof
US20130071397A1 (en) * 2009-12-29 2013-03-21 Yale University Inhibitors of vascular endothelial growth factor (vegf) receptors and methods of use thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110311538A1 (en) * 2007-06-05 2011-12-22 Yale University Inhibitors of receptor tyrosine kinases and methods of use thereof
US20130071397A1 (en) * 2009-12-29 2013-03-21 Yale University Inhibitors of vascular endothelial growth factor (vegf) receptors and methods of use thereof

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