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Inhibitors of receptor tyrosine kinases and methods of use thereof

A technology of tyrosine kinase and tyrosine kinase cell, applied in receptor tyrosine kinase inhibitor and its application field, can solve the problem of incomprehensible capacitative mutation and the like

Active Publication Date: 2010-08-11
YALE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite good evidence that capacitation mutations in the JM and PTK domains lead to constitutive activation of Kit by releasing autoinhibitory constraints (Mol et al. (2004) J Biol Chem. 279:31655-31663), there is still Molecular mechanisms underlying capacitation mutations in D5 of the ectodomain are not understood

Method used

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  • Inhibitors of receptor tyrosine kinases and methods of use thereof
  • Inhibitors of receptor tyrosine kinases and methods of use thereof
  • Inhibitors of receptor tyrosine kinases and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 22-25

[0295] Moieties of the invention can be screened for RTK inhibitory activity using any of the assays described herein and those well known in the art. For example, assays that measure receptor internalization, receptor autophosphorylation, and / or kinase signaling can be used to identify components that prevent activation of a target RTK (eg, a Kit receptor). can be obtained by using standard methods known in the art, for example by using phosphoELISA TM method (available at Invitrogen) to determine the phosphorylation status of RTKs or downstream molecules, enabling the screening of new inhibitor components. The phosphorylation state of the receptor (such as the Kit receptor) can be obtained using a commercially available kit, such as C-Kit[pY823]ELISA KIT, HU (BioSource TM ; CatalogNumber-KHO0401); c-KIT[TOTAL]ELISA KIT, HU (BioSource TM ; Catalog Number-KHO0391), to be determined. Antibodies, small molecules and other components of the invention can be screened using such...

Embodiment 1

[0359] Example 1: Expression, purification and crystallization of SCF and Kit

[0360] The complete ectodomain of Kit consisting of five Ig-like domains called D1, D2, D3, D4 and D5 was expressed in insect cells using the baculovirus expression system. Purified Kit ectodomain monomers or SCF-induced Kit ectodomain homodimers (SCF-Kit2:2 complexes) were each subjected to large-scale screening for crystal growth and optimization, followed by determination of their crystal structures.

[0361] Protein Expression and Purification

[0362] The soluble Kit ectodomain (amino acids 1-519) containing a polyhistidine tag at the C-terminus was expressed in insect cells (Sf9) using the baculovirus expression system. The Kit ectodomain was purified by Ni chelation followed by size exclusion chromatography (Superdex 200, GE Healthcare). After partial deglycosylation using endoglycosidase F1, the extracellular domain was further purified by anion exchange chromatography (MonoQ, GE Health...

Embodiment 2

[0368] Example 2: Structure Determination

[0369] By using multiple isomorphic substitution with anomalous scattering (MIRAS) and by multiwavelength anomalous scattering (MAD) at 3.0 The resolution was calculated for the experimental phase (Table 1A). The resulting electron density map shows a continuous electron density of the β-sandwich structure and a clear solvent-protein boundary. The molecular model of the monomeric Kit ectodomain was artificially constructed into the experimental electron density map. The structure was optimized to 3.0 using a raw data set of 25.4% crystalline R-factor and 29.6% free R-factor resolution (Table 1B). SCF-Kit was resolved by molecular replacement using the monomeric form and SCF structure described in this report (Zhang et al. (2000) Proc Natl Acad Sci US A 97:7732-7737; available code: 1EXZ obtained from the Protein Data Bank) as search models Structure of the 2:2 complex. The structure was optimized to 3.5 using the original data...

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Abstract

The present invention provides moieties that bind to an Ig-like domain, e.g., D4 or D5, of a human receptor tyrosine kinase, e.g., the human Kit RTK or the PDGFR RTK, or the D7 domain of a type V receptor tyrosine kinase wherein the moieties lock the ectodomain of the receptor tyrosine kinase in an inactive state thereby antagonizing the activity of the receptor tyrosine kinase.

Description

[0001] Cross References to Related Applications [0002] This application is related to, and claims priority to, U.S. Provisional Application Serial No. 61 / 070,506, filed March 24, 2008, U.S. Provisional Application Serial No. 61 / 009,482, filed December 28, 2007, and U.S. Provisional Application Serial No. 61 / 009,482, filed December 28, 2007, and U.S. Provisional Application Serial No. 60 / 933,238 filed on . The entire content of each of the aforementioned applications is incorporated into this application by such reference. [0003] Statement Regarding Federally Sponsored Research or Development [0004] This invention was made with Government support under Contracts R01-AR 051448, R01-AR 051886, and P50 AR054086 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] Stem cell factor (SCF) is a cytokine that mediates its diverse cellular responses by binding to and activating the receptor tyrosine kin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395
CPCC07K2317/34C07K16/2803C07K2317/77C07K2316/96C07K2317/76A61P19/02A61P25/04A61P27/02A61P29/00A61P35/00A61P35/02A61P43/00A61P9/10
Inventor J·施莱辛格I·拉克斯汤泽悟Y·奥帕汤斯基杨艳
Owner YALE UNIV
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