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Compounds that inhibit hsp90 protein-protein interactions with iap proteins

A protein interaction and compound technology, applied in the direction of apoptosis-related proteins, mammalian proteins, peptide/protein components, etc., can solve the problem of undetectable

Inactive Publication Date: 2007-10-31
UNIV OF MASSACHUSETTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Survivin is a small molecule 16.5kDa mammalian member of the IAP family, which is widely expressed in embryonic and fetal organs but barely detectable in most terminally differentiated normal tissues

Method used

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  • Compounds that inhibit hsp90 protein-protein interactions with iap proteins
  • Compounds that inhibit hsp90 protein-protein interactions with iap proteins
  • Compounds that inhibit hsp90 protein-protein interactions with iap proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Example 1: Survivin interacts with Hsp90

[0205] Several experiments were performed to demonstrate the protein-protein interaction between survivin and Hsp90. Human cervical carcinoma HeLa cells and B lymphoma Raji cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and maintained in culture medium according to the supplier's instructions. For Western blotting, 12% sodium dodecyl sulfate (SDS) gels were transferred to nylon membranes and incubated with 1-5 μg of primary antibody. Rabbit polyclonal antibody against survivin was obtained from NOVUS Biologicals (Littleton, CO); monoclonal antibody (mAb) against Hsp90 was obtained from BD-Transduction Laboratories (catalogue # H38220; Lexington, KY); monoclonal anti-tubulin Antibodies were obtained from Sigma. Primary antibodies were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham, Piscataway, NJ) and a chemiluminescent kit (Amersham).

[0206] Figure...

Embodiment 2

[0209] Example 2: Proper folding of survivin is required for binding to Hsp90

[0210] Recombinant survivin (r-survivin) (10 mg / ml) or recombinant Hsp90 (r-Hsp90) (10 μg / ml) was mixed with bicarbonate buffer pH 9.5 (100 μl / well) in wells of a plastic microtiter plate (Immulon TM -2, Dynatech Laboratories, Chantilly, VA) at 4° C. for 18 hours for ELISA experiments. Bound proteins were blocked with 3% gelatin at 37°C for 1 hour, washed with washing buffer (TBS pH 7.4, 0.1% Tween TM , 0.1% BSA), and then incubated with different concentrations of the test protein at 37° C. for 1 hour to determine whether the test protein binds to the solid-phase protein.

[0211] GST-Hsp90 (amino acids 1-732), recombinant GST-survivin, GST-survivin (C84A) were expressed in E. coli and bound to glutathione beads (Sigma). Hsp90α was cloned into pGex-4T3 (Amersham Pharmacia Biotech.) by PCR. Purified r-survivin lacking the GST box was treated with thrombin (Sigma, 20 U / ml in 50 mM Tris TM pH 7....

Embodiment 3

[0215] Example 3: Survivin binds to the N-terminus of Hsp90

[0216] For full-length Hsp90α (SEQ ID NO: 21; amino acids 1-732) or three fragments: N-Hsp90 (amino acids 1-272 of SEQ ID NO: 21), M-Hsp90 (amino acids 273- 617), and C-Hsp90 (amino acids 629-732 of SEQ ID NO: 21), the nucleotide sequences encoding them were cloned into pGex-4T3 (Pharmacia Biotech.) and pFLAG-CMV 6c by PCR using the BamHI cloning site (Sigma). GST fusions of full-length Hsp90 or individual Hsp90 fragments were expressed in E. coli and bound to glutathione beads (Sigma). Purified recombinant survivin lacking the GST box was obtained as described in Example 2.

[0217] Figure 3A shows the results of the survivin / Hsp90 pull-down experiment in vitro. Increasing concentrations of r-survivin (30, 100 and 300ng / 50μl reaction) were mixed with Sepharose TM - GST-Hsp90 (10 μg / 50 μl reaction) or incubated with GST fused to N-Hsp90, M-Hsp90 or C-Hsp90 region. The reaction was centrifuged to make Sepharose ...

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Abstract

Disclosed herein are compounds that inhibit Hsp90 interactions with IAP proteins, such as Survivin, XIAP, cIAP1, or cIAP2, and methods for identifying and using such compounds.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application 60 / 590,584, filed July 23,2004. The content of this prior application is incorporated herein by reference in its entirety. [0003] Statement of Federally Funded Research [0004] This invention was made with support from National Institutes of Health Grant Nos. 2R01CA078810, 5R01HL54131, and 5R01CA90917, and thus the Government has certain rights in this invention. technical field [0005] The present invention relates to compounds that inhibit the protein-protein interaction between heat shock protein Hsp90 and inhibitor of apoptosis (IAP) proteins, and methods of identifying and utilizing such compounds, such as peptides and peptide derivatives, said IAP proteins such as Survivin, XIAP, cIAP1 or cIAP2. Background technique [0006] Tumor cells have an enhanced ability to survive and proliferate in a highly hostile environment. For example, tumor c...

Claims

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Application Information

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IPC IPC(8): A61K38/04
CPCA61K38/00A61K39/39558G01N33/574G01N2510/00C07K14/4747A61P35/00A61P35/02A61P43/00
Inventor 达里奥·C·奥尔蒂里珍妮特·普莱西亚惠特妮·萨尔兹
Owner UNIV OF MASSACHUSETTS
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