a. HER-kinase expression inhibitors: Identification of compounds that promote Her2 degradation has required cumbersome in vitro analyses involving tissue culture with individual drugs followed by detergent lysis of samples, polyacrylamide gel electrophoresis of cellular proteins, and Western blotting to determine Her2 levels, the methodology being decidedly unsuitable for rapid, high-throughput screening of compound libraries. We developed a microtiter method, which relies on whole-cell immunodetection of the proteins in question, utilizes a minimal number of cells yet is sufficiently sensitive and reproducible to permit quantitative determinations (published by our group in Chemistry&Biology 2003; represented on the issue cover). We intend to use this assay for a blinded HTS to identify agents that down-regulate kinase expression via action on transcription or translation and/or effect on RNA or protein half-life.
b. Hsp90 binding assay
: Measurements of ligand binding to Hsp90 have required the use of laborious methods and of large quantities of recombinant protein. We have developed a homogenous and quick fluorescence polarization Hsp90 assay (published in Journal of Biomolecular Screening; cover of August 2004 issue). The advantage of our assay is that it measures real-time interactions of inhibitors with cellular Hsp90 found in cell-specific multi-chaperone complexes. In this method the protein and ligand are added together in solution and a response is directly read, a great advantage over previous methods that require immobilization of reagents or radioactive ligands. The assay is currently used by both the HTS facility of MSKCC and the NIH to perform a screen to identify novel Hsp90 inhibitors.c,d,e. Microtiter Hsp70 binding assay; microtiter assay to determine agents with selective anti-mitotic activity in Rb-defective cells; novel microtiter apoptosis read-out assay; microtiter assay to identify/study Hsp70 chaperone expression modulators
f. Reagents for dissecting/understanding the function of proteins involved in oncogenesis: fluorescently labeled Hsp90 inhibitors; biotinylated reagents, tissue-specific targeted Hsp90 inhibitors, selective Hsp90 inhibitors to be used as dissectors of biological processes regulated by specific Hsp90-client proteins, DNA-PK inhibitors, disruptors of MDM2/p53 interaction.