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.
