Abstract 3881: BacMam-enabled biochemical and cellular assays to assess inhibitors of full-length receptor tyrosine kinases

Abstract

Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881