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P2050
Template-Directed Self-Assembly Imparts Biological Relevance to Standard In vitro Assays
Presenter Anthony Shrout, P.A. Technologies, LLC, USA
Additional Authors: Edward A. Esposito, and Robert M. Weis (P. A. Technologies, LLC and the University of Massachusetts Amherst, Chemistry Department)
Many of the important drug discovery targets are RTKs or other membrane associated proteins involved in complex signaling pathways. Millions of small molecule candidates are screened against these targets each year via high-throughput screening (HTS) assays. Many of these assays use recombinant proteins which no longer function as they do in the cell. Template-Directed Assembly (TDA) technology employs the same cloned catalytic domain fragments used in today’s assays, but places them in a membrane environment that promotes organization, allowing native protein activity and function. Template-assembled catalytic domains of the IGF-1R and Tie2 exhibited substantially larger autophosphorylation levels compared to standard assays. IGF-1R also demonstrated significant increases in substrate phosphorylation, and did not require the addition of manganese when assembled. The increased level of autophosphorylation of Tie2 led to the inhibition of substrate phosphorylation which is consistent with published reports suggesting that autophosphorylation of the TK domain is autoinhibitory. These results and the underlying similarity in organization of transmembrane signaling systems suggest that template-directed assembly of signaling pathways will provide widespread benefits to basic research and drug discovery. We also demonstrate that TDA technology is compatible with standard HTS platforms, such as the Caliper LC-3000 instrument and the Molecular Devices IMAP detection reagents, both commonly used in the discovery process.