Abstract 2842: Development of a novel TrkA HTS assay using template directed self assembly (TDA)

Abstract

Abstract Tropomyosin-related kinases (Trks A, B, and C) are a family of membrane-associated receptor tyrosine kinases (RTKs) activated by neurotrophins. RTKs are shown to be implicated in Alzheimer's disease, depression, pain sensation and various cancers including tumor cell growth and survival signaling. Inhibitors of Trk receptor kinases might provide targeted treatments for neurodegenerative diseases, pain and cancer. One strategy to indentify Trk isoform-specific inhibitors is to develop an assay where compound libraries can be screened in a more-native membrane associated lipid environment. Template-Directed Self Assembly (TDA) is a biomimetic technology designed to restore membrane-associated proteins to more relevant physiological conditions. This technology utilizes recombinant histidine-tagged proteins bound to the surface of nickel-chelated liposomes. Recombinant HIS-tagged proteins are engineered on the correct terminus of the protein to reflect the polarity of the enzyme with respect to the membrane. This allows the HIS-tagged proteins to bind to the liposome creating an environment much like a cellular membrane. The lipids in TDA are fully fluid within the surface of the liposome and associated proteins can rotate freely. Furthermore it promotes the formation of high-order structures such as homo- or hetro-dimers and shown to recruit accessory factors. Coupling of membrane associsted kinases to liposomes reconstitutes an environment that confers proper polarity, stereochemistry, topology and other critical properties, thereby creating a more relevant biochemical assay. To date, we have shown that several RTKs have enhanced biochemical specific activity and substrate specificity that resembles physiological conditions when coupled to TDA. The TDA approach restores TrkA activity to a more-native environment; therefore, we hypothesize that we will be able to identify compounds that are more specific and selective. To address the need of an enzyme assay near a lipid membrane environment, we successfully developed a robust TrkA assay shown to exhibit up to 20-fold increase in specific activity and a reduction in Km for ATP relative to TrkA in a conventional assay. Furthermore, TDA-associated TrkA exhibited a rapid and robust trans-activation, obviating the need for a pre-activation step. Thus, assays of TrkA coupled to TDA have the potential to identify more chemically diverse and specific inhibitors. We are currently (1) validating the TDA assay with known inhibitors (2) screening a focus library to identify novel hits (3) testing the primary hits against a small kinase selectivity panel and (4) designing and developing a TrkA specific reporter cell line to confirm the primary hits. The advantages of the TDA technology will be also discussed against other therapeutically relevant RTKs. 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 2842. doi:1538-7445.AM2012-2842