The following proposal is in response to the program announcement PA-10-213 titled "Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery (R01)." Integrin cell adhesion receptors play essential roles in normal physiologic process and have been implicated in neoplasia, autoimmunity, inflammation, and cardiovascular disease. Indeed, they are validated drug targets. Present integrin-based therapeutics has proven invaluable in the treatment of patients but is limited by side-effects related to immunosuppression and the induction of autoimmune reactions. There is need for a new strategy to target integrins that would eliminate side-effects. Integrins bind a variety of extracellular ligands such as serum fibrinogen, extracellular matrix (ECM) components, and cell-surface counter receptors. Their activity is tightly regulated by intracellula scaffolding molecules such as Talin-1 and Kindlins that can bind to integrin cytoplasmic domains and in a process termed "inside-out" signaling increase integrin ectodomain affinity for ligand. Integrins also serve as signaling receptors, where "outside-in" signaling is mediated by direct interactions between integrin cytoplasmic domains and intracellular effectors such as the Syk family of nonreceptor tyrosine kinases. We have developed a cell-free assay platform for high throughput screening (HTS) of small molecule compound libraries for antagonists of the interactions between integrin cytoplasmic domains and intracellular effector molecules. This system utilizes the amplified luminescent proximity homogenous assay (ALPHA) screen format. In our first aim, we propose to develop a suite of primary HTS assays targeting the binding of integrin beta3 cytoplasmic domains with Syk, Kindlin-3, and Galpha13. A secondary screen to triage false positive hits will also be developed. In a second aim, we propose a number of orthogonal assays ranging from Elisa-based approaches to isothermal titration calorimetry and surface plasmon resonance, which will serve to validate hit specificity and selectivity. Finally, a third aim is proposed to test antagonist mechanism of action in primary cells, examining integrin activation of Syk, integrin affinity regulation, and platelet function. All primary ALPHA screens will be optimized according to HTS metrics as guided by the NIH Chemical Genomics Center. Pilot screens of a small molecule compound library will be performed to demonstrate primary and secondary screen tractability. Small molecule antagonist identified here may represent excellent starting points for the development of novel classes of anti-platelet and anti-inflammatory drug candidates, which could target integrin function at the intracellular level. Furthermore, small molecule compounds derived from the HTS assays proposed here could be important novel tools in the analysis of both "inside-out" and "outside-in" integrin signal transduction. Public Health Relevance: The screening platforms developed in this proposal will lead to discovery of novel small molecule antagonists that could represent a new direction in the development of adhesion molecule therapeutics for cardiovascular diseases and others. Also, small molecule antagonists identified will be important tools for basic research into the molecular mechanisms regulating cell adhesion molecule function both in vitro and in vivo.
The screening platforms developed in this proposal will lead to discovery of novel small molecule antagonists that could represent a new direction in the development of adhesion molecule therapeutics for cardiovascular diseases and others. Also, small molecule antagonists identified will be important tools for basic research into the molecular mechanisms regulating cell adhesion molecule function both in vitro and in vivo.
|Bakthavatsalam, Deenadayalan; Soung, Roh Hun; Tweardy, David J et al. (2014) Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor. FEBS Lett 588:2133-40|