This competitive renewal proposes to continue our investigation of allosteric enhancers (AEs) of the A1 adenosine receptor (A^R). These compounds bind to an allosteric site on the receptor to increase the affinity of adenosine to the orthosteric site on the A^R. We have identified new and improved aminothiophene AEs and we have discovered a new chemical class of more potent and effective aminothiazole AEs. We also have discovered that some disulfides and H2O2 have AE-like effects on the AiAR and the A^R. To learn more about the molecular mechanisms of AE action we investigated receptor mutants and chimera. We have developed a novel system to """"""""score"""""""" enhancer activity based on the ability of these compounds to prevent rapid dissociation of an agonist radioligand, 125I-ABA, from the A^R in response to GTPyS. We propose newstudies to achieve three specific aims.
Aim 1 is to synthesize new enhancer structures in two chemical classes and to characterize them based on radioligand binding to receptors and guanine nucleotide exchange from G proteins.
This aim i s guided by conformational molecular field analysis (CoMFA) of existing structures and the use of new synthetic techniques.
Aim 2 is to efficiently evaluate enhancer function in intact cells and to evaluate selected compounds for activity in modulating adenosine A1 receptor function in brain slices and stimulation of angiogenesis.
Aim 3 will investigate the role and disulfide bond formation in AE and H2O2 action through analysis of thiol alkylating agents, epitope- tagged receptors, site-directed mutagenesis and protein sequencing. We will also investigate the possibility the H2O2 is a physiological regulator of A^R signaling. We hypothesize that cysteines in the extracellular loops of the AiAR participate in disulfide cross linking reactions. AEs are significant therapeutic candidates that may reduce chronic pain or promote site-specific angiogenesis. Moreover, our investigation of the role of thiols/disulfides in enhancer and H2O2 action may reveal important new information about general mechanisms of GPCR G protein coupling and trafficking.
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