Discovery of Adenosine Receptor Allosteric Modulators for Cardiovascular Disease and Inflammation

项目总结

PROJECT SUMMARY Cardiovascular disease is the leading cause of death in the United States for both men and women, costing $351.2 billion dollars and accounting for over 840,000 deaths annually. The A3 adenosine receptor (A3AR) is a Gi protein-coupled receptor that is highly expressed in several types of inflammatory cells, including neutrophils. Increased adenosine levels following tissue injury/inflammation leads to activation of A3AR signaling, which aids to limit inflammation and to promote repair, in part by impairing immune cell chemotaxis and activation. Agonists of the A3AR are being investigated for a multitude of inflammatory diseases including cardiac ischemia and chemotherapy-induced cardiotoxicity. Unfortunately, dose-limiting side effects have been reported in early clinical trials potentially limiting their usefulness. Compounds that potentiate signaling of endogenous ligands, termed positive allosteric modulators (PAMs), allow for spatiotemporal specificity, and reduced off-target effects. It is anticipated that PAMs targeting the A3AR will offer a superior treatment approach. In these proposed studies, I aim to characterize and further develop A3AR PAMs that will serve as small molecule probes and useful therapeutics for cardiac injury and inflammatory disorders. Prior structure-activity-relationship studies (SAR) identified the 1H- imidazo[4,5]quinoline-4-amine, LUF6000, and the 2,4-disubstituted quinoline, LUF6096, as exhibiting PAM activity at the A3AR, where these modulators enhance orthosteric agonist efficacy upwards of 2-fold. Unfortunately, they also have the undesirable tendency to decrease agonist potency. In addition, none of the PAMs we have investigated thus far exhibit PAM activity versus rodent receptors, preventing us from assessing biological activity in preclinical rodent models of disease. In Aim 1, I will expand on prior SAR studies by characterizing two new series of derivatives, based off the structures of LUF6000 and LUF6096 with the goal of identifying improved A3AR PAMs that dually enhance orthosteric agonist efficacy and potency. As part of this aim, I will investigate whether our PAM ligands support biased (G protein-dependent vs G protein-independent) signaling and assess for activity versus the mouse A3AR. In Aim 2, I will exploit species differences and generate human/mouse chimeric and mutant A3ARs to facilitate identification of the binding pocket for LUF6000 and LUF6096. Lastly, in Aim 3 I will investigate the biological effects of LUF6000 and LUF6096 on two critical neutrophil functions - superoxide production and chemotaxis - utilizing a human neutrophilic cell line (HL60 cells). Upon completion of these proposed studies, under the guidance of my mentor, a diverse team of collaborators, and my dissertation committee, I will gain experience in experimental design, execution of biochemical assays, molecular cloning, and basics of rational drug design, which will result in publishable data and prepare me to successfully compete for a position as a research scientist at a biopharmaceutical company. Completion of this work will advance our understanding of A3AR allosteric pharmacology and potentially lead to the development of a new therapeutic for the treatment of ischemic heart disease and other inflammatory diseases.