Function- and interaction-based discovery of negative allosteric modulators of the A2A Receptor

Molecular Medicine Research Institute, Sunnyvale CA

Investigators

Abstract

Project Summary Immune checkpoints are critical for maintaining immune homeostasis. They prevent overactivation of the immune response. However, aberrant activation of immune checkpoints pathways in certain cancers reduces anti-tumor immunity allowing tumors to proliferate. Therapies targeting immune checkpoints CTLA4, PD-1 and PDL-1 have been successful in treating a wide variety of refractory cancers. However, their efficacy is limited to a small percentage of patients highlighting the need for targeting additional immune checkpoint pathways. In this regard, the blockade of adenosine-adenosine A2A receptor (A2AR) immune checkpoint activation with high-affinity antagonists has shown promise in preclinical studies. As antagonists block adenosine-A2AR immune checkpoint globally, adverse events are anticipated. In contrast, negative allosteric modulators (NAM) conceivably block the activation of adenosine-A2AR immune checkpoint only at tumor sites where adenosine levels are elevated. We envision that blocking the adenosine-A2AR immune checkpoint activation with NAMs is disease-site specific and thus a more directed approach to enhance anti-tumor immunity. We have demonstrated previously that adenosine-A2AR immune checkpoint is amenable to positive allosteric modulation through synthetic small molecules. However, negative allosteric modulation of the adenosine-A2AR immune checkpoint has not been reported. To test our notion, we have designed and synthesized a library of over 300 compounds potentially containing adensoine-A2AR NAMs. In this proposal, we will identify A2AR NAMs utilizing in vitro screening paradigms established in our laboratory. Aim 1: Identify NAMs of the adenosine-A2AR immune checkpoint. (a) The compound library will be screened in a cell- based cAMP assay utilizing CHO cells stably expressing the A2AR. NAMs will be identified on the basis of adenosine-mediated inhibition of cAMP production. (b) NAMs will be confirmed in a radiolabeled agonist binding assay utilizing CHO-A2AR membranes. A reduction in the binding affinity of the A2AR selective agonist CGS 21680 is indicative of NAMs. Aim 2: Identify NAMs with best immune-enhancing activity and A2AR selectivity. (a) NAMs will be screened for their ability to reverse the adenosine-A2AR- mediated inhibition of IFN-g production by a-CD3/CD28-stimulated CD8+ T cells. (b) NAMs with the best immune enhancing activity will be evaluated for A1R, A2BR and A3R activity in cell-based cAMP assays utilizing cell lines stably expressing the receptors. If successful, this will be the first identification of NAMs of the adenosine-A2AR immune checkpoint and demonstration of a novel approach targeting the same.