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

> **NIH NIH R03** · MOLECULAR MEDICINE RESEARCH INSTITUTE · 2022 · $97,500

## 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.

## Key facts

- **NIH application ID:** 10355152
- **Project number:** 1R03AI166365-01
- **Recipient organization:** MOLECULAR MEDICINE RESEARCH INSTITUTE
- **Principal Investigator:** EDWARD P AMENTO
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $97,500
- **Award type:** 1
- **Project period:** 2022-05-23 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10355152

## Citation

> US National Institutes of Health, RePORTER application 10355152, Function- and interaction-based discovery of negative allosteric modulators of the A2A Receptor (1R03AI166365-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10355152. Licensed CC0.

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