A2AR plays a crucial role in regulating brain function, the immune system, and cardiovascular health. Its significance extends to potential treatments for Parkinson's disease and cancer immunology, making it a prime target for drug development. The ability to work with wild-type A2AR opens new avenues for identifying novel compounds, including allosteric modulators that could overcome the selectivity issues often associated with orthosteric ligands.
In the rapidly advancing field of drug discovery, G protein-coupled receptors (GPCRs) have emerged as pivotal targets for pharmaceutical innovation. These integral membrane proteins, known for their conformational flexibility, have long posed challenges in protein production and crystallographic studies. However, a groundbreaking development in the field has emerged, centering on the adenosine A2A receptor (A2AR), a member of the GPCR class-A family.
Unlike alternative methods that result in mutated and truncated versions of the Adenosine receptor A2A, Eurofins CALIXAR's approach highlights the preservation of the receptor's functional integrity. With conventional methods, the Adenosine receptor A2A, like all GPCRs, is inherently unstable and challenging to produce in a native state. Previous approaches could only lock the receptor in a specific conformation, leading to a solution that was impure, non-native (truncated and mutated), and lacking post-translational modifications. However, Eurofins CALIXAR has overcome this challenge with native full-length A2AR.
Our native A2AR were screened with 200k compounds for agonistic and antagonistic effect by Affinity Selection - Mass Spectrometry (AS-MS) and Saturation Transfer Difference - Nuclear Magnetic Resonance (STD-NMR). Epitope mapping showed its potential binding sites. This comprehensive screening approach yielded interesting results, demonstrating the power and versatility of these techniques in identifying potential ligands for GPCRs. The STD-NMR experiments proved particularly valuable, allowing us to observe and characterize the binding of both known antagonists and agonists to A2AR. Notably, we were able to detect the binding of caffeine and adenosine, and perform competition experiments with antagonist (ZM241385) and agonist (CGS-21680), respectively. These experiments not only validated the STD-NMR method for GPCRs but also revealed intriguing insights into the binding behavior of these compounds. For instance, the persistent STD signal of adenosine in the presence of agonist (CGS-21680) suggested the existence of multiple binding sites on A2AR, a valuable finding for further investigation. Following this initial validation, we conducted a fragment screening using a library of 100 compounds, identifying 19 fragments with significant binding affinity to A2AR. Subsequent functional validation through cAMP cell-based assays revealed that several of these fragments exhibited antagonistic effects on A2AR, with no agonistic activity observed. This combination of STD-NMR screening and cellular assays proved to be a powerful approach for discovering functionally relevant fragments, highlighting the potential of this method for future drug discovery efforts targeting GPCRs.
Eurofins CALIXAR’s Adenosine receptor A2A facilitates reliable fragment-based drug design (FBDD), structure-based drug discovery (SBDD) and antibody discovery corresponding to this specific target. Explore our case studies and discover how Eurofins CALIXAR can contribute to your next scientific breakthrough. For custom inquiries or collaborations, please contact us.
References :
Igonet S et al. 2018
Desuzinges Mandon E. et al. 2017
