Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Kinetic characterization of allosteric modulation at different conformational states of a GPCR: a novel application of SPR (#216)

Jianrong Xu 1 2 , Hongzhuan Chen 1 3 , Hao Wang 1 , Arthur Christopoulos 2 , David M. Thal 2
  1. Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  2. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia
  3. Institute of Interdisciplinary Integrative Biomedical Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China

G protein-coupled receptors (GPCRs) constitute the largest family of proteins targeted by approved drugs. Allosteric modulation can achieve selectivity between closely related receptors or receptor subtypes, and allosteric modulators are frequently utilized as chemical probes to investigate GPCR biology. A major limitation of the traditional approaches to monitor ligand binding and function is that it is difficult to determine the binding kinetics of allosteric modulators, particularly on receptors in different conformational states (e.g. active/inactive). Surface plasmon resonance (SPR) is one of the primary biophysical methods for the characterization of binding kinetics, and provides an opportunity to better understand how allosteric modulators bind to GPCRs. Here, we have used the M4 muscarinic acetylcholine receptor (M4 mAChR) as a model GPCR target for SPR experiments. We have utilized different experimental strategies to immobilize the receptors onto the SPR sensor to generate robust data. After optimization, we found that receptors could be immobilized at desirable level with high stability by using NTA chip to capture receptors in a detergent mix of 0.01% LMNG and 0.001% CHS. We then measured the kinetics of negative allosteric modulators (NAMs) binding to the M4 mAChR in different conformational states: apo (no ligand), inactive (antagonist bound), active (agonist bound). Our results show that NAMs display higher affinity for the apo conformation, while the dissociation kinetics became much faster concomitant with a decrease in affinity for both the active and inactive conformations. Moreover, we observed a conformational change of the receptor that was induced by the allosteric modulator during the binding process, which was consistent with the two-state model in BIACORE kinetic models. In conclusion, we have developed an approach to investigate the binding kinetics of GPCRs with SPR that could bring new insights into the underlying mechanisms of allosteric modulation, while also provide the potential for SPR based screening approaches.