P2X3-selective mechanism of Gefapixant, a drug candidate for the treatment of refractory chronic cough

Wen-Wen Cui; Si-Yu Wang; Yu-Qing Zhang; Yao Wang; Ying-Zhe Fan; Chang-Run Guo; Xing-Hua Li; Yun-Tao Lei; Wen-Hui Wang; Xiao-Na Yang; Motoyuki Hattori; Chang-Zhu Li; Jin Wang; Ye Yu

doi:10.1016/j.csbj.2022.03.030

P2X3-selective mechanism of Gefapixant, a drug candidate for the treatment of refractory chronic cough

Comput Struct Biotechnol J. 2022 Mar 31:20:1642-1653. doi: 10.1016/j.csbj.2022.03.030. eCollection 2022.

Authors

Wen-Wen Cui^{1

2}, Si-Yu Wang¹, Yu-Qing Zhang¹, Yao Wang³, Ying-Zhe Fan⁴, Chang-Run Guo¹, Xing-Hua Li¹, Yun-Tao Lei¹, Wen-Hui Wang¹, Xiao-Na Yang¹, Motoyuki Hattori³, Chang-Zhu Li⁵, Jin Wang¹, Ye Yu^{1

2}

Affiliations

¹ School of Basic Medicine and Clinical Pharmacy and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.
² Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
³ State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Shanghai Key Laboratory of Bioactive Small Molecules, Department of Physiology and Neurobiology, School of Life Sciences, Fudan University, Shanghai 200438, China.
⁴ Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200025, China.
⁵ State Key Laboratory of Utilization of Woody Oil Resource, Hunan Academy of Forestry, Changsha 410004, China.

Abstract

Gefapixant/AF-219, a selective inhibitor of the P2X3 receptor, is the first new drug other than dextromethorphan to be approved for the treatment of refractory chronic cough (RCC) in nearly 60 years. To date, seven P2X subtypes (P2X1-7) activated by extracellular ATP have been cloned, and subtype selectivity of P2X inhibitors is a prerequisite for reducing side effects. We previously identified the site and mechanism of action of Gefapixant/AF-219 on the P2X3 receptor, which occupies a pocket consisting of the left flipper (LF) and lower body (LB) domains. However, the mechanism by which AF-219 selectively acts on the P2X3 receptor is unknown. Here, we combined mutagenesis, chimera construction, molecular simulations, covalent occupation and chemical synthesis, and find that the negative allosteric site of AF-219 at P2X3 is also present in other P2X subtypes, at least for P2X1, P2X2 and P2X4. By constructing each chimera of AF-219 sensitive P2X3 and insensitive P2X2 subtypes, the insensitive P2X2 subtype was made to acquire the inhibitory properties of AF-219 and AF-353, an analog of AF-219 with higher affinity. Our results suggest that the selectivity of AF-219/AF-353 for P2X3 over the other P2X subtypes is determined by a combination of the accessibility of P2X3 binding site and the internal shape of this pocket, a finding that could provide new perspectives for drug design against P2X3-mediated diseases such as RCC, idiopathic pulmonary fibrosis, hypertension and overactive bladder disorder.

Keywords: Binding sites; DF, dorsal fin; Gefapixant/AF-219; IC50, the concentration yielding half of the maximal inhibition; LB, lower body; LF, left flipper; MD, molecular dynamics; NPM, N-phenylmaleimide; P2X3 receptors; RCC, refractory chronic cough; Refractory chronic cough; Subtype selectivity.