Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Using cryo-EM to solve the P2X1 receptor structure – a target for male contraception (#139)

Felix M Bennetts 1 , Jesse I Mobbs 1 2 , Alisa Glukhova 2 3 , Sab Ventura 1 , David M Thal 1 2
  1. Drug Discovery Biology, Monash Institute Of Pharmaceutical Sciences, Melbourne, Victoria, Australia
  2. Monash Institute Of Pharmaceutical Sciences, ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Melbourne, Victoria, Australia
  3. Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

331,000 unintended pregnancies are conceived every day yet, there are only two male contraceptives available. A novel target for male contraception is the P2X1 receptor which is genetically validated but further studies are held back by a lack of potent P2X1 receptor antagonists. To accelerate drug discovery efforts at the P2X1 receptor this project aims to solve a high-resolution structure of the P2X1 receptor and use this information to guide the design of highly potent P2X1 receptor antagonists. Full-length human P2X1 receptor was purified using a membrane purification preparation. Cryogenic transmission electron microscopy (cryo-EM) was used to solve the P2X1 receptor structure. P2X1 receptor antagonists were validated using a HEK293 P2X1 expressing cell line in an intracellular calcium mobilisation assay and a radioligand binding assay. Initial cryo-EM images of the P2X1 receptor revealed severe preferred orientation of the receptor in vitreous ice. The addition of a secondary detergent, fluorinated FOS-Choline-8, significantly reduced preferred orientation which assisted in obtaining a 1.96 Å structure of the P2X1 receptor in an ATP-bound state. The activity of P2X1 receptor antagonists were validated in pharmacology assays. The next step is to generate novel and more effective P2X1 receptor antagonists leveraging our high-resolution P2X1 receptor structure and optimised cryo-EM workflow.