Oral Presentation 49th Lorne Conference on Protein Structure and Function 2024

Structural insights into the activation and modulation of a class B1 GPCR by small-molecule ligands (#15)

Xin Zhang 1 2 , Hariprasad Venugopal 3 , Samantha McNeill 1 , Matthew J Belousoff 1 2 , Giuseppe Deganutti 4 , Christopher A Reynolds 4 , Patrick M Sexton 1 2 , Denise Wootten 1 2
  1. Drug Discovery Biology, Monash Univeristy, Melbourne, Vic, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane , Monash University, Melbourne, Vic, Australia
  3. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, Vic, Australia
  4. Centre for Sport, Exercise and Life Sciences, Coventry University, Coventry, UK

Membrane receptors are critical for regulating diverse physiological processes in the human body upon activation by extracellular stimuli. Among these, the glucagon-like peptide 1 receptor (GLP-1R), a class B1 G protein-coupled receptor (GPCR) plays essential roles in regulating insulin secretion, carbohydrate metabolism and appetite, making it a well-established clinical target for the treatment of type II diabetes and obesity [1].

GLP-1R is activated by two endogenous peptides, GLP-1 and oxyntomodulin, and primarily signals through the stimulatory G protein Gs. While multiple synthetic peptide agonists, such as semaglutide and tirzepatide, have been clinically approved, their use is hampered by side effects and challenges in administration. Hence, there is a growing interest in developing orally available, non-peptidic small-molecule drugs. Several small-molecule drugs have been discovered, but the structural basis of ligand binding and receptor modulation remains elusive. In this study, we determined 11 different cryo-electron microscopy structures of GLP-1R-Gs complexes bound to three endogenous agonists (GLP-1, its principle metabolite GLP-1(9-36)NH2, oxyntomodulin), two non-peptidic agonists (PF 06882961 and CHU-128) [2-3] and three positive allosteric modulators (compound 19, BETP and compound 2, unpublished data) at global resolution of 2.1Å - 3.0Å.

Notably, the binding site for PF 06882961 exhibits substantial overlap with that of endogenous peptide agonists within the receptor core. In contrast, CHU-128 displays limited overlap, which aligns with its divergent pharmacological properties [2]. Surprisingly, the allosteric modulator compound 19 engages at the extracellular side of the receptor, while compound 2 and BETP bind to the intracellular end. Besides the unique ligand binding modes, these diverse ligands induce distinct complex conformations and receptor conformational dynamics, as uncovered by MD simulation and 3D variance analysis. Together with functional characterization in vitro, the observed structural and dynamic differences induced upon binding different ligands provide crucial insights into non-peptide ligand interactions and allosteric modulation. This work sets the foundation for designing novel small-molecule therapeutics for GLP-1R and other class B1 GPCRs.

  1. Graaf, C, Donnelly, D, Wootten, D, Lau, J, Sexton, PM, Miller, LJ, Ahn, JM, Liao, J, Fletcher, MM, Yang, D, Brown, AJH, Zhou C, Deng, J, Wang, MW, 2016. Glucagon-like Peptide-1 and its class B G protein-coupled receptors: A long march to therapeutic success. Pharmacol Rev, 68, 954-1013.
  2. Zhang, X, Belousoff, MJ, Zhao, P, Kooistra, AJ, Truong, TT, Ang, SY, Underwood, CR, Egebjerg, T, Senel, P, Stewart, GD, Liang, YL, Glukhova, A, Venugopal, H, Christopoulos, A, Furness, SGB, Miller, LJ, Reedtz-Runge, S, Langmead, CJ, Gloriam, DE, Danev, R, Sexton, PM, Wootten, D, 2020. Differential GLP-1R binding and activation by peptide and non-peptide agonists. Mol Cell, 80, 1–16.
  3. Deganutti, G*, Liang, YL*, X. Zhang*. Khoshouei, ML, Clydesdale, MJ, Venugopal, H, Truong, TT, Glukhova, A, Keller, AN, Gregory, KJ, Leach, K, Christopoulos, A, Danev, R, Reynolds, CA, Zhao, P, Sexton, PM, Wootten, D, 2022. Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation. Nat. Commun. 13: 92.