Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a ‘molecular glue’. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.
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We are grateful to B. Anderson, A. Castano, B. Czeskis, C. Corkins, T. Gopalappa, D. Jett, C.R. Logan, Y. Qian, M. Russell, H. Wang, J. Wyss and R. Zink for advice and technical support. We also appreciate long-standing support for the project by C. Montrose-Rafizadeh, G. Zhu, J. Moyers and R. Gimeno. The cryo-EM data were collected at NanoImaging Service.
A.B.B., F.S.W., J.D.H., D.B.W., A.D.S., M.V., Q.C., C.S., B.C., J.F., F.J.A., G.R.C., A.J., I.R. and K.W.S. are employees of Eli Lilly and Company and may own company stock. B.S., D.F., T.S.K. and B.K.K. are employees of or consultants for ConfometRx. T.S.K. and B.K.K. cofounded ConfometRx.
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a, The potency of unlabeled GLP-1(9-36) to displace [125I]GLP-1(7-36) binding to the GLP-1R was measured at different concentrations of LSN3160440. b, Data were replotted to generate a modified Schild plot and LSN3160440 KB (5.7 µM) and α (407) parameters were calculated using the allosteric Schild equation. Data are a representative example of eight independent experiments for which summary statistics are mean (SD): pKB 5.14 (0.2) and log α 2.64 (0.4).
Extended Data Fig. 2 LSN3160440 enhances insulin secretion in a glucose-, ligand-, and GLP-1R-dependent manner.
a-b, Insulin secretion in islets isolated from wild-type (WT, grey bars) and Glp-1r knockout (KO, white bars) mice at high or low glucose. Insulin levels were quantified in media from cultures of WT and Glp-1r KO mouse (C57/Bl6 background) islets treated with GLP-1(7-36), GIP(1-42), GLP-1(9-36), LSN3160440, or GLP-1(9-36) plus LSN3160440 in the presence of 11.2 mM glucose (a) or 2.8 mM glucose (b). For each treatment, mean (SEM) insulin concentrations were determined in six individual wells containing three islets per well. * p = 0.0002 and ** p < 0.0001 using one-way ANOVA followed by Dunnett’s comparison versus the vehicle response. Data are representative of three (a) or two (b) independent experiments. c, Time course of plasma insulin concentrations in fasted, anesthetized Wistar rats treated with either vehicle, GLP-1(7-36) (3 nmol/kg), LSN3160440 (5 mg/kg), GLP-1(9-36) (50 nmol/kg), or LSN3160440 (5 mg/kg) plus 5, 20, or 50 nmol/kg of GLP-1(9-36), dosed immediately before intravenous administration of a glucose bolus (0.5 g/kg). Data are presented as the mean (SEM) from five animals per treatment group and are representative of three independent experiments.
a, Cryo-EM data processing flow chart. b, Gold standard Fourier shell correlation (FSC) curves of two individual half maps indicating an average resolution of 3.3 Å at 0.143 FSC threshold. c, Cross-validation of model to cryo-EM density map. The model was refined against one half map, and FSC curves were calculated between this model and the final cryo-EM map (full dataset, blue) of the outcome of model refinement with a half map versus the same map (orange), and of the outcome of model refinement with a half map versus the other half map (green). d, Density map colored by local resolution. e, Local resolution near the binding site for LSN3160440. LSN3160440 density is indicated by white dashed circle.
Extended Data Fig. 4 Model of the GLP-1R/GLP-1/LSN3160440/Gs/Nb35 complex in the cryo-EM density map.
Cryo-EM density map and the model are shown for all seven transmembrane (TM) helices and helix 8 of GLP-1R, GLP-1(7-37), and LSN3160440 (zoomed in). All the residues in GLP-1(7-37) are resolved.
Potentiation of the cAMP accumulation produced by GLP-1(9-36) in combination with various fixed concentrations of positive allosteric modulator using mutant GLP-1R transiently transfected into HEK293 cells. LSN3160440: a, L142A, b, Y145A, c, K202A, and d, L142A, Y145A, K202A. GLP-1R with BETP: g, L142A, h, Y145A, i, K202A, and j, L142A, Y145A, K202A. Each graph is a single experiment. Replicate data was generated in a concentration response format with altered compound testing concentrations but gave analogous data. N values for replication: a = 2, b = 2, c = 2, d = 3, g = 2, h = 2, i = 2, j = 3. The magnitude of allosteric modulator affinity and efficacy cooperativity could not be quantified using the operational model of allosterism due to the efficacy of GLP-1(9-36) alone, being below the noise level in these experiments. Therefore, we used an empirical approach to quantify allosterism. Concentration response curves were individually fit to the 4-parameter logistic model to obtain potency and efficacy for GLP-1(9-36). The maximal efficacy obtained at any single concentration of modulator is plotted for e, LSN3160440 and k, BETP. EC50 values at different concentrations of modulators were calculated and are plotted versus modulator concentrations for f, LSN3160440, and l, BETP.
a, GLP-1R (WT), b, GLP-1R triple mutant (L142A, K202A, Y145A), c, GLP-1R (K202A), d, GLP-1R (Y145A), e, GLP-1R (Y145F). Competitive binding of GLP-1(9-36) in the presence and absence of 10 µM positive allosteric modulator (PAM) LSN3160440 was measured using 4 nM labeled exendin-4. Data are presented as the mean (SEM) of three independent experiments.
Interaction of ligand with protein based on initial 50 ns simulation. Interaction of Lys202 with N3 of benzimidazole is bridged with water 45% of simulation time. Stacking interaction with Tyr145 is present 98% of simulation time. Benzimidazole oscillates 1 Å in RMSD from its original position from the cryo-EM structure. a, 2D ligand protein interaction diagram over the initial 50 ns simulation. b, 3D representation of the MMGBSA energetically Representative MD frame at 47 ns of simulation. Waters bridging interactions with ligand are shown as ball-and-stick model. Piperidine is also interacting with waters which at times bridge interaction with POPC (shown as maroon sticks) phosphates.
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Bueno, A.B., Sun, B., Willard, F.S. et al. Structural insights into probe-dependent positive allosterism of the GLP-1 receptor. Nat Chem Biol 16, 1105–1110 (2020). https://doi.org/10.1038/s41589-020-0589-7
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