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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Marso, S. P. et al. Liraglutide and cardiovascular outcomes in type 2 diabetes. N. Engl. J. Med. 375, 311–322 (2016).
Marso, S. P. et al. Semaglutide and cardiovascular outcomes in patients with type 2 diabetes. N. Engl. J. Med. 375, 1834–1844 (2016).
Hernandez, A. F. et al. Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial. Lancet 392, 1519–1529 (2018).
Gerstein, H. C. et al. Dulaglutide and cardiovascular outcomes in type 2 diabetes (REWIND): a double-blind, randomised placebo-controlled trial. Lancet 394, 121–130 (2019).
Wootten, D. et al. Allosteric modulation of endogenous metabolites as an avenue for drug discovery. Mol. Pharmacol. 82, 281–290 (2012).
Mentlein, R., Gallwitz, B. & Schmidt, W. E. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829–835 (1993).
Knudsen, L. B. & Pridal, L. Glucagon-like peptide-1-(9-36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor. Eur. J. Pharmacol. 318, 429–435 (1996).
Chen, X. et al. Substituted pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrazines as inhibitors of c-Met and RON tyrosine kinase receptor and their preparation, pharmaceutical compositions and use in the treatment of cancer. World patent WO2010059771 (2010).
May, L. T., Leach, K., Sexton, P. M. & Christopoulos, A. Allosteric modulation of G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 47, 1–51 (2007).
Sloop, K. W. et al. Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets. Diabetes 59, 3099–3107 (2010).
Bueno, A. B. et al. Positive allosteric modulation of the glucagon-like peptide-1 receptor by diverse electrophiles. J. Biol. Chem. 291, 10700–10715 (2016).
Gallwitz, B. et al. Structure/activity characterization of glucagon-like peptide-1. Eur. J. Biochem. 225, 1151–1156 (1994).
Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).
Che, Y., Gilbert, A. M., Shanmugasundaram, V. & Noe, M. C. Inducing protein–protein interactions with molecular glues. Bioorg. Med. Chem. Lett. 28, 2585–2592 (2018).
Schreiber, S. L. Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell 70, 365–368 (1992).
Liu, J. et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807–815 (1991).
Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017).
Venkatesan, K. et al. An empirical framework for binary interactome mapping. Nat. Methods 6, 83–90 (2009).
Arkin, M. R., Tang, Y. & Wells, J. A. Small-molecule inhibitors of protein–protein interactions: progressing toward the reality. Chem. Biol. 21, 1102–1114 (2014).
Jochim, A. L. & Arora, P. S. Assessment of helical interfaces in protein–protein interactions. Mol. Biosyst. 5, 924–926 (2009).
Wells, J. A. & McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 450, 1001–1009 (2007).
Beck, B. et al. in Assay Guidance Manual (eds Sittampalam, G. S. et al.) (Eli Lilly & Co., 2004).
Willard, F. S. et al. Small molecule allosteric modulation of the glucagon-like Peptide-1 receptor enhances the insulinotropic effect of oxyntomodulin. Mol. Pharmacol. 82, 1066–1073 (2012).
Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).
Sattler, M. et al. A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase. Cancer Res. 63, 5462–5469 (2003).
Christopoulos, A. & Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharm. Rev. 54, 323–374 (2002).
Scheres, S. H. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, https://doi.org/10.7554/eLife.42166 (2018).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. 66, 486–501 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. 66, 213–221 (2010).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).
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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