Structural basis of peptidomimetic agonism revealed by small- molecule GLP-1R agonists Boc5 and WB4-24

Abstract

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.

Keywords: cryoelectron microscopy; glucagon-like peptide-1 receptor; peptidomimetic agonism; type 2 diabetes mellitus.

Fig. 1.

Overall cryo-EM structures of the Boc5- and WB4-24–bound GLP-1R–G_s complexes. (A) The cryo-EM map with a disk-shaped micelle (Left) and cartoon representation (Middle) of the Boc5-bound complex. The top view shows the binding pose of Boc5. (Right) chemical structure of Boc5 (molecular weight: 1,018) with two 3-methoxy-4-((thiophene-2-carbonyl)oxy)phenyl groups (blue shade) named A-1 and A-2, respectively, and two 4-((tert-butoxycarbonyl)amino)phenyl groups (orange shade) named B1-1 and B1-2, respectively. (B) The cryo-EM map with a disk-shaped micelle (Left) and cartoon representation (Middle) of the WB4-24–bound complex. The top view shows the binding pose of WB4-24. (Right) chemical structure of WB4-24 (molecular weight: 1,074) with two 3-methoxy-4-((thiophene-2-carbonyl)oxy)phenyl groups (blue shade) that are the same as A-1 and A-2 of Boc5, and two 4-((tert-butoxycarbonyl)amino)phenyl groups (pink shade) named B2-1 and B2-2, respectively. Moieties that are different between Boc5 and WB4-24 are highlighted in red circles, Boc5-bound GLP-1R in purple, WB4-24–bound GLP-1R in dodger blue, Ras-like domain of Gα_s in gold, Gβ subunit in deep pink, Gγ subunit in green, Nb35 in gray, Boc5 in dark green, and WB4-24 in magenta.

Fig. 2.

Structural analysis of the WB4-24–bound GLP-1R. (A) Structural comparison of the WB4-24–bound GLP-1R with inactive and GLP-1–bound GLP-1R complexes (PDB code: 6X18 and 6LN2). Arrows show the transition from the inactive conformation to the WB4-24–induced active state. (B) Interactions of WB4-24 within the TM binding cavity. WB4-24 and its interaction residues are shown by sticks. (C) Overlay of the WB4-24– and GLP-1–bound receptors reveals that WB4-24 has a partial overlap with the N terminus of GLP-1 in the TM binding cavity. (D) Superimposition of the WB4-24– and LY3502970-bound receptors reveals a similar binding pose and a large degree of overlap in the GLP-1R TM cavity. (E) Superimposition of the WB4-24– and TT-OAD2-bound receptors reveals a partial overlap in the TM2-TM3 cleft.

Fig. 3.

Comparison of GLP-1R conformation and ligand binding pocket stabilized by Boc5 and WB4-24. (A) Overlay of Boc5- and WB4-24–bound GLP-1R structures. (B and C) Close-up of the TM bundles viewed from the extracellular side reveals conformational differences in TM2/TM3 (B) and TM1/TM7 (C) backbones when stabilized by different arms of Boc5 and WB4-24. (D) WB4-24 makes more extensive contacts with TM bundles than Boc5 by interacting with residues L144, K197, and R380, which contribute to the agonism of GLP-1. Coloring for A–D denotes the segments as highlighted (GLP-1 in cyan, GLP-1–bound GLP-1R in sandy brown, Boc5 in dark green, Boc5-bound GLP-1R in purple, WB4-24 in magenta, WB4-24–bound GLP-1R in dodger blue). The interactions of the residues with WB4-24 and GLP-1 are indicated by black and red dashed lines, respectively. (E) Mutagenesis analysis of residues L144, K197, and R380 for GLP-1, Boc5, and WB4-24 show the requirement of these contacts for receptor activation. Data are shown as means ± SEM from at least three independent experiments.

Fig. 4.

Peptidomimetic binding mode of Boc5 and WB4-24. (A) Structural comparison of the binding conformations of peptidic and nonpeptidic agonists of GLP-1R. The receptor is shown in surface representation and colored from dodger blue for the most hydrophilic region to white, and to orange-red for the most hydrophobic region. Small-molecule agonists and GLP-1 are shown as sticks and ribbon, respectively. (B) Schematic diagram of the interactions between peptidic and nonpeptidic agonists and GLP-1R. (C) Scatterplot of agonist binding in GLP-1R. All structures were superimposed on the GLP-1–bound GLP-1R coordinates from the OPM database (PDB: 6X18) using the TMD Cα atoms. The x and y axis represent into the depth of the agonist that penetrated the GLP-1R TMD pocket and the buried surface area between the agonist and TMD, respectively. The former is defined as the minimum vertical distance between the heavy atoms of a certain agonist and the carboxyl oxygen atom of E9^P in GLP-1 (the deepest atom of GLP-1 that inserted into the TMD), while the latter was calculated using freeSASA. (D) Interface comparison across peptidic and nonpeptidic agonists of GLP-1R. Different regions of the GLP-1R TMD pocket including TM1/7, TM2/3, ECL1 (residues S206 to D222), and the deep pocket (defined as the collection of the residues Y148^1.43b-F195^2.65b, M233^3.36b-V281^4.57b, R310^5.40b-F367^6.56b, and L388^7.43b-W420^8.61b) were subjected to the interface area calculation.

Fig. 5.

Systematic comparison of ligand recognition patterns of different nonpeptidic GLP-1R agonists. (A) Binding poses of the reported nonpeptidic agonists, GLP-1 (residues H7-L20 are shown), and a positive allosteric modulator, LSN3160440. Each ligand is displayed with a different color (GLP-1, cyan; PF-06882961, salmon; RGT1383, gray; Boc5, dark green; WB4-24, magenta; LY3502970, light green; CHU-128, yellow; TT-OAD2, sky blue; and LSN3160440, orange red). The binding sites of these ligands are located mainly in six regions represented by color dots, including the clefts of TM1-TM2 (orange), TM2-TM3 (red), and TM1-TM7 (yellow); ECLs 1 (blue) and 3 (pink); and the deep orthosteric pocket (green). (B) Positions of the binding sites identified among published GLP-1R structures. The dashed boxes show the binding modes of the agonists in specific regions. Colors are consistent with binding sites in panel (A).