Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Abstract

Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.

Keywords: GPCR; affinity; allosteric modulators; cooperativity; ensemble docking.

Fig. 1.

Overview flowchart for discovering allosteric modulators of the M₂ mAChR. Starting from X-ray structures of the inactive QNB-bound and active IXO-nanobody-bound M₂ receptor, aMD-enhanced sampling simulations were carried out to construct structural ensembles that account for receptor flexibility. Meanwhile, a compound library was prepared from the NCI Diversity Set (∼1,600 compounds) by using LigPrep in the Schrödinger package. Ensemble docking was then performed to identify potential allosteric modulators. Glide HTVS+IFD was applied and provided much improved enrichment factors in retrospective docking, allowing the selection of 38 top-ranked compounds for experimental testing. Of this set, 19 compounds that significantly slowed the dissociation of the antagonist radioligand [³H]NMS were selected for further characterization. Finally, 12 allosteric modulators exhibited binding affinity of ≤30 μM.

Fig. 2.

Allosteric binding properties of NCI compounds at the M₂ mAChR. (A) The dissociation rates of the radioligand, [³H]NMS (k_off), were revealed by adding 100 μM atropine alone or in the presence of each of the 38 computationally selected lead compounds at a concentration of 100 μM. Included are also the k_off values, with three known allosteric modulators (LY-2033298, gallamine, and strychnine) for comparison. Dashed lines show the threshold, k_off ≤ 0.06 min⁻¹ (light gray) and k_off ≤ 0.04 min⁻¹ (dark gray), that were used to select the 19 or 12 lead compounds for further investigation, respectively. As a negative control, IXO at a concentration of 1 μM (∼300 x K_I) was also tested. (B–D) Binding affinity (pK_B) of each of the 12 selected NCI compounds (B) and cooperativity (Logα) between each NCI compound and [³H]NMS (C) and IXO (D). n.a., not applicable.

Fig. 3.

Chemical structures of the 12 NCI compounds that were validated experimentally as allosteric ligands of the M₂ mAChR. These compounds slowed down the dissociation rates of the [³H]NMS antagonist by ≥50%. In addition, they exhibited significant binding affinities in the range of ∼3−30 μM (4.50 ≤ pK_B ≤ 5.50).

Fig. 4.

Functional signaling effects of the six selected NCI compounds on agonist-mediated pERK1/2 responses at the M₂ mAChR. (A–E) Functional interactions between 0.03 nM IXO and increasing concentrations of four NCI compounds in a titration interaction format are shown as follows. (A) NSC-322661. (B) NSC-121868. (C) NSC-13316. (D) NSC-147866. (E) NSC-99657. (F) NSC-93427. (G and H) Functional interactions between IXO and increasing concentrations of NSC-322661 (G) and NSC-13316 (H) in a full interaction format, to validate the distinctive allosteric properties of the two selected NCI compounds.

Fig. 5.

The predicted most favorable binding poses of functional allosteric modulators of the M₂ mAChR obtained from ensemble docking calculations. (A and B) NSC-322661, a PAM that causes only slight conformational changes in the receptor (A) and NSC-13316, a NAM that binds deeper into the receptor and induces larger structural rearrangements of the TM helices with significant conformational changes in residues Y426^7.39, W422^7.35, Y83^2.64, and F181^ECL2 (B). The ligands are shown as spheres with carbons in yellow for IXO and purple for the allosteric modulators. The receptor is represented by orange ribbons, and residues found within 3 Å of the bound allosteric modulators are labeled and shown as sticks. The X-ray structure of the active M₂ mAChR (Protein Data Bank ID code 4MQS) is also shown in gray as reference. (C and D) Comparison of binding poses for the NSC-322661 (pink), NSC-13316 (yellow), NSC-121868 (purple), NSC-147866 (red), and NSC-99657 (green) compounds in the side (C) and extracellular (D) views. The compounds are shown as thick sticks and the interacting residues as thin sticks. The receptor is represented by ribbons colored by the sequence conservation across five subtypes of human mAChRs. Blue means high conservation 1, and red means low conservation 0.

Fig. 6.

Functional signaling effects of the six selected NCI compounds on agonist-mediated pERK1/2 responses at the M₃ mAChR. (A and B) Effect of 10 μM concentration of each NCI compound on IXO potency (A) and IXO maximal response (B). (C and D) Full interaction curves between IXO and NSC-147866 (C) or NSC-99657 (D) at a concentration of 10 μM. Statistical analyses were performed by one-way ANOVA between the control value, IXO potency, or Emax, using Prism (Version 7.01), and statistical significance was taken as P < 0.05.

Fig. 7.

Functional signaling effects of the six selected NCI compounds on agonist-mediated pERK1/2 responses at the M₁ mAChR. (A and B) Effect of 10 μM concentration of each NCI compounds on IXO potency (A), and IXO maximal response (B). (C and D) Full interaction curves between IXO and NSC-147866 (C) or NSC-99657 (D) at 10 μM. Statistical analyses were performed by one-way ANOVA between the control value, IXO potency or Emax, using Prism (Version 7.01), and statistical significance was taken as P < 0.05.