Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor

Abstract

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.

Keywords: Allosteric Regulation; Cell Signaling; Drug Discovery; G Protein-coupled Receptor (GPCR); Muscarinic Acetylcholine Receptor; Mutagenesis.

FIGURE 1.

A, chemical structure of M₁ mAChR-selective positive allosteric modulators. Left panel, BQCA. Right panel, benzoquinazolinone 12. B, BQCA and benzoquinazolinone 12 inhibit the equilibrium binding of [³H]NMS. Data points represent the means ± S.E. of three independent experiments performed in triplicate.

FIGURE 2.

Pharmacological characterization of benzoquinazolinone 12, a high affinity positive allosteric modulator of the M₁ mAChR. A, whole cell radioligand competition binding between [³H]NMS and increasing concentrations of ACh in the absence or presence of increasing concentrations of benzoquinazolinone 12 in CHO FlpIn cells stably expressing WT M₁ mAChR. B, interaction between ACh and benzoquinazolinone 12 in an IP₁ accumulation assay in CHO FlpIn cells stably expressing WT M₁ mAChR. C, whole cell radioligand competition binding between [³H]NMS and increasing concentrations of ACh in the absence or presence of increasing concentrations of benzoquinazolinone 12 in CHO FlpIn cells stably expressing WT M_2–5 mAChR. The curves in A and C represent the best fit of an allosteric ternary complex model (Equation 1). The curves in B represent the best fit of an operational allosteric model (Equation 2).

FIGURE 3.

Effect of M₁ mAChR Ala mutations on the binding and function of BQCA and benzoquinazolinone 12. The bars represent the difference for each mutant receptor in allosteric ligand affinity (pK_B, top panel), binding cooperativity value (Log α, middle panel), or functional cooperativity value (Log αβ, bottom panel) of BQCA and benzoquinazolinone 12 relative to WT. The values are derived from interaction experiments in [³H]NMS radioligand binding and IP₁ accumulation (Tables 3 and 4). *, significantly different (p < 0.05) from WT as determined by one-way ANOVA with Dunnett's post hoc test. nd, not determined (no allosteric modulation).

FIGURE 4.

Structural homology model of the M₁ mAChR complex co-bound with CCh-BQCA (orange) or ACh-benzoquinazolinone 12 (blue). The overall structure of the BQCA-bound M₁ mAChR model (orange) is similar to that of the benzoquinazolinone 12-bound M₁ mAChR model (blue). ACh (pink), BQCA (orange), and benzoquinazolinone 12 (blue) are shown as spheres colored according to element. Top inset, overlay of the BQCA (orange) and benzoquinazolinone 12 (blue) poses represented as stick structures. Bottom inset, ACh binding site in benzoquinazolinone 12-bound M₁ mAChR complex model. Important residues are shown as sticks.

FIGURE 5.

Proposed arrangement of the binding sites of BQCA and benzoquinazolinone 12 at the M₁ mAChR. A, extracellular view of the binding sites of BQCA and benzoquinazolinone 12. BQCA (orange) and benzoquinazolinone 12 (blue) are shown as spheres colored according to element. Important residues are shown as sticks. Insets, positions of Tyr-2^2.61, Tyr-85^2.64, Tyr-404^7.39, Tyr-179, Phe-182, and Trp-400^7.35 in the ACh-benzoquinazolinone 12-bound M₁ mAChR model. B, extracellular view of the BQCA-bound (orange) and benzoquinazolinone 12-bound (blue) M₁ mAChR models showing a tighter pocket for benzoquinazolinone 12 as compared with that predicted for BQCA.

FIGURE 6.

Effect of M₁ mAChR conservative mutations on the binding and function of BQCA and benzoquinazolinone 12. The bars represent the difference for each mutant receptor in allosteric ligand affinity (pK_B, top panel), binding cooperativity value (Log α, middle panel), or functional cooperativity value (Log αβ, bottom panel) of BQCA and benzoquinazolinone 12 relative to WT. The values are derived from interaction experiments in [³H]NMS radioligand binding and IP₁ accumulation (Tables 3 and 4). *, significantly different (p < 0.05) from WT as determined by one-way ANOVA with Dunnett's post hoc test.