Probing the metabotropic glutamate receptor 5 (mGlu₅) positive allosteric modulator (PAM) binding pocket: discovery of point mutations that engender a "molecular switch" in PAM pharmacology

Abstract

Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu₅) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu₅ modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu₅ served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain (TM) regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine, a prototypical NAM. Three conserved amino acids (Y658, T780, and S808) and two nonconserved residues (P654 and A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TMs 6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs.

Fig. 1.

Probing the common allosteric binding site on mGlu₅ with the NAM MPEP. (A) At the wild-type rat mGlu₅ receptor, MPEP inhibits glutamate-mediated mobilization of intracellular Ca²⁺, depressing the maximal response. (B) Single point mutations of mGlu₅ were screened for their ability to impact inhibition of the maximal response to glutamate in the presence of 10 nM MPEP. (C) Comparison of MPEP affinity estimates at mutants with wild type. Data represent the mean ± S.E.M. of 3–6 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 2.

At two point mutations, MPEP did not fully depress the maximal response to glutamate. MPEP inhibition of glutamate-mediated mobilization of intracellular Ca²⁺ at mGlu₅ P654F (A) and W784A (B). In the presence of MPEP at concentrations up to 100 μM, glutamate retained some activity in both cell lines. Data represent the mean ± S.E.M. of 3–5 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 3.

The NAM MPEP docked into the mGlu₅ comparative model. MPEP was docked into the mGlu₅ comparative model in two separate experiments, centered at P654 and S808. (A) The lowest-energy conformation for MPEP from the largest cluster docked at P654 is shown in green and at S808 is shown in cyan. Highlighted are the residues that caused decreases in MPEP affinity when mutated, colored by graded effect compared with wild type. (B) The lowest-energy models from the largest three clusters for MPEP docked at P654. (C) The lowest-energy models from the largest five clusters for MPEP docked at S808. Predicted hydrogen bonds between the nitrogen on the pyridine ring and S808 are depicted by dotted blue lines.

Fig. 4.

Potentiation of glutamate-mediated Ca²⁺ mobilization by nicotinamide and picolinamide acetylene PAMs at wild-type mGlu₅. The six PAMs included in this study potentiate the response to glutamate at mGlu₅ wild type in a Ca²⁺ mobilization assay with varying degrees of cooperativity, as evidenced by increased glutamate potencies in the presence of PAMs. Nicotinamide acetylene PAMs are shown on the left (A–C), with the corresponding picolinamide acetylene PAM on the right (D–F). Data represent the mean ± S.E.M. of 3–7 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 5.

Effect of mutations on the fold-shift caused by a single concentration of PAM. Nicotinamide acetylene PAMs are shown on the left, with the corresponding picolinamide acetylene PAM on the right as indicated. The increase in glutamate potency in the presence of PAM, or fold-shift, at each mutant is expressed relative to that observed for the same concentration at the wild-type receptor. Specifically, PAM concentrations used were 10 μM VU0360173 (A), 1 μM VU0360172 (B), 100 nM VU0415051 (C), 100 nM VU0405398 (D), 10 nM VU0403602 (E), and 10 nM VU0405386 (F). ^#No detectable PAM activity; *P < 0.05 vs. wild type, one-way analysis of variance (ANOVA), Dunnett’s post-test. Data represent the mean ± S.E.M. of 3–7 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 6.

Effect of mutations on PAM affinity estimates. Nicotinamide acetylene PAMs are shown in the top three panels (A–C), with the corresponding picolinamide acetylene PAM in the bottom three panels (D–F). Affinity estimates (pK_B) were derived using an operational model of allosterism (Leach et al., 2007; Gregory et al., 2012) from progressive fold-shifts of the glutamate concentration-response curve for Ca²⁺ mobilization. The difference between the pK_B for the mutant versus wild type is plotted. ^#No detectable PAM activity; *P < 0.05 vs. wild type, one-way analysis of variance (ANOVA), Dunnett’s post-test. Data represent the mean ± S.E.M. of 3–7 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 7.

Computational docking of three pairs of nicotinamide and picolinamide acetylene PAMs into mGlu₅. (A) VU0360173 in blue and VU0405398 in green, (B) VU0360172 in blue and VU0403602 in green, and (C) VU0415051 in blue and VU0405386 in green. Residues that when mutated caused a significant decrease in modulator affinity in mGlu₅ are highlighted in the respective color of the modulator. Residues that affect both the nicotinamide and picolinamde in the pair are highlighted in purple. A predicted hydrogen bond network involving the modulators, Y658, T780A, and W784 is represented by the dashed black lines. Highlighted in gray are residues that influence the cooperativity of certain modulators.

Fig. 8.

Effect of mutations on PAM cooperativity factors. Nicotinamide acetylene PAMs are shown in the top three panels (A–C), with the corresponding picolinamide acetylene PAM in the bottom three panels (D–F). Cooperativity estimates (logβ) were derived using an operational model of allosterism (Leach et al., 2007; Gregory et al., 2012) from progressive fold-shifts of the glutamate concentration-response curve for Ca²⁺ mobilization. The difference between the logβ for the mutant versus wild type is plotted. ^#No detectable PAM activity; *P < 0.05 vs. wild type, one-way analysis of variance (ANOVA), Dunnett’s post-test. Data represent the mean ± S.E.M. of 3–7 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 9.

Characterization of mutations that engender a molecular switch in PAM pharmacology. (A) At mGlu₅ Y658V, 10 μM and 30 μM VU0405398 inhibited the response to maximal glutamate. (B) The interaction between glutamate and VU0415051 at the T780A mutant is negative, with inhibition approaching a limit as defined by the cooperativity. (C) At S808A, VU0405398 causes a reduction in glutamate potency and depresses the maximal response to glutamate. (D) Concentration-response curves for VU0405398 inhibition of an ∼EC₈₀ of glutamate in the absence and presence of the indicated concentrations of VU0405386. (E) Schild regression of the interaction between VU0405386 and VU0405398 at S808A. Data represent the mean ± S.E.M. of 3–6 experiments performed in duplicate. Error bars not shown lie within the dimensions of the symbol.

Fig. 10.

Mutations engendering a molecular switch for mGlu₅ allosteric modulators. (A) VU0405398 docked into wild-type mGlu₅ (green) and the S808A mutant (magenta). (B) VU0415051 docked into wild-type mGlu₅ (blue) and the T780A mutant (magenta). (C) VU0405386 docked into wild-type mGlu₅ (blue) and the S808A mutant (magenta). Mutated residues are colored by element. Key affinity determinants are highlighted to show conformational changes in the binding pocket.