Investigating metabotropic glutamate receptor 5 allosteric modulator cooperativity, affinity, and agonism: enriching structure-function studies and structure-activity relationships

Abstract

Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu(5)) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu(5) to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu(5)-mediated Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

Fig. 1.

Influences of agonist concentration, modulator affinity, and cooperativity on allosteric modulator potency. A and B, simulations of the effects of different agonist concentrations on the potencies of a negative allosteric modulator (A) and a positive allosteric modulator (B). EC100**, agonist concentration 30-fold in excess of that required to elicit a maximal response. C and D, literature findings on mGlu₅ NAM potency and affinity estimates (C) and mGlu₅ PAM potency and affinity estimates (D) (Huang et al., 2004; Poon et al., 2004; Roppe et al., 2004; Chua et al., 2005; Tehrani et al., 2005; de Paulis et al., 2006; Kulkarni et al., 2006, 2009; Jaeschke et al., 2007; Milbank et al., 2007; Liu et al., 2008; Vanejevs et al., 2008; Felts et al., 2009, 2010; Galambos et al., 2010; Rodriguez et al., 2010; Wágner et al., 2010; Zhang et al., 2010; Alagille et al., 2011; Gilbert et al., 2011; Lindemann et al., 2011; Sams et al., 2011; Weiss et al., 2011; Zou et al., 2011; Mueller et al., 2012). Dashed lines, unity; dotted lines, potency and affinity within 3-fold of each other; solid lines, 10-fold difference.

Fig. 2.

Structures of mGlu₅ allosteric modulators included in this study. VU0366248 and VU0366249 were originally reported as compounds 41 and 42, respectively (Felts et al., 2010).

Fig. 3.

Inhibition of [³H]methoxy-PEPy binding to wild-type mGlu₅-expressing HEK293A cell membranes. Data represent the mean ± S.E.M. from at least three independent determinations.

Fig. 4.

Allosteric modulation of glutamate concentration-response curves for Ca²⁺ mobilization in the low-level mGlu₅-expressing HEK293A cell line. A and B, in the low-level, wild-type mGlu₅-expressing HEK293A cell line, CPPHA (A) and CDPPB (B) induced leftward shifts in the glutamate concentration-response curve for intracellular Ca²⁺ mobilization, with no change in the maximal response. C to E, MPEP (C), VU0366249 (D), and M-5MPEP (E) inhibited glutamate-stimulated intracellular Ca²⁺ mobilization. F, modulator affinity estimates that were calculated by determining the composite cooperativity parameter αβ for PAMs (●) and allowing α to float for NAMs (○) (x-axis) or assuming α = 1 and calculating logβ (y-axis) were compared. G, for calculated cooperativity estimates, the assumption of α = 1 had no effect on the apparent cooperativity between glutamate and PAMs (●) and weak NAMs (○). H, affinity estimates for PAMs (●) and NAMs (○) determined from radioligand binding assays (x-axis) and Ca²⁺ mobilization assays (y-axis) in low-level mGlu₅-expressing HEK293A cells were compared. Dashed line, unity. Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols.

Fig. 5.

Allosteric modulation of glutamate concentration-response curves for Ca²⁺ mobilization in the high-level mGlu₅-expressing HEK293A cell line. A and B, in the high-level mGlu₅-expressing HEK293A cell line, increased agonist activity was seen for CPPHA (A) and CDPPB (B), as well as induction of a leftward shift in the glutamate concentration-response curve for Ca²⁺ mobilization. C to E, MPEP (C), VU0366249 (D), and M-5MPEP (E) inhibited glutamate stimulation of Ca²⁺ mobilization. F, affinity estimates for NAMs (○) and PAMs (●) determined from Ca²⁺ mobilization assays in low-level (x-axis) versus high-level (y-axis) mGlu₅-expressing cell lines were compared. Dashed line, unity. Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols.

Fig. 6.

Allosteric modulation of glutamate concentration-response curves for ERK1/2 phosphorylation in the low-level mGlu₅-expressing HEK293A cell line. A and B, in the low-level mGlu₅-expressing cell line, CPPHA (A) and CDPPB (B) displayed agonist activity and potentiated the glutamate concentration-response curve for phosphorylation of ERK1/2. C to E, MPEP (C), VU0366249 (D), and M-5MPEP (E) inhibited the glutamate-stimulated phosphorylation of ERK1/2. F, affinity estimates for PAMs (●) and NAMs (○) determined from ERK1/2 phosphorylation assays (y-axis) and Ca²⁺ mobilization assays (x-axis) in the low-level mGlu₅-expressing cell line were compared. Dashed line, unity. Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols.

Fig. 7.

Effects of single point mutations of mGlu₅ on MPEP inhibition of mGlu₅-mediated Ca²⁺ mobilization in response to glutamate. Translocation of glutamate concentration-response curves in the presence of the indicated concentrations of MPEP in polyclonal HEK293A cells expressing wild-type (A), Y658V (B), L743V (C), or A809V (D) mGlu₅ was determined. Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols.

Fig. 8.

Effects of single point mutations of mGlu₅ on potentiation by VU29 and CPPHA of mGlu₅-mediated Ca²⁺ mobilization in response to glutamate. A and B, translocation of glutamate concentration-response curves in the presence of the indicated concentrations of VU29 in polyclonal HEK293A cells expressing wild-type (A) or A809V (B) mGlu₅ was determined. C and D, potentiation of glutamate concentration-response curves for Ca²⁺ mobilization by the indicated concentrations of CPPHA in polyclonal HEK293A cells expressing wild-type (C) or F585I (D) mGlu₅ was determined. Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols.

Fig. 9.

Estimation of allosteric modulator cooperativity and affinity values from potency curves. A and B, allosteric modulator potency curves were determined for the indicated positive allosteric modulators in the presence of an EC₂₀ concentration of glutamate. CPPHA and VU0364289 both achieved maximal responses to glutamate; therefore, logβ values were constrained to equal the average maximal leftward shift in the glutamate concentration-response curves (Table 6) for estimations of pK_B. For all other PAMs, both logβ and pK_B values were determined through nonlinear regression analyses. C, negative allosteric modulators were assessed for their ability to inhibit a submaximal glutamate response. D, affinity estimates from potency curves (y-axis) and from progressive fold-shift analyses (x-axis) for PAMs (●) and NAM (○) showed strong correlation. E, strong correlation was observed between logβ values estimated through nonlinear regression from modulator potency curves (y-axis) and from progressive fold-shift analyses (x-axis). Data represent the mean ± S.E.M. from at least three independent determinations. Error bars not shown lie within the dimensions of the symbols. Dashed line, unity.