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 (mGlu 5 ) 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 mGlu 5 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 mGlu 5 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.

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

“…Before this, researchers relied on family A 7TM templates to perform homology modeling and interpret site-directed mutagenesis data. Despite low sequence homology (,20%), these approaches proved fruitful, localizing allosteric binding pockets of mGlu 1 , mGlu 2, mGlu 4 , and mGlu 5 to a region analogous to that of the biogenic amine binding sites in family A GPCRs (Ott et al, 2000;Pagano et al, 2000;Malherbe et al, 2003a,b;Lundstrom et al, 2011;Molck et al, 2012;Gregory et al, 2013bGregory et al, , 2014Rovira et al, 2015). In light of the new crystal structures, although alignments and secondary structure predictions of TMs 5-7 deviated from that observed in the mGlu 1 and mGlu 5 structures, the general localization of the pocket was consistent with earlier reports.…”

“…Across the four subtypes multiple residues have repeatedly been reported as contributing to allosteric modulation. For all four subtypes, residues in positions 3.36, 5.43, and 6.48 are crucial for both positive and negative allosteric modulators (Pagano et al, 2000;Malherbe et al, 2003aMalherbe et al, , 2006Muhlemann et al, 2006;Fukuda et al, 2009;Lundstrom et al, 2011;Gregory et al, 2012Gregory et al, , 2013bGregory et al, , 2014Molck et al, 2012;Turlington et al, 2014;Wu et al, 2014;Rovira et al, 2015). Position 7.45 has also been implicated in mGlu 4 and mGlu 5 allosteric modulation (Molck et al, 2012;Gregory et al, 2013bGregory et al, , 2014Turlington et al, 2014;Rovira et al, 2015); there are no reports investigating the influence of this amino acid on selective allosteric modulators of mGlu 1 or mGlu 2 .…”

“…For all four subtypes, residues in positions 3.36, 5.43, and 6.48 are crucial for both positive and negative allosteric modulators (Pagano et al, 2000;Malherbe et al, 2003aMalherbe et al, , 2006Muhlemann et al, 2006;Fukuda et al, 2009;Lundstrom et al, 2011;Gregory et al, 2012Gregory et al, , 2013bGregory et al, , 2014Molck et al, 2012;Turlington et al, 2014;Wu et al, 2014;Rovira et al, 2015). Position 7.45 has also been implicated in mGlu 4 and mGlu 5 allosteric modulation (Molck et al, 2012;Gregory et al, 2013bGregory et al, , 2014Turlington et al, 2014;Rovira et al, 2015); there are no reports investigating the influence of this amino acid on selective allosteric modulators of mGlu 1 or mGlu 2 . Between group I mGlu receptors and mGlu 2 , three residues are common: 5.44, 5.47, and 6.55 (Knoflach et al, 2001;Malherbe et al, 2003bMalherbe et al, , 2006Schaffhauser et al, 2003;Hemstapat et al, 2006;Muhlemann et al, 2006;Rowe et al, 2008;Fukuda et al, 2009;Lundstrom et al, 2011;Gregory et al, 2012Gregory et al, , 2013bGregory et al, , 2014Molck et al, 2012).…”

“…These mutagenesis studies pointed to a common location for allosteric binding pockets for group I mGlu receptors. Additional studies have shown that residues at positions 3.40 and 7.38 are important for allosteric modulation of both mGlu 1 and mGlu 5 [Ballesteros-Weinstein numbering (Ballesteros and Weinstein, 1995) adopted from mGlu 1 structural alignment to family A GPCRs; Malherbe et al, 2003aMalherbe et al, ,b, 2006Muhlemann et al, 2006;Suzuki et al, 2007;Fukuda et al, 2009;Gregory et al, 2012Gregory et al, , 2013bGregory et al, , 2014Turlington et al, 2014;Wu et al, 2014]. Subsequently, key residues for the potency and/or binding of mGlu 1 , mGlu 2 , mGlu 4 , and mGlu 5 allosteric modulators from diverse chemical scaffolds have been mapped to TMs 3-7.…”

Do Plants Contain G Protein-Coupled Receptors?

“…In this respect, only GCR1 is useful, as the fold recognition studies indicate that GCR1 is the most likely candidate to have a GPCR fold while the evidence for other plant GPCRs is at best minimal. While many methods have been used to align GPCRs from different classes (Frimurer and Bywater 1999;Sheikh et al, 1999;Bissantz et al, 2004;Miedlich et al, 2004;Eilers et al, 2005;Kratochwil et al, 2005;Dong et al, 2007;Coopman et al, 2011;Gregory et al, 2013), it has not been possible to validate these methods on GPCRs until recently. However, with the recent publication of the structure of the class B glucagon receptor , the class B corticotropinreleasing factor1 receptor (Hollenstein et al, 2013), and the class F human smoothened receptor (Wang et al, 2013) and the associated structural alignments between class A and these remote homologs, we have been able, to our knowledge for the first time, to successfully test our new method.…”