Transposition of Three Amino Acids Transforms the Human Metabotropic Glutamate Receptor (mGluR)-3-Positive Allosteric Modulation Site to mGluR2, and Additional Characterization of the mGluR2-Positive Allosteric Modulation Site

Abstract

Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

“…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). Group 1 receptors and mGlu 4 share a common determinant in F6.51 (Malherbe et al, 2003a(Malherbe et al, ,b, 2006Muhlemann et al, 2006;Suzuki et al, 2007;Fukuda et al, 2009;Gregory et al, 2014;Rovira et al, 2015).…”

Allosteric Modulation of Family C G-Protein-Coupled Receptors: from Molecular Insights to Therapeutic Perspectives

“…Although the orthosteric ligand binding site of group II mGlu receptors has been highly conserved during evolution, some key differences between residues in the 7TM region allow for the discrimination of the two by allosteric agents . In fact, substitution of Ser688 and/or Gly689 in transmembrane region 4 along with Asn735 in transmembrane region 5 with the homologous amino acids of the mGlu3 receptor abolished allosteric modulation of mGlu2 receptors by 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide (LY487379) and other positive modulators (Rowe et al, 2008) (see below). Of a great number of positive allosteric group II modulators known to date, only a very few enhance both mGlu2 and mGlu3 receptor responses with more or less similar potencies Govek et al, 2005), whereas most of them are selective for the mGlu2 receptor subtype.…”

Positive Allosteric Modulators for mGluR2 Receptors: A Medicinal Chemistry Perspective

“…These ligands do not activate the mGluR2 receptor per se but act at an allosteric binding site on the receptor to potentiate glutamate-induced activation of this receptor. Since a potentiator with no inherent agonist activity would only function in the presence of the endogenous agonist, the receptor would not be activated continuously, avoiding receptor desensitization which often occurs after repeated dosing of orthosteric agonists [19][20]. In addition, the allosteric binding sites on glutamate receptors might sufficiently be different as to make subgroup selectivity achievable [21].…”