Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP

Abstract

Fenobam [N-(3-chlorophenyl)-N¢-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a clinically validated non-benzodiazepine anxiolytic, has been shown to be a potent and non-competitive metabotropic glutamate (mGlu)-5 receptor antagonist. In the present study, we have used the site-directed mutagenesis coupled with three-dimensional receptor-based pharmacophore modelling to elucidate the interacting mode of fenobam within the seven-transmembrane domain (7TMD) of mGlu5 receptor and its comparison with that of 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototype antagonist. The common residues involved in the recognition of MPEP and fenobam include Pro654 3.36 , Tyr658 3.40 , Thr780 6.44 , Trp784 6.48 , Phe787 6.51 , Tyr791 6.55 and Ala809 7.47 . The differentiating residues between both modulators' interacting modes are Arg647 3.29 , Ser657 3.39 and Leu743 5.47 . Our data suggest that these chemically unrelated mGlu5 antagonists act similarly, probing a functionally unique region of the 7TMD. Using [ 3 H]inositol phosphates accumulation assay, we have also identified the critical residues involved in the inverse agonist effect of MPEP. The mutation W784 6.48 A completely blocked the inverse agonist activity of MPEP; two mutations F787 6.51 A and Y791 6.55 A, caused a drastic decrease in the MPEP inverse agonism. Furthermore, these three mutations led to an increased efficacy of quisqualate without having any effect on its potency. The fact that the residues Trp784 6.48 and Phe787 6.51 are essential equally in antagonism and inverse agonism effects emphasizes again the key role of these residues and the involvement of a common transmembrane network in receptor inactivation by MPEP. Keywords: fenobam, inverse agonist, metabotropic glutamate receptor, MPEP, pharmacophore modelling, phosphoinositide hydrolysis. The major excitatory neurotransmitter in the brain, glutamate, acts at two distinct classes of receptors, ionotropic (NMDA, AMPA and kainate) and metabotropic (mGlu; Pin and Acher 2002). On the basis of sequence homology, second messenger coupling and pharmacology, eight mGlu receptors are divided into three groups: group I (mGlu1 and -5) receptors couple to the activation of phospholipase C leading to phosphoinositide hydrolysis and elevation of intracellular Ca 2+ levels ([Ca 2+ ] i ), group II (mGlu2 and -3) and group III (mGlu4, are negatively coupled to cAMP production (Pin and Acher 2002).

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

“…For these reasons, big chemical efforts have been undertaken to find metabolically stable and safe acetylenic mGlu5 receptor antagonists, to remove the acetylene group by designing analogs maintain the relative geometry of the two aryl rings in MPEP and MTEP (examples shown in Table 3), or to identify completely new scaffolds by high-throughput screening campaigns. A special case in this context is the compound fenobam (Table 3), which was tested in clinical trials as an anxiolytic agent in the late 1970s and much later turned out to be a potent, negative mGlu5 receptor modulator binding to the same allosteric site as MPEP and MTEP (Porter et al, 2005;Malherbe et al, 2006). Fenobam has also become an important starting point for chemistry programs.…”

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).…”

“…Multiple selective tritiated and positron emission tomography (PET) allosteric ligands have been developed for mGlu 1 , mGlu 2 , and mGlu 5 (Anderson et al, 2002(Anderson et al, , 2003Gasparini et al, 2002;Cosford et al, 2003b;Lavreysen et al, 2003;Kohara et al, 2005;Malherbe et al, 2006;Ametamey et al, 2007;Treyer et al, 2007;Baumann et al, 2010). Through the use of inhibition binding assays with these allosteric ligands a direct assessment of competitive versus noncompetitive binding modes can be performed (Lavreysen et al, 2003(Lavreysen et al, , 2004Kinney et al, 2005;Malherbe et al, 2006;Hemstapat et al, 2006;Chen et al, 2007;Lundstrom et al, 2011). Such experiments are often the first step toward identifying allosteric modulators that bind to alternative allosteric sites [ (Chen et al, 2008;Hammond et al, 2010) discussed in further detail later].…”

“…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 .…”

Pharmacological Characterization and Modeling of the Binding Sites of Novel 1,3-Bis(pyridinylethynyl)benzenes as Metabotropic Glutamate Receptor 5-Selective Negative Allosteric Modulators

“…With the lack of target crystal structures, mutational studies combined with homology modeling and/or ligand analog series represent a powerful tool for obtaining important structural information regarding the identity and spatial arrangement of key residues essential for ligand-receptor interactions (Ballesteros and Palczewski, 2001;Gregory et al, 2011). This approach has previously been used for mGluR5 in delineation of the binding site of 2-methyl-6-(phenylethynyl)pyridine (MPEP), a selective negative allosteric modulator (NAM) (Pagano et al, 2000;Malherbe et al, 2003), as well as for other allosteric modulators Malherbe et al, 2006;Chen et al, 2007Chen et al, , 2008.…”