Identification of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Acting at Site Distinct from 2-Methyl-6-(phenylethynyl)-pyridine Binding

As part of the G-protein coupled receptor (GPCR) family, metabotropic glutamate (mGlu) receptors play an important role as drug targets of cognitive diseases. Selective allosteric modulators of mGlu subtype 5 (mGlu₅) have the potential to alleviate symptoms of numerous central nervous system disorders such as schizophrenia in a more targeted fashion. Multiple mGlu₅ positive allosteric modulators (PAMs), such as 1-(3-fluorophenyl)-N-((3-fluorophenyl)-methylideneamino)-methanimine (DFB), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (CDPPB), and 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (VU-29), exert their actions by binding to a defined allosteric site on mGlu₅ located in the seven-transmembrane domain (7TM) and shared by mGlu₅ negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Actions of the PAM N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) are mediated by a distinct allosteric site in the 7TM domain different from the MPEP binding site. Experimental evidence confirms these findings through mutagenesis experiments involving residues F585 (TM1) and A809 (TM7). In an effort to investigate mGlu₅ PAM selectivity for this alternative allosteric site distinct from MPEP binding, we employed in silico quantitative structure-activity relationship (QSAR) modeling. Subsequent ligand-based virtual screening prioritized a set of 63 candidate compounds predicted from a library of over 4 million commercially available compounds to bind exclusively to this novel site. Experimental validation verified the biological activity for seven of 63 selected candidates. Further, medicinal chemistry optimizations based on these molecules revealed compound VU6003586 with an experimentally validated potency of 174 nM. Radioligand binding experiments showed only partial inhibition at very high concentrations, most likely indicative of binding at a non-MPEP site. Selective positive allosteric modulators for mGlu₅ have the potential for tremendous impact concerning devastating neurological disorders such as schizophrenia and Huntington's disease. These identified and validated novel selective compounds can serve as starting points for more specifically tailored lead and probe molecules and thus help the development of potential therapeutic agents with reduced adverse effects.