Mechanisms of Orthosteric Agonist and Allosteric Modulator Activity at mGlu 7 .

Metabotropic glutamate receptor 7 (mGlu ) is a dimeric, group III metabotropic glutamate (mGlu) receptor. It is a G protein-coupled receptor that acts to modulate neurotransmission across many brain structures. mGlu is most highly expressed presynaptically in neurons and is widely distributed in the central nervous system (CNS) and body. Mutations, deletions, or decreases in mGlu result in symptoms and phenotypes of neurodevelopmental disorders in humans and mice, including Rett syndrome. Our group is focused on developing new ligands that interact with mGlu to understand the therapeutic potential of the receptor in various CNS disorders. However, in vitromolecular pharmacology studies show that the orthosteric ligand, glutamate, has a low affinity for mGlu that limits its ability to be routinely used for drug screening. An alternative agonist, L-AP4, is a synthetic compound that, while not produced naturally in vivo, activates mGlu with higher affinity than glutamate. However, our preliminary research indicates that there are differences in the profiles of positive allosteric modulators (PAMs) when assessed using glutamate versus L-AP4. This difference creates a challenge for the development of mGlu -selective PAMs, and the focus of our current studies is to understand the difference between these two agonists in activating the mGlu receptor. The mGlu receptors function as constitutive dimers. Here, we hypothesize that glutamate and L-AP4 activate mGlu either via a different number of agonist binding sites or via distinct effector interactions; additionally, we hypothesize that mGlu potentiation by structurally distinct PAMs may differ based on these agonists. We have found that two agonist binding sites are required for mGlu activation for both glutamate and L-AP4, and two G-protein/effector binding sites are required for any glutamate-dependent activation. L-AP4, however, can partially activate the dimer with one functioning G-protein binding site, and two G-protein binding sites fully activate mGlu with L-AP4. The PAM VU0422288 requires two agonist binding sites to potentiate mGlu ; in contrast, VU0155094 and VU6005469 show partial potentiation of mGlu if one of the agonist binding sites is mutated. VU0422288 induces partial potentiation of mGlu with only one functional G-protein binding site and requires two G-protein binding sites to induce full potentiation. In contrast, other PAMs (VU0155094 and VU6005469) can fully potentiate mGlu activity with one functioning G-protein binding site. Additionally, VU0422288 also shows elevated efficacy with L-AP4 compared to glutamate while the other two PAMs do not show differences in efficacy between these two agonists. These studies suggest that mGlu can be differentially regulated by various agonists and PAMs and provide insight into considerations for chemical optimization campaigns to generate new allosteric ligands for the receptor.