Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia.
Basic Clin Pharmacol Toxicol. 2020 Jun;126 Suppl 6:104-115. doi: 10.1111/bcpt.13239. Epub 2019 May 7.
Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of "selective" allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu demonstrated that monellin agonism was not mediated via the "common" allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu agonism was positively modulated by the mGlu pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.
变构调节剂结合的位点与正位配体不同，从而可以更好地控制受体的时空分布，并提高亚型选择性。然而，我们最近发现，以前被归类为选择性作用于某些 C 类 G 蛋白偶联受体（GPCR）的变构配体，在某些情况下对该类中的其他非靶标受体具有未被认识到的活性，亲和力更高。在这里，我们扩展了对“选择性”变构配体对甜味受体的非靶标活性的研究。使用代谢型谷氨酸受体 5（mGlu5）作为 C 类 GPCR 的代表，我们评估了甜味蛋白莫内林和小分子人工甜味剂 NHDC。我们发现，莫内林而非 NHDC 是 mGlu5 的激动剂。在表达 N 端截断 mGlu5 的细胞中进行的放射性配体结合和功能测定表明，莫内林激动作用不是通过跨膜域中的“共同”变构结合位点介导的，而是需要 mGlu5 的大细胞外 N 端域的存在。莫内林与正位配体显示出中性功能协同作用。然而，莫内林在细胞内钙测定中正向调节 mGlu 正向变构调节剂 VU0424465 的活性，但在肌醇磷酸盐积累测定中相互作用为中性。此外，莫内林正向调节 mGlu 纯变构调节剂 VU0360172 的活性。总之，这些数据表明，莫内林是 mGlu5 的变构激动剂，与 N 端的变构结合位点结合，该结合位点以偏向的方式与共同的 C 类 GPCR 变构结合位点在功能上相关。这是第一个证明天然来源的蛋白变构配体对 mGlu 受体家族的证据。
