Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu₅) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu₅ PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu₅ PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu₅ using Ca²⁺ mobilization, inositol monophosphate (IP₁) accumulation, extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca²⁺ mobilization over IP₁ accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca²⁺ mobilization over IP₁ accumulation. Our characterization of three structurally diverse mGlu₅ PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu₅ internalization.

中文翻译：

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代谢型谷氨酸受体5的正变构调节剂作为研究信号偏倚的工具化合物

代谢型谷氨酸亚型5（mGlu ₅）受体的正变构调节已成为治疗精神分裂症和认知障碍的潜在新治疗策略。但是，积极的变构调节剂（PAM）激动剂活性已与不良副作用相关联，并且也已观察到纯PAM的神经毒性。mGlu ₅ PAM对体内的治疗作用与不良作用的结构和药理基础仍然未知。因此，深入了解信号指纹，以及结构多样的mGlu ₅ PAM的结合动力学，可能有助于合理设计具有所需特性的化合物。我们评估了N的结合和信号传导特征-甲基-5-（苯基乙炔基）嘧啶-2-胺（MPPA），3-氰基N-（2,5-二苯基吡唑-3-基）苯甲酰胺（CDPPB）和1- [4-（4-氯- 2-氟-苯基）哌嗪-1-基] -2-（4-吡啶甲氧基）乙烯酮[化合物2c，1-（4-（2-氯-4-氟苯基）哌嗪-1-基）-的相似物人胚肾293A细胞中的2-（吡啶-4-基甲氧基）乙酮]通过Ca ²⁺动员肌醇一磷酸（IP _1）稳定表达mGlu ₅）积累，细胞外信号调节激酶1/2（ERK1 / 2）磷酸化和受体内在化分析。在这三个变构配体中，只有CDPPB具有内在的激动剂功效，并且它还具有最长的受体停留时间和最高的亲和力。MPPA是一种偏向PAM，在IP ₁积累和受体内在化方面，ERK1 / 2磷酸化和Ca ²⁺动员方面，与正构激动剂表现出更高的正协同性。在原代皮层神经元中，所有三个PAM在IP ₁积累上的Ca ²⁺动员中均表现出与（S）-3,5-二羟基苯基甘氨酸（DHPG）较强的正协同性。我们对三种结构多样的mGlu _5的表征PAM提供了进一步的分子药理学见解，并提出了对PAM介导的mGlu ₅内在化的首次评估。