Molecular basis defining the selectivity of substituted isoquinolinones for the melatonin MT2 receptor
Molecular basis defining the selectivity of substituted isoquinolinones for the melatonin MT2 receptor
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DOI：
10.1016/j.bcp.2020.114020
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发表时间：
2020-07-01
影响因子：
5.8
通讯作者：
Wong, Yung H.
中科院分区：
文献类型：
--
作者：
Chan, King H.;Tse, Lap H.;Wong, Yung H.
褪黑素 MT1 和 MT2 受体代表了治疗各种疾病的有吸引力的药物靶点。然而，褪黑激素结合袋的高度保守性阻碍了亚型选择性化合物的开发。通过利用最近解析的 MT1 和 MT2 受体的晶体结构，本研究旨在阐明一组异喹啉酮衍生物的 MT2 选择性的结构基础。采用分子建模和配体对接方法来预测参与与 MT2 选择性异喹啉酮形成相互作用的残基。选择七个保守残基（Asn（175）、His（208）、Trp（264）、Asn（268）、Gly（271）、Tyr（294）和Tyr（298））作为定点诱变的靶标。进行 Ca2+ 动员、cAMP 抑制、细胞外信号调节激酶的磷酸化和配体结合测定，以对转染的 CHO 细胞中的受体突变体进行功能表征。与褪黑激素不同，带有 3-甲氧基苄氧基取代基的异喹啉酮不受 MT2 的 His(208) 处丙氨酸取代的影响。尽管 Tyr(294) 或 Tyr(298) 的丙氨酸取代降低了褪黑激素和一些异喹啉酮对 MT2 的效力，但 MT1 上的类似突变允许五种迄今为止无效的异喹啉酮充当激动剂。带有 4-甲氧基苄氧基部分的异喹啉酮拮抗剂在 His(208)、Tyr(294) 或 Tyr(298) 处被丙氨酸取代的 MT2 突变体中转变为激动剂。残基的子集显然参与形成疏水性结合腔以赋予异喹啉酮化合物的芳香族取代基选择性。跨膜螺旋 7 上的两个保守酪氨酸残基可能赋予 MT1 和 MT2 受体配体选择性，而跨膜螺旋 5 上的保守组氨酸显然参与受体激活。
Melatonin MT1 and MT2 receptors represent attractive drug targets for the treatment of various disorders. However, the high conservation of the melatonin binding pocket has hindered the development of subtype-selective compounds. By leveraging on the recently resolved crystal structures of MT1 and MT2 receptors, this study aims to elucidate the structural basis of MT2-selectivity of a panel of isoquinolinone derivatives. Molecular modelling and ligand docking approaches were employed to predict residues involved in forming interactions with the MT2-selective isoquinolinones. Seven conserved residues (Asn(175), His(208), Trp(264), Asn(268), Gly(271), Tyr(294) and Tyr(298)) were selected as targets for site-directed mutagenesis. Ca2+ mobilization, cAMP inhibition, phosphorylation of extracellular signal-regulated kinase, and ligand binding assays were performed to functionally characterize the receptor mutants in transfected CHO cells. Unlike melatonin, isoquinolinones bearing a 3-methoxybenzyloxyl substituent were unaffected by alanine substitution at His(208) of MT2. Although alanine substitutions at Tyr(294) or Tyr(298) reduced the potency of melatonin and some isoquinolinones on MT2, similar mutations on MT1 allowed five hitherto ineffective isoquinolinones to act as agonists. An isoquinolinone antagonist bearing a 4-methoxybenzyloxyl moiety turned into an agonist at MT2 mutants with alanine substitutions at His(208), Tyr(294) or Tyr(298). A subset of residues is apparently involved in forming a hydrophobic binding cavity to confer selectivity upon the aromatic substituent of isoquinolinone compounds. Two conserved tyrosine residues on transmembrane helix 7 may confer ligand selectivity at MT1 and MT2 receptors, while a conserved histidine on transmembrane helix 5 is apparently involved in receptor activation.