Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence

IF 8.3 1区 医学 Q1 ENDOCRINOLOGY & METABOLISM Journal of Pineal Research Pub Date : 2024-01-31 DOI:10.1111/jpi.12941
Annalida Bedini, Gian Marco Elisi, Fabiola Fanini, Michele Retini, Laura Scalvini, Silvia Pasquini, Chiara Contri, Katia Varani, Gilberto Spadoni, Marco Mor, Fabrizio Vincenzi, Silvia Rivara
{"title":"Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence","authors":"Annalida Bedini,&nbsp;Gian Marco Elisi,&nbsp;Fabiola Fanini,&nbsp;Michele Retini,&nbsp;Laura Scalvini,&nbsp;Silvia Pasquini,&nbsp;Chiara Contri,&nbsp;Katia Varani,&nbsp;Gilberto Spadoni,&nbsp;Marco Mor,&nbsp;Fabrizio Vincenzi,&nbsp;Silvia Rivara","doi":"10.1111/jpi.12941","DOIUrl":null,"url":null,"abstract":"<p>The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[<sup>125</sup>I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure–activity relationships, as the <i>K</i><sub>i</sub> values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[<sup>125</sup>I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT<sub>1</sub> and MT<sub>2</sub> receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[<sup>125</sup>I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT<sub>2</sub> receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured <i>K</i><sub>i</sub> values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure–activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT<sub>2</sub> receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT<sub>2</sub>-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor–ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure–activity relationships and with interactions taken at the receptor binding site.</p>","PeriodicalId":198,"journal":{"name":"Journal of Pineal Research","volume":null,"pages":null},"PeriodicalIF":8.3000,"publicationDate":"2024-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Pineal Research","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/jpi.12941","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
引用次数: 0

Abstract

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure–activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure–activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor–ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure–activity relationships and with interactions taken at the receptor binding site.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
强效褪黑素受体配体的结合与解除结合:机理模拟和实验证据
褪黑激素受体配体竞争结合研究中常用的标记配体 2-[125I]碘褪黑激素在两种受体亚型上的半衰期不同,解离速度缓慢。这可能会影响亲和力常数的操作测量,因为在短孵育时间内无法在平衡条件下获得亲和力常数;也可能会影响结构-活性关系,因为测试配体的 Ki 值可能取决于结合位点的相互作用或解离路径。为了解决这些问题,我们在短孵育时间(2 小时)和长孵育时间(20 小时)下测量了 2-[125I]碘美拉宁的动力学和饱和结合参数以及一系列代表性配体的竞争常数。同时,我们通过分子建模模拟了 2-iodomelatonin 与 MT1 和 MT2 受体的解离路径,并研究了结合位点上的相互作用对亚型选择性配体 UCM1014 对映体立体选择性的影响。我们发现,2-[125I]碘美拉宁的结合只有在较长的孵育时间下才能达到平衡条件,特别是对于 MT2 受体亚型,20 小时的孵育时间近似于这一条件。另一方面,一组配体(包括激动剂、拮抗剂、非选择性和亚型选择性化合物)的测定 Ki 值并未受到孵育时间长短的显著影响,这表明基于较短时间收集的数据的结构-活性关系反映了结合位点的不同相互作用。分子建模模拟证明,2-碘美拉宁与 MT2 受体的解离速度较慢,这可能与守门酪氨酸在从结合位点到膜双分子层的亲脂路径上的流动性受限有关。我们分别合成并测试了强效、MT2 选择性激动剂 UCM1014 的对映体。受体-配体复合物的分子动力学模拟为其立体选择性提供了一种解释,即在结合位点的对映体偏好配体在溶液中采用的最丰富的轴向构象。这些结果表明,尽管标记配体的结合动力学较慢,但在较短的孵育时间内进行的亲和力测量结果可靠,与已知的结构-活性关系和受体结合位点的相互作用相一致。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
Journal of Pineal Research
Journal of Pineal Research 医学-内分泌学与代谢
CiteScore
17.70
自引率
4.90%
发文量
66
审稿时长
1 months
期刊介绍: The Journal of Pineal Research welcomes original scientific research on the pineal gland and melatonin in vertebrates, as well as the biological functions of melatonin in non-vertebrates, plants, and microorganisms. Criteria for publication include scientific importance, novelty, timeliness, and clarity of presentation. The journal considers experimental data that challenge current thinking and welcomes case reports contributing to understanding the pineal gland and melatonin research. Its aim is to serve researchers in all disciplines related to the pineal gland and melatonin.
期刊最新文献
Cover image RETRACTION: Effects of Melatonin on Fatty Liver Disease: The Role of NR4A1/DNA-Pkcs/P53 Pathway, Mitochondrial Fission, and Mitophagy From Seasonality to Species Conservation: Chronobiological Research on European Hamsters in Strasbourg, France Melatonin Inhibits ET-1 Production to Break Crosstalk Between Prostate Cancer and Bone Cells: Implication for Osteoblastic Bone Metastasis Treatment Issue Information
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1