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 K_i 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 K_i 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.

中文翻译：

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有效褪黑激素受体配体的结合和解除结合：机制模拟和实验证据

褪黑激素受体配体竞争结合研究中常用的标记配体，2-[ ¹²⁵ I]碘褪黑素，在两种受体亚型上表现出缓慢的解离，半衰期不同。这可能会影响亲和常数的操作测量（在短孵育时间内无法在平衡条件下获得）和结构-活性关系，因为测试配体的K _i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，在短（2小时）和长（20小时）孵育时间下测量了2-[ ¹²⁵ I]碘褪黑素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-碘褪黑素从 MT ₁和 MT ₂受体的解离路径，并研究了结合位点的相互作用对亚型选择性配体 UCM1014 的对映体观察到的立体选择性的作用。我们发现，2-[ ¹²⁵ I]碘褪黑激素结合的平衡条件只有在较长的孵育时间下才能达到，特别是对于MT ₂受体亚型，20 小时的时间接近该条件。另一方面，测量的一组配体（包括激动剂、拮抗剂、非选择性和亚型选择性化合物）的K _i值并未受到孵育时间的显着影响，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。分子模型模拟证明，2-碘褪黑激素从 MT ₂受体解离速度较慢可能与看门人酪氨酸沿着从结合位点到膜双层的亲脂路径的受限移动性有关。单独合成并测试了有效的MT ₂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解释，因为结合位点的优异构体对溶液中配体采用的最丰富的轴向构象表现出偏好。这些结果表明，尽管标记配体的结合动力学缓慢，但在较短的孵育时间内进行的亲和力测量给出了与已知的结构-活性关系以及在受体结合位点处发生的相互作用一致的稳健结果。