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首页> 外文期刊>Biochemical Pharmacology >Impact of allosteric modulation: Exploring the binding kinetics of glutamate and other orthosteric ligands of the metabotropic glutamate receptor 2
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Impact of allosteric modulation: Exploring the binding kinetics of glutamate and other orthosteric ligands of the metabotropic glutamate receptor 2

机译：变构调节的影响：探索谷氨酸谷氨酸的结合动力学和代谢谷氨酸受体2的其他矫形配体2

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While many orthosteric ligands have been developed for the mGlu2receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e.konandkoff, of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric modulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay.We found that dissociation rate constants of orthosteric mGlu2ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu2ligands iskon-drivenin vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on- and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency.In conclusion, we found that affinity- or potency-based orthosteric ligand optimization primarily results in ligands with highkonvalues. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasingkoff, which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacyin vivo.
机译：虽然已经为MGLU2重复开发了许多骨癌配体，但对于它们的靶结合动力学知之甚少，并且这些含有结合动力学以及与内源激动剂谷氨酸的那些人有何相关。这里，首次测定谷氨酸的动力速率常数，即谷氨酸的谷氨酸盐，其次是合成激动剂Ly354740和拮抗剂Ly341495。为了提高对末端调节的结合机制和对变构调制的影响，在颠覆性调节剂存在下重复动力学实验。进行功能测定以进一步研究矫形和变构结合位点之间的相互作用，包括阻抗的形态测定。我们发现矫形率mglu2ligands的解离速率常数在小6倍范围内，而关联率常数是测距的超过三个数量级，与亲和力和效力相关。后者表明，靶标mglu2ligands iskon-drivenin的靶啮合。此外，仅通过阳性变构调制器（PAM）降低了两个激动剂的速率，从而增加了它们的亲和力。有趣的是，PAM增加了谷氨酸诱导的细胞反应的持续时间。阴性变形调节剂（NAM）在不改变其亲和力的情况下增加谷氨酸和偏移和偏移，同时它不会影响LY354740的这些参数，表明探针依赖性。在结论中，我们发现，我们发现亲和力或效力的矫形症状配体优化主要导致配体具有高kon值。此外，阳性变构调节剂主要通过减少来改变正向激动剂的结合动力学，其能够与延长的细胞反应相关。这项研究在一起表明，在早期药物发现中研究结合动力学的重要性，这可能对改善效率的体内提供重要的见解。

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