Avlani Vimesh A, McLoughlin David J, Sexton Patrick M, Christopoulos Arthur
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
J Pharmacol Exp Ther. 2008 Jun;325(3):927-34. doi: 10.1124/jpet.108.136978. Epub 2008 Mar 5.
Radioligand binding assays remain a common method for quantifying the effects of allosteric modulators at G protein-coupled receptors. The allosteric ternary complex model (ATCM) is the simplest model applied to derive estimates of modulator affinity (K(B)) and cooperativity (alpha), which are necessary for understanding structure-activity relationships. However, the increasing drive toward assay miniaturization in modern drug discovery may lead to conditions where appreciable ligand depletion occurs in the assay. Theoretical simulations investigating the impact of orthosteric radioligand depletion on the estimation of ATCM parameters revealed the following. 1) For allosteric inhibitors, application of the standard ATCM to data obtained under depletion conditions leads to an underestimation of pK(B) and an overestimation of log alpha. 2) For allosteric enhancers, the opposite was noted, but not always; the nonlinear regression algorithm is more likely to struggle to converge to a satisfactory solution of (nondepletion) ATCM parameters in this situation. 3) Application of a novel ATCM that explicitly incorporates orthosteric ligand depletion will yield more reliable model estimates, provided the degree of depletion is not high (< approximately 50%). Subsequent experiments investigated the interaction between [3H]N-methylscopolamine and the allosteric enhancer, alcuronium, or inhibitor, gallamine, in the presence of increasing concentrations of M(2) muscarinic acetylcholine receptor and showed that application of an ATCM that explicitly incorporates radioligand depletion can indeed give more robust estimates of modulator affinity and cooperativity estimates than the standard model. These results have important implications for the quantification of allosteric modulator actions in binding-based discovery assays.
放射性配体结合测定法仍然是一种用于量化变构调节剂对G蛋白偶联受体作用的常用方法。变构三元复合物模型（ATCM）是用于推导调节剂亲和力（K(B)）和协同性（α）估计值的最简单模型，而这些对于理解构效关系是必不可少的。然而，现代药物发现中对检测小型化的需求日益增加，可能会导致检测中出现明显的配体耗竭情况。研究正构放射性配体耗竭对ATCM参数估计影响的理论模拟结果如下：1）对于变构抑制剂，在耗竭条件下将标准ATCM应用于所获得的数据会导致pK(B)的低估和logα的高估。2）对于变构增强剂，情况则相反，但并非总是如此；在这种情况下，非线性回归算法更有可能难以收敛到（非耗竭）ATCM参数的满意解。3）应用一种明确纳入正构配体耗竭的新型ATCM将产生更可靠的模型估计值，前提是耗竭程度不高（<约50%）。随后的实验研究了在M(2)毒蕈碱型乙酰胆碱受体浓度增加的情况下，[3H]N-甲基东莨菪碱与变构增强剂阿库氯铵或抑制剂加拉明之间的相互作用，结果表明，与标准模型相比，应用明确纳入放射性配体耗竭的ATCM确实可以对调节剂亲和力和协同性估计值给出更可靠的结果。这些结果对于基于结合的发现检测中变构调节剂作用的量化具有重要意义。
