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CELLULAR AND MOLECULAR

The Impact of Orthosteric Radioligand Depletion on the Quantification of Allosteric Modulator Interactions

Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (V.A.A., P.M.S., A.C.); and High Throughput Screening Centre of Emphasis, Pfizer, Sandwich Laboratories, Sandwich, United Kingdom (D.J.M.)

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 (), 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 . 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 (<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 M₂ 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.

Address correspondence to: Dr. Arthur Christopoulos, National Health and Medical Research Council Senior Research Fellow, Department of Pharmacology, Monash University, Clayton, 3800 Victoria, Australia. E-mail: arthur.christopoulos{at}med.monash.edu.au