Equilibrium competition binding assay: inhibition mechanism from a single dose response.

Inhibition of a receptor by a small-molecule compound in many cases is achieved via a competitive, uncompetitive or non-competitive mechanism. The receptor-inhibitor interaction is often probed through the displacement of a ligand in an equilibrium competition binding experiment. The previous solutions to receptor inhibition mechanisms were borrowed from steady-state enzyme inhibition mechanisms. The inhibition mechanism is determined by a visual inspection or a global fit of ligand dose response curves at a series of inhibitor concentrations. However these solutions only apply to situations when both the ligand and the inhibitor are not significantly depleted by the receptor. In most published equilibrium receptor binding studies, only the relative potency of the inhibitor is calculated. Ranking inhibitors tested under differing experimental conditions is often not possible. In the current paper, we offer exact mathematical solutions to uncompetitive and non-competitive inhibition, and demonstrate that in most cases both the inhibition mechanism and absolute potency of an inhibitor can be simultaneously determined from a single dose response of the inhibitor at a fixed concentration of the ligand. Therefore, an equilibrium competition assay provides a quick and facile method to determine the inhibition mechanism of a large number of inhibitors. The theory herein described is applicable to equilibrium competition binding experiments such as radioligand assays and fluorescence polarization assays.