Analyzing Kinetic Binding Data

Excerpt

Biological and therapeutic agents exert their actions by interacting with specific molecular targets. This process of ligand binding takes time, for the ligand to associate with the target, forming the target-ligand complex, and for the complex to break down. These processes are quantified by the association and dissociation rate constants. Knowledge of these rates can be important in the optimization of new therapeutics and in understanding the biological behavior of target proteins. Experimentally the rates are determined by measuring the time course of test compound binding to the target, directly or indirectly. This chapter describes how to analyze these time course data to determine the binding kinetic rate constant values. Data are analyzed by curve fitting using familiar nonlinear regression data analysis programs (exemplified here with Prism from GraphPad Software). The concepts are introduced using simple, direct target-ligand binding assays. Recently, indirect competition binding methods have become popular for evaluating binding kinetics of the large numbers of compounds encountered in drug discovery. Data analysis for this “Competition kinetics” approach is presented using a step-by-step guide, together with instruction on troubleshooting, limits of sensitivity and experimental artifacts. More complex scenarios are then introduced, including multistep binding interactions and the use of functional assays to quantify ligand binding kinetics. By describing how to quantify ligand binding kinetics and interpret the data, this chapter enables investigators to evaluate the role of the temporal dimension of binding activity to their targets of interest.