The purpose of this chapter is to describe how to measure the kinetics of target-ligand interaction, specifically how to analyze binding kinetic data to measure the binding rate constants. Useful introductory webinars are available here and here. They also impact quantification of other drug parameters, such as affinity, resulting in erroneous estimates of activity if the rates are not considered appropriately ( 17- 19). They can impact the therapeutic action and side effect profile of the ligand, as described in numerous recent reviews ( 1- 16). The binding rates are fundamental properties of the drug-target interaction. 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. More complex scenarios are then introduced, including multistep binding interactions and the use of functional assays to quantify ligand binding kinetics. 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.
Recently, indirect competition binding methods have become popular for evaluating binding kinetics of the large numbers of compounds encountered in drug discovery. The concepts are introduced using simple, direct target-ligand binding assays. Data are analyzed by curve fitting using familiar nonlinear regression data analysis programs (exemplified here with Prism from GraphPad Software). This chapter describes how to analyze these time course data to determine the binding kinetic rate constant values. Experimentally the rates are determined by measuring the time course of test compound binding to the target, directly or indirectly. Knowledge of these rates can be important in the optimization of new therapeutics and in understanding the biological behavior of target proteins. These processes are quantified by the association and dissociation rate constants. 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. Biological and therapeutic agents exert their actions by interacting with specific molecular targets.
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