Moderate to high throughput in vitro binding kinetics for drug discovery.

Abstract

This review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds. For seconds to hours timescale kinetics we discuss surface plasmon resonance-based biosensor, global progress curve analysis for high throughput kinetic profiling of enzyme inhibitors and activators, and filtration plate-based radioligand or fluorescent binding assays for receptor binding kinetics. Jump dilution after pre-incubation is the preferred method for very slow kinetics lasting for days. The basic principles, best practices and simulated data for these technologies are described. Finally, the application of a universal label-free technology, liquid chromatography coupled tandem mass spectrometry (LC/MS/MS), is briefly reviewed. Select literature referencesare highlighted for in-depth understanding. A new reality is dawning wherein binding kinetics is an integral and routine part of mechanism of action elucidation and translational, quantitative pharmacology for drug discovery.