Ligand Screening Using Enzymatic Assays

Abstract

An important aspect of enzymatic assays is that the effect of a ligand on enzyme activity is readily apparent and quantifiable. Enzyme-based assays are, therefore, highly amenable to high-throughput ligand screening, which profiles the effect of a panel of small molecules on a designated target. In order for enzyme assays to provide useful screening data, the kinetics, assay components, readout signal, and overall stability of the assay are optimized and adapted to the equipment prior to the screen. For the screen itself, careful consideration is given to the number of replicates, the plate layout, the compound concentration, and the details of assay assembly. Lastly, in the post-screen stages, the ligand screening data is processed and analyzed using various strategies, and the resulting preliminary hits are subjected to a series of secondary and tertiary assays to eliminate false positives and poor quality hits. The various stages of screening are described, using a viral protease, NS3/4A from Hepatitis C virus, as an example of an enzyme target.