Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived...

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4_S × 4_I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC₅₀-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC₅₀-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.