Experimental validation of in silico target predictions on synergistic protein targets

Abstract

Two relatively recent trends have become apparent in current early stage drug discovery settings: firstly, a revival of phenotypic screening strategies and secondly, the increasing acceptance that some drugs work by modulating multiple targets in parallel (‘multi-target drugs’). The work presented here combines both those aspects by integrating experimental phenotypic screening for cytotoxic compounds with an experimental validation of individual protein targets predicted in silico. In this first step of this work, in silico target predictions for a dataset comprising cytotoxic compounds showed an enrichment of enzymes involved in cell cycle progression (such as Topoisomerase I, Bcl-X and Protein Kinase C alpha) as well as in the defense against xenobiotic compounds (such as P-gp 1 and the CYPs). Ten compounds predicted to be active on each of two of the enriched targets, P-glycoprotein 1 and Topoisomerase I, were tested in vitro to validate (or invalidate) the predicted mode of action. Hoechst 33342 dye uptake, P-gp ATPase activity and Topoisomerase I DNA relaxation assays were able to identify two inhibitors of P-gp with IC50 values of 37 ± 5 and 28 ± 2 μM, respectively (comparable to the activity of Verapamil of 12 μM measured with the same assay) as well as five moderate inhibitors of Topoisomerase I. Furthermore, we also screened combinations of compounds with different modes of action to evaluate possible synergistic effects. When evaluating compound synergies, four of the five compounds exhibit synergistic effects in HeLa cell cultures in the presence of the two P-gp inhibitors identified (two independent samples t-test, p < 0.01). Hence, this appears to be one of the first studies where multiple aspects of compound action as predicted by in silico models are prospectively validated, namely phenotypic effect as well as on-target activities, and where synergies between compound combinations could also be experimentally confirmed.