A discovery funnel for nucleic acid binding drug candidates

Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. Although in silico screens targeting proteins have proved successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many noncanonical DNA structures such as G-quadruplexes, increased efforts are under way to discover new small molecules that can bind selectively to DNA structures. In the present work, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high-throughput 96-well thermal denaturation assay to yield the top 10 candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy, and competition dialysis. This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti-cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed.