SpPurC Hit Selection Optimization and Evaluation of Benzimidazole-based Inhibitors of S. aureus FabI

This dissertation includes work on three projects. The first project was focused on conducting a high-throughput screen (HTS) with a 25000 compound library to discover inhibitors of the PurC enzyme from S. pneumoniae. In addition to optimizing the malachite green assay for high throughput screening, a compound interference assay was also used to eliminate false positive HTS hits with a single step. This method was also validated by a 93% agreement in results from the interference assay and the continuous pyruvate kinase/lactate dehydrogenase assay, which was used as a secondary assay for hit validation. The second project describes the characterization of a previously published series of benzimidazole-based inhibitors of the FabI enzyme from F. tularensis (FtFabI) to evaluate their potential for anti-staphylococcal drug discovery. A preliminary structure-activity relationship (SAR) study showed that some of the benzimidazole-based compounds are potent inhibitors of S. aureus FabI (SaFabI). Important SaFabI residues that are critical for the inhibitory activity of these compounds have been identified through mutagenesis studies. This work also confirms that the observed antibacterial activity of three of the benzimidazole-based SaFabI inhibitors from this preliminary series, is a result of direct FabI inhibition (on-target effect). The third project evaluated the potential of the benzimidazole-based compounds for developing anti-malarials. Contrary to the hypothesis, these benzimidazole-based inhibitors of FtFabI and SaFabI were found to be inactive against the FabI enzyme from Plasmodium falciparum. A potential reason for this lack of observed activity has been discussed to drive this project further in the future.