Discovery of novel inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH)

Fatty acid biosynthesis by type II fatty acid synthase (FAS) is a fundamental metabolic pathway in bacteria. The FAS components exist as discrete monofunctional proteins which contrast distinctively with the type I FAS in humans, where all the enzyme activities are encoded in a single multifunctional polypeptide. The essentiality of type II FAS for cell viability and its distinguishing organizational characteristics make it an attractive target for the development of novel broad-spectrum antibiotics. In the type II system, β-ketoacyl-acyl carrier protein synthase III, also known as KASIII or FabH, is an essential condensing enzyme central to the initiation and regulation of the entire pathway. In the face of rising worldwide multidrug-resistant infections, this enzyme has emerged as a prime target for novel antibacterial agents.; Most of the initial studies were focused on the model FabH enzyme from E. coli. In this study we carried out research on a series of FabHs from clinically relevant microorganisms such as Mycobacterium tuberculosis, Staphylococcus aureus and Plasmodium falciparum. These investigations have greatly expanded our knowledge on the FabH substrate specificities. Thiolactomycin (TLM) and its analogues are the only known class of specific FabH inhibitors. The limited number of FabH inhibitors and their poor potency prompted us to initiate a program to discover more potent and novel inhibitors. Taking advantage of crystal structure information of E. coli and the complex of thiolactomycin with a FabH homologue, both ligand and receptor-based in silico screenings were performed on National Cancer Institute (NCI) database. A number of FabH inhibitors with novel structures were identified. Enzyme assays demonstrated that they had much improved inhibitory activities over TLM. Molecular modeling studies were applied to probe the interactions between the most potent inhibitors and FAR FabH mutants of the important residues identified in these studies were generated. Subsequent experiments with the inhibitors and these mutants provided strong evidence supporting the predicted binding model. These compounds represent promising lead compounds for further FabH inhibitor design. A scintillation proximity assay (SPA), readily amenable to automation for the high-throughput screening of novel FabH inhibitors, was also developed.