Mechanistic studies of potential drug targets against methicillin-resistant Staphylococcus aureus

Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the most common causative agents for hospital-acquired infections. The recent emergence of MRSA strains that are resistant to all current antibiotics, has caused a further increase in the pressing need to discover new drugs against this bacterium.;Fatty acids are essential components of the bacterial cell membrane. saFabI, the enoyl ACP reductase from S. aureus, catalyzes the rate-limiting step in the fatty acid biosynthesis pathway, and is a validated target for drug discovery. Detailed kinetic studies reveal that saFabI is NADPH-dependent, and has a preference for ACP-linked substrates with a long acyl chain. The preference for NADPH is determined by residues R40 and K41, while product inhibition studies indicate that the reaction proceeds via an ordered sequential mechanism. According to the preincubation inhibition analysis, triclosan and two related diphenyl ethers are all nanomolar slow-onset inhibitors of saFabI, and bind preferentially to the enzyme-NADP+ complex. Three saFabI mutations, A95V, I193S, and F204S, were identified upon selection for resistance. Strains containing these mutations have MIC values 100-fold higher than that of the wildtype strain, whereas the mutant enzymes have Ki values 5 to 3000-fold larger than that of wild-type enzyme, supporting the hypothesis that saFabI is the intracellular target of the inhibitors.;The redox active lipid soluble cofactor menaquinone is an essential component of the electron transport chain and oxidative phosphorylation in some prokaryotes, including S. aureus. Disruption of menD, one of the genes hypothesized to function in the S. aureus menaquinone biosynthetic pathway, resulted in a small colony variant (SCV) phenotype, suggesting that menD functions in menaquinone biosynthesis. Ultrastructural studies of this mutant strain reveal that menaquinone may participate directly or indirectly in lipoteichoic acid. It is reasonable since the synthesis of phosphatidyl glycerol, a precursor of lipoteichoic acid, requires electron transport. The mechanistic properties of MenD from S. aureus (saMenD), a thiamin-diphosphate enzyme, have been explored. 2-Succinyl-5-enoylpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid is shown to be the product of the enzymatic reaction, instead of the earlier proposed 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid. In addition, Mn 2+ inhibits the saMenD reaction at high concentrations while Mg 2+ does not, probably due to different effects of the metal ions on the enzyme secondary structure, as revealed by the CD spectra. Finally, the role of the residues in the substrate-binding pocket has been explored by site-directed mutagenesis and enzyme kinetics.;Mechanistic studies of MenB, the naphthoate synthase from S. aureus, have also been performed. Previous work identified mutations in menB genes in clinical S. aureus SCV isolates. Kinetic analysis of these mutant saMenB suggests that the mutations abolish enzyme activity, indicating that the menquinone biosynthesis is blocked in clinical SCVs. Inhibitory activities of a series of O-succinyl benzoic acid (OSB) analogues against saMenB and S. aureus has been studied. Daptomycin, a novel antibiotic, which contains an OSB-like moiety, may target saMenB, since the morphological changes on S. aureus caused by daptomycin treatment and defect in menaquinone biosynthesis are similar.