Wednesday, August 1, 2007 - 10:30 AM
S146
Novel approaches for antibiotic discovery
S. Singh, Merck Research Laboratories, Rahway, NJ
FabH and FabF are essential enzymes in type II fatty acid synthesis and are promising targets for antibacterial drug discovery and development. A new approach using a xylose inducible plasmid to express antisense RNA (AS-RNA) in Staphylococcus aureus has been recently described. In order to identify FabF/FabH target specific cell permeable inhibitors from natural products, we developed an agar-diffusion two-plate differential sensitivity assay. Because both the fabH and fabF genes share the same operon, the increase in fabF AS-RNA levels decreases the expression of FabH and FabF proteins, making the cells more sensitive to FabF and/or FabH inhibitors. In this assay, natural product extracts are applied on two plates, one seeded with S. aureus cells with expression of fabF AS-RNA (AS plate) and the other without expression of AS-RNA (control plate). The extracts that contain selective inhibitors for either of the two targets will form a larger inhibition zone on the AS plate compared to the control plate. The assay was validated with ~80 known antibiotics including fatty acid synthesis inhibitors. Using this assay, we screened over 250,000 natural product extracts followed by confirmation in biochemical assays, giving a hit rate of 0.1%. We discovered a number of novel natural products as FabF inhibitors including platensimycin and platencin. The details of discovery process, structures, biological activities, in vivo efficacy, mechanism of action and inhibitor bound X-ray crystal structure will be discussed.