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Discovery of FabH/FabF Inhibitors from Natural Products

Katherine Young, Hiranthi Jayasuriya, John G. Ondeyka, Kithsiri Herath, Chaowei Zhang, Srinivas Kodali, Andrew Galgoci, Ronald Painter, Vickie Brown-Driver, Robert Yamamoto, Lynn L. Silver, Yingcong Zheng, Judith I. Ventura, Janet Sigmund, Sookhee Ha, Angela Basilio, Francisca Vicente, José Rubén Tormo, Fernando Pelaez, Phil Youngman, Doris Cully, John F. Barrett, Dennis Schmatz, Sheo B. Singh, and Jun Wang^*

Merck Research Laboratories, Rahway, New Jersey 07065

Received 26 September 2005/ Returned for modification 2 November 2005/ Accepted 30 November 2005

Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5' mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.

Present address: Rx3 Pharmaceuticals, 6310 Nancy Ridge Dr. #105, San Diego, Calif.

Present address: LL Silver Consulting, 3403 Park Place, Springfield, N.J.

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