Young Katherine, Jayasuriya Hiranthi, Ondeyka John G, Herath Kithsiri, Zhang Chaowei, Kodali Srinivas, Galgoci Andrew, Painter Ronald, Brown-Driver Vickie, Yamamoto Robert, Silver Lynn L, Zheng Yingcong, Ventura Judith I, Sigmund Janet, Ha Sookhee, Basilio Angela, Vicente Francisca, Tormo José Rubén, Pelaez Fernando, Youngman Phil, Cully Doris, Barrett John F, Schmatz Dennis, Singh Sheo B, Wang Jun
Merck Research Laboratories, Rahway, NJ 07065, USA.
Antimicrob Agents Chemother. 2006 Feb;50(2):519-26. doi: 10.1128/AAC.50.2.519-526.2006.
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.
缩合酶在II型脂肪酸合成中至关重要，是抗菌药物研发的潜在靶点。最近，有人描述了一种利用木糖诱导型质粒在金黄色葡萄球菌中表达反义RNA的新方法；然而，其实际机制尚未阐明。在本文中，利用Northern印迹法研究了反义RNA表达导致靶蛋白产量降低的机制。结果表明，反义RNA通过靶向mRNA在转录后发挥作用，导致mRNA 5'端降解。利用该技术，开发了一种双平板测定法，通过筛选天然产物提取物来鉴定FabF/FabH靶标特异性细胞可渗透抑制剂。对超过250,000种天然产物发酵液进行了筛选，然后通过生化测定进行确认，命中率为0.1%。所有已知的天然产物FabH和FabF抑制剂，包括浅蓝菌素、硫代乳霉素、硫代四霉素和Tü3010，都是使用这种基于全细胞机制的筛选方法发现的。还发现了作为FabF新抑制剂的腐殖酸。这些新抑制剂在凝胶延伸测定和基于全细胞的双平板测定中表现出靶标选择性。腐殖酸C对金黄色葡萄球菌显示出良好的抗菌活性，比硫代乳霉素和浅蓝菌素高约20倍。它对包括耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌和流感嗜血杆菌在内的临床重要病原体具有抗菌活性谱。
