Identification of a small-molecule inhibitor of bacterial AraC family activators.
Identification of a small-molecule inhibitor of bacterial AraC family activators.
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DOI：
10.1177/1087057112474690
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发表时间：
2013-06
影响因子：
--
通讯作者：
Egan SM
中科院分区：
文献类型：
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作者：
Skredenske JM;Koppolu V;Kolin A;Deng J;Kettle B;Taylor B;Egan SM
细菌转录激活剂AraC家族的蛋白成员在新型抗菌药物的开发中具有很大的前景。在这里，我们描述了一种体内高通量筛选，以确定AraC家族激活蛋白RHAS的抑制剂。该筛查使用了两种大肠杆菌报告融合；一种用于识别潜在的RHAS抑制剂，另一种用于从考虑中消除非特异性抑制剂。选择了一种选择性较好的化合物OSSL051168进行进一步的研究。OSSl_051168对rhaS DNA结合域的体内转录激活的抑制作用与全长蛋白相同，表明该结构域是其抑制的靶点。生长曲线显示，在本研究使用的浓度范围内，OSSl_051168对细菌细胞的生长没有影响。体外与纯化蛋白的结合分析表明，OSSl_051168可抑制rHAs与DNA的结合。此外，我们还发现它抑制了第二个AraC家族蛋白Rhar与DNA的结合，Rhar与RHAS有30%的氨基酸同源性。OSSL_051168对非AraC家族蛋白CRP和LaCI的结合没有显著影响，表明这种抑制可能是RHAS、RHAR和其他AraC家族激活蛋白所特有的。
Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two E. coli reporter fusions; one to identify potential RhaS inhibitors, and a second to eliminate non-specific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not impact bacterial cell growth at the concentrations used in this study. In vitro DNA binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.