Wang Liqin, Xu Miao, Southall Noel, Zheng Wei, Wang Shuishu
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd. Bethesda, MD 20814, USA.. United States.
Comb Chem High Throughput Screen. 2016;19(10):855-864. doi: 10.2174/1386207319666161010163249.
Tuberculosis (TB) kills over 1.5 million people per year despite the available anti-TB drugs. The long duration needed to treat TB by the current TB drugs, which target the essential cellular activities, inevitably leads to the emergence of drug-resistance. The emergence of drug-resistant TB prompts for an urgent need for new and more effective drugs.
The response regulator PhoP, an essential virulence factor of Mycobacterium tuberculosis (MTB), is an attractive target for developing novel anti- TB drugs. This study aims to develop a robust high-throughput screening assay to identify PhoP inhibitors that disrupt the PhoP-DNA binding.
Guided by the crystal structure of the PhoP-DNA complex, we designed and developed an assay based on Foster resonance energy transfer (FRET) by labeling Cy3 on the DNA and Cy5 on PhoP. We screened compound libraries for inhibitors that dissociated the PhoP-DNA complex by detection of the FRET signal. Hits were confirmed for their direct binding to PhoP by thermal shift assays.
From a test screening of ~6,000 bioactive compounds and approved drugs, three active compounds were identified that directly bound to PhoP and inhibited the PhoP-DNA interactions. These three PhoP inhibitors can be further developed to improve potency and are useful to study the mechanism of inhibition.
Our results demonstrated that this FRET-based PhoP-DNA binding assay is valid for additional compound library screening to identify new leads for developing novel TB drugs that target the virulence of MTB.
尽管有抗结核药物可用，但结核病每年仍导致超过150万人死亡。目前用于治疗结核病的药物针对基本细胞活动，所需治疗时间长，不可避免地导致耐药性的出现。耐多药结核病的出现促使迫切需要新的、更有效的药物。
应答调节因子PhoP是结核分枝杆菌（MTB）的一种必需毒力因子，是开发新型抗结核药物的一个有吸引力的靶点。本研究旨在开发一种强大的高通量筛选方法，以鉴定破坏PhoP-DNA结合的PhoP抑制剂。
以PhoP-DNA复合物的晶体结构为指导，我们设计并开发了一种基于荧光共振能量转移（FRET）的检测方法，通过在DNA上标记Cy3和在PhoP上标记Cy5来实现。我们通过检测FRET信号筛选化合物库，寻找能使PhoP-DNA复合物解离的抑制剂。通过热位移分析确认命中化合物与PhoP的直接结合。
在对约6000种生物活性化合物和已批准药物的测试筛选中，鉴定出三种活性化合物，它们直接与PhoP结合并抑制PhoP-DNA相互作用。这三种PhoP抑制剂可进一步开发以提高效力，并有助于研究抑制机制。
我们的结果表明，这种基于FRET的PhoP-DNA结合检测方法对于额外的化合物库筛选是有效的，可用于识别开发针对MTB毒力的新型抗结核药物的新先导物。
