Yadav Nisha, Khanam Taran, Shukla Ankita, Rai Niyati, Hajela Kanchan, Ramachandran Ravishankar
From Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Sector10, Jankipuram Extension, Sitapur Road, Uttar Pradesh, Lucknow-226031, India.
Org Biomol Chem. 2015 May 21;13(19):5475-87. doi: 10.1039/c5ob00439j.
DNA ligases are critical components for DNA metabolism in all organisms. NAD(+)-dependent DNA ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA ligase or Mycobacterium tuberculosis NAD(+)-dependent DNA ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA ligase I & T4 DNA ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.
DNA连接酶是所有生物体中DNA代谢的关键组成部分。仅在细菌和某些昆虫痘病毒中发现的依赖NAD(+)的DNA连接酶（LigA）作为治疗靶点正受到越来越多的关注，因为它们在辅因子需求方面与依赖ATP的真核同源物不同。由于这两类DNA连接酶在辅因子结合位点上的相似性，有必要找到能够区分它们的决定因素，以便将LigA开发为抗菌靶点。在目前的研究中，我们合成并评估了一系列三环二氢苯并恶唑嗪和四环吲哚衍生物区分细菌和人类DNA连接酶的能力。体内抑制试验采用分别由依赖ATP的T4 DNA连接酶或结核分枝杆菌依赖NAD(+)的DNA连接酶（Mtb LigA）拯救的LigA缺陷型大肠杆菌GR501和鼠伤寒沙门氏菌LT2菌株，结果表明这些化合物可以特异性抑制细菌LigA。使用纯化的MtbLigA、人类DNA连接酶I和T4 DNA连接酶进行的体外酶抑制试验表明，在低微摩尔范围内对MtbLigA有特异性抑制作用。我们的结果表明，三环二氢苯并恶唑嗪和四环吲哚衍生物能够以约5倍的差异区分细菌和人类DNA连接酶。计算机对接和酶抑制试验确定这些化合物与辅因子结合位点结合并与辅因子竞争。溴化乙锭置换和凝胶迁移试验表明，这些抑制剂与底物DNA没有任何不必要的一般相互作用。这些结果为详细探索这类化合物作为抗菌药物的开发奠定了基础。
