Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase
Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase
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DOI：
10.1016/j.bbapap.2015.05.003
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发表时间：
2015-10-01
影响因子：
3.2
通讯作者：
Yoon, Moon-Young
中科院分区：
文献类型：
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作者：
Baig, Irshad Ahmed;Moon, Ji-Young;Yoon, Moon-Young
结核分枝杆菌乙酰羟酸合酶（AHAS）是一类新型抗结核药物的潜在靶点。在这项研究中，我们确定短（30聚体）单链DNA适体作为一种新型的有效抑制剂Mtb-AHAS通过体外DNA-SELEX方法。在所有检测的适体中，两种候选适体（Mtb-Apt 1和Mtb-Apt 6）表现出对Mtb-AHAS活性的最大抑制潜力，IC 50值在低纳摩尔范围内（分别为28.94 +/- 0.002和2235 +/- 0.001 nM）。有趣的是，这些适体的抑制动力学分析显示了不同的酶抑制模式（分别为竞争性和混合型抑制）。Mtb-Apt 1和Mtb-Apt 6的二级结构指导的突变修饰分析确定了负责其抑制作用的最小区域，并因此导致17-mer和20-mer缩短的适体保留了相等或更大的抑制潜力。值得注意的是，建模和对接练习研究了这两种有效抑制性适体在靶蛋白上的结合位点，并显示AHAS的一些关键催化二聚体界面残基可能参与导致其有效抑制的DNA-蛋白质相互作用。重要的是，这两种短的候选适体Mtb-Apt 1（17-mer）和Mtb-Apt 6（20-mer）也表现出对结核病的多药耐药（MDR-TB）和广泛耐药（XDR-TB）菌株的显著生长抑制，其MIC分别为536 μ g/ml和6.24 μ g/ml，并且对哺乳动物细胞系没有显著的细胞毒性。这是针对Mtb-AHAS的功能性抑制性适体的首次报道，并为开发这些适体作为新的和强的抗结核药物提供了基础。(C)2015爱思唯尔B. V.保留所有权利。
Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94 +/- 0.002 and 2235 +/- 0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 536 mu g/ml and 6.24 mu g/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents. (C) 2015 Elsevier B.V. All rights reserved.