Dhiman Abhijeet, Kumar Chanchal, Mishra Subodh Kumar, Sikri Kriti, Datta Ishara, Sharma Pradeep, Singh Tej P, Haldar Sagarika, Sharma Neera, Bansal Anjali, Ahmad Yusra, Kumar Amit, Sharma Tarun Kumar, Tyagi Jaya Sivaswami
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India; Faculty of Pharmacy, Uttarakhand Technical University, Dehradun 248007, Uttarakhand, India.
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India.
Mol Ther Nucleic Acids. 2019 Dec 6;18:661-672. doi: 10.1016/j.omtn.2019.09.026. Epub 2019 Oct 4.
The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [K] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC of 251.1 nM and inhibitor constant (K) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.
结核病（TB）的成功管理需要有效的诊断和治疗。此外，耐药结核病的患病率不断上升，凸显了开发针对药物敏感和耐药形式疾病的新型抑制剂的迫切需求。苹果酸合酶（MS）是乙醛酸途径的一种酶，在分枝杆菌的持续存在中起着至关重要的作用，因此它被认为是新型抗结核药物开发的一个有吸引力的靶点。最近的研究还赋予了MS黏附素功能，并将其确立为一种有效的诊断生物标志物。在本研究中，通过指数富集配体系统进化（SELEX）鉴定了一组结核分枝杆菌（Mtb）MS特异性单链DNA适配体。性能最佳的形成G-四链体的44聚体适配体MS10在SELEX后进行了优化，以生成一个11聚体适配体MS10-Trunc。该适配体通过各种生化、生物物理和计算机技术进行了表征。使用酶抑制、宿主细胞结合和侵袭试验确定了其对Mtb的诊疗活性。MS10-Trunc适配体对MS表现出高亲和力（平衡解离常数[K]~19 pM），并对MS酶活性表现出强大的抑制作用，IC为251.1 nM，抑制常数（K）为230 nM。该适配体通过与表面相关的MS结合，阻止分枝杆菌进入宿主细胞。此外，我们还证明了其在检测结核性脑膜炎（TBM）患者中的应用，敏感性和特异性均>97%。
