Targetting InhA For Anti-TB Drug Discovery

描述（申请人提供）：结核分枝杆菌是HIV/AIDS患者的主要机会致病菌。目前的结核病治疗方案受到耐多药结核分枝杆菌（MDR-TB）菌株的严重阻碍，迫切需要开发新的化疗药物。我们将设计和合成来自结核分枝杆菌（MTB）烯基还原酶InhA抑制剂。InhA催化脂肪酸延伸途径的最后一步，是异烟肼（INH）一种一线抗结核药物的活化形式的靶标。重要的是，绝大多数INH耐药菌株是由激活INH的酶KatG突变引起的，而不是由最终药物靶点突变引起的。因此，假设InhA抑制剂可以规避KatG激活的需要，将形成治疗耐多药结核病的有效基础。首先发现的抑制剂是三氯生（5-氯-2-（2,4-二氯苯氧基）苯酚），该化合物抑制InhA的Ki值为0.22 mu/M，对MTB的MIC99为5 mu/g/mL （17 mu/M）。建议的研究包括以下具体目标：

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is a major opportunistic pathogen in patients with HIV/AIDS. Current tuberculosis treatment regimes are severely hampered by the occurrence of multidrug resistant strains of M. tuberculosis (MDR-TB) and there is a critical need for the development of novel chemotherapeutics. We will design and synthesize inhibitors of InhA, the enoyl-reductase enzyme from M. tuberculosis (MTB). InhA, which catalyzes the final step in the fatty acid elongation pathway, is a target for the activated form of isoniazid (INH) a front-line anti-TB drug. Importantly, the large majority of INH-resistant isolates result from mutations in KatG, the enzyme that activates INH, rather than from mutations in the ultimate drug target(s). Consequently, it is hypothesized that inhibitors of InhA, which circumvent the need for KatG activation, will form an effective basis for tackling MDR-TB. The lead compound for inhibitor discovery is triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), a compound that inhibits InhA with a Ki value of 0.22 mu/M and which has an MIC99 for MTB of 5 mu/g/mL (17 mu/M). The proposed research includes the following specific aims: (1) We will synthesize a library of triclosan analogs using directed synthesis and parallel synthetic methods. (2) We will determine the inhibitory potency of the analogs and analyze the interaction of promising compounds with InhA using techniques such as kinetics, X-ray crystallography and Raman spectroscopy. (3) We will determine MIC99 values against sensitive and resistant TB strains. To evaluate whether InhA is the target for these compounds we will (i) determine MIC99 values in an MTB strain overexpressing InhA and (ii) use promoter-specific reporter fusions coupled with radiolabeled uptake studies and photoaffinity labeling to investigate the mechanism of compound action in live bacteria. The in vivo activity of promising InhA inhibitors will be further analyzed through the NIAID-funded Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF). Information derived from Aims 2-3 will direct the synthesis of additional compounds which will then be submitted for further rounds of screening and evaluation.