Inhibitors of sulfate assimilation as new therapy for TB

描述(申请人提供)：结核分枝杆菌(结核分枝杆菌)是一种具有全球重要性的主要病原体。尽管有了化疗和卡介苗，但结核分枝杆菌夺走的生命仍然比其他任何单一的感染性病原体都多。近年来，发展中国家和工业化国家的结核病发病率都有所增加，出现了广泛的抗药性菌株，与人类免疫缺陷病毒产生了致命的协同作用。目前治疗的局限性源于抗生素耐药菌株的广泛出现，以及对抗生素长期不间断使用的要求。因此，迫切需要新类别的抗生素。含硫代谢产物，包括必需氨基酸半胱氨酸和蛋氨酸，以及毒力因子霉硫醇和硫脂-1，对结核分枝杆菌的感染性至关重要。它们的生物合成涉及硫酸盐同化途径，这在人类中没有对应的途径。由于这一途径中的酶组成了新陈代谢的中心枢纽，并且是细菌所特有的，我们认为这些酶是抗分枝杆菌治疗的主要靶点。本建议将5‘-腺苷磷酸根(APS)还原酶(CysH)作为小分子药物开发的靶点。在初步工作中，我们1)利用基因互补在大肠杆菌中证实了结核分枝杆菌CysH的APS还原酶活性，2)证明了结核分枝杆菌致病株CysH的基因缺失导致突变株无法在结核病小鼠模型中建立感染。在本提案的具体目标1中，我们将使用大肠杆菌互补试验筛选结核分枝杆菌硫酸盐同化抑制物化合物文库。在具体目标2中，我们将建立重组结核分枝杆菌CysH的无细胞均质检测方法，从初步筛选中检查我们的引线直接抑制该酶的能力，并通过药物化学优化这些引线。在具体目标3中，将利用结核分枝杆菌临床分离株的体外敏感性研究来检验最佳化合物。在这项研究的第二阶段，来自我们可行性研究的抑制剂将在小鼠结核病模型中检验其抑菌和杀菌效果。这项研究的长期目标是开发新的抗生素，以更有效地治疗人类结核病和其他分枝杆菌疾病。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (M. tuberculosis) is a major pathogen of global importance. Despite the availability of chemotherapy and the Bacille Calmette-Guerin vaccine, M. tuberculosis continues to claim more lives than any other single infectious agent. Recent years have seen increased incidence of tuberculosis in both developing and industrialized countries, the widespread emergence of drug-resistant strains and a deadly synergy with the human immunodeficiency virus. The limitation of current therapy originates from the widespread occurrence of antibiotic resistant strains and also the requirement for prolonged and uninterrupted administration of antibiotics. Thus, new classes of antibiotics are desperately needed. Sulfur-containing metabolites, including the essential amino acids cysteine and methionine, and the virulence factors mycothiol and sulfolipid-1, are crucial for the infectivity of M. tuberculosis. Their biosynthesis involves the sulfate assimilation pathway, which has no counterpart in humans. Since the enzymes in this pathway comprise a central hub of metabolism and are unique to bacteria, we propose that these enzymes are prime targets for anti-mycobacterial therapy. This proposal focuses on 5'-adenosinephosphosulfate (APS) reductase (CysH) as a target for small molecule drug development. In preliminary work, we have 1) confirmed the APS reductase activity of M. tuberculosis CysH using genetic complementation in E. coli, and 2) demonstrated that genetic deletion of CysH from a pathogenic strain of M. tuberculosis renders the mutant unable to establish infection in a mouse model of tuberculosis. In specific aim 1 of this proposal we shall screen a compound library for inhibitors of M. tuberculosis sulfate assimilation using an E. coli complementation assay. In specific aim 2 we shall set up cell-free homogenous assays for recombinant M. tuberculosis CysH, examine our leads from the primary screen for their ability to directly inhibit this enzyme, and optimize these leads through medicinal chemistry. In specific aim 3 the best compounds will be examined using in vitro susceptibility studies of clinical isolates of M. tuberculosis. In phase II of this study, the inhibitor leads from our feasibility study will be examined for bacteriostatic and bacteriocidic efficacy in mouse models of tuberculosis. The long-term objective of this study is to develop novel antibiotics for more effective treatment of human tuberculosis and other mycobacterial diseases.