Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450 CYP121
Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450 CYP121
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DOI：
10.1002/anie.201202544
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Abell, Chris
中科院分区：
文献类型：
--
作者：
Hudson, Sean A.;McLean, Kirsty J.;Abell, Chris
耐药结核分枝杆菌（Mtb）的出现推动了对具有新作用模式的新型一线结核病（TB）药物的迫切需求。[1]据估计，每年有超过65万例耐多药结核病病例出现，到2015年将有130万例需要治疗，预算费用超过160亿美元。[2]世界卫生组织已宣布这一流行病为全球卫生紧急情况。[3]抑制生物合成细胞色素P450酶（CYP）的药物（如阿比特龙，来曲唑和伏立康唑）的成功推动了研究，以了解在Mtb H37 Rv基因组中发现的异常高数量的CYP（20）。[4]特别令人感兴趣的是必需的CYP 121亚型，它最近成为一个诱人的新的抗结核药物靶点。[4a-c这种生物合成酶似乎是Mtb独有的，并且只有当存在携带CYP 121的互补载体时，Mtb染色体CYP 121敲除突变体的构建才是可能的。[4b最近还显示CYP 121催化环二酪氨酸（cYY）中两个酪氨酸的邻位之间的不寻常的分子内CycloC键形成反应以形成霉环素。[5a]虽然cYY和mycyclosin的生理作用仍有待确定，但我们相信CYP 121的独特催化作用将导致选择性抑制剂的产生。特异性抑制剂也可以用作化学探针，以显示这种关键酶与结核分枝杆菌感染，生长和持久性的关系。目前已知的CYP 121的唯一高亲和力配体是唑类抗真菌剂（传统的真菌CYP 51抑制剂，其通过II型唑-血红素配位作用）。[5b这些化合物表现出有效的体外/体内抗分枝杆菌活性，其中它们对Mtb H37 Rv的MIC（最小抑制浓度）值与它们对CYP 121的结合亲和力相关。[5b然而，大的柔性抗真菌剂也具有广泛的重叠的抗真菌抑制特性，因此对于开发特异性抑制剂和一线TB药物候选物来说是差的支架。[4a临床上施用的那些抗真菌剂，例如氟康唑和伏立康唑，也仅与CYP 121弱结合。[5b 6a，8a]此外，已经分离出抗性Mtb突变体，其显示被认为充当唑类外排泵的跨膜转运蛋白的上调。[9]基于片段的方法代表了开发小分子配体作为药物开发的化学工具和先导物领域的新方法。[10]这种强大的方法涉及结构指导的设计和合成有效的配体从弱结合的低分子量片段分子（通常< 250 Da）。[10]在此，我们报告了一个片段为基础的方法，以靶向结核杆菌CYP 121，试图确定新的抑制分子，并探讨活性位点的酶的特性。通过最初的片段筛选级联涉及热位移，NMR光谱，和X-射线晶体学，发现四个片段结合在CYP 121活性位点，在两个重叠的组。实施了直接片段-片段合并策略，从而发现了一种新型II型氨基喹啉抑制剂，其具有高配体效率（LE=配体中结合/非氢原子（NHA）的ΔG）和比天然CYP 121底物cYY高四倍的亲和力。该先导化合物提供了CYP 121特异性抑制模式，并证实了CYP 121的潜在可药用性。这项研究代表了第一次成功应用基于片段的方法的细胞色素P450。
The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) drives a critical need for new front-line tuberculosis (TB) drugs with a novel mode of action.[1] It is estimated that there are over 650000 cases of multidrug-resistant tuberculosis emerging every year, and that 1.3 million cases will need to be treated by 2015, at a budgeted cost of over 16 billion US dollars.[2] The World Health Organization has declared this epidemic a global health emergency.[3] The success of drugs that inhibit biosynthetic cytochrome P450 enzymes (CYPs), such as abiraterone, letrozole, and voriconazole, has propelled research towards understanding the unusually high number of CYPs (20) found encoded in the Mtb H37Rv genome.[4] Of particular interest is the essential CYP121 isoform, which has recently come into focus as an enticing new anti-TB drug target.[4a–c, 5] This biosynthetic CYP appears to be exclusive to Mtb, and construction of an Mtb chromosomal CYP121 knock-out mutant was only possible when a complementary vector carrying CYP121 was present.[4b, 5b] CYP121 has also recently been shown to catalyze an unusual intramolecular CÀC bond-forming reaction between the ortho-positions of two tyrosines in cyclodityrosine (cYY) to form mycocyclosin.[5a] While the physiological roles of cYY and mycocyclosin remain to be determined, we believe that the unique catalytic action of CYP121 will lead to selective inhibitors. Specific inhibitors could also be used as chemical probes to show how this pivotal enzyme relates to Mtb infection, growth and persistence. The only high-affinity ligands of CYP121 currently known are azole antifungals (traditional fungal CYP51 inhibitors, which act by way of type-II azole–heme coordination).[5b, 6] These compounds exhibit potent in vitro/in vivo antimycobacterial activity, where their MIC (minimal inhibitory concentration) values for Mtb H37Rv correlate with their binding affinities to CYP121.[5b, 7] However, the large flexible antifungals also have broad overlapping CYP inhibition profiles and are thus poor scaffolds for developing specific inhibitors and front-line TB drug candidates.[4a, 6b, 8] Those antifungals that are administered clinically, for example, fluconazole and voriconazole, also only bind weakly to CYP121.[5b, 6a, 8a] Furthermore, resistant Mtb mutants have already been isolated that show upregulation of a transmembrane transporter protein believed to act as an azole efflux pump.[9]Fragment-based approaches represent a new method in the field of developing small-molecule ligands as chemical tools and leads for drug development.[10] This powerful method involves the structure-guided design and synthesis of potent ligands from weaker-binding low-molecular-weight fragment molecules (typically< 250 Da).[10] Herein, we report a fragment-based approach to targeting Mtb CYP121 in an attempt to identify new inhibitory molecules and to explore the active-site properties of the enzyme. Through an initial fragment-screening cascade involving thermal shift, NMR spectroscopy, and X-ray crystallography, four fragments were found to bind within the CYP121 active site, in two overlapping groups. A direct fragment–fragment merging strategy was implemented, leading to the discovery of a novel type-II aminoquinoline inhibitor with high ligand efficiency (LE= ÀΔG of binding/non-hydrogen atoms (NHA) in the ligand) and fourfold greater affinity than the natural CYP121 substrate cYY. This lead provides a pattern for CYP121-specific inhibition and confirms the potential druggability of CYP121. This study represents the first successful application of fragment-based approaches to a cytochrome P450.