Application of Fragment Growing and Fragment Linking to the Discovery of Inhibitors of Mycobacterium tuberculosis Pantothenate Synthetase
Application of Fragment Growing and Fragment Linking to the Discovery of Inhibitors of Mycobacterium tuberculosis Pantothenate Synthetase
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DOI：
10.1002/anie.200903821
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Abell, Chris
中科院分区：
文献类型：
--
作者：
Hung, Alvin W.;Silvestre, H. Leonardo;Abell, Chris
尽管每年有近200万人死于结核分枝杆菌（TB），[1]我们仍然缺乏强大的潜在疗法来应对这一负担。[2]基于片段的方法[3-5]为开发高质量的化学配体提供了一种新的范例，并具有作为开发抗结核药物的起点的巨大潜力。[6]泛酸合成酶（PS）是panC基因的产物，催化泛酸与β-丙氨酸的三磷酸腺苷镁（ATP）依赖性缩合以形成泛酸（维生素B5）。[7，8] M.发现panC和panD基因缺陷的结核病在该疾病的小鼠模型中高度减毒。[9]这一观察结果为使用PS作为开发新TB疗法的靶点提供了一定程度的遗传学验证。迄今为止，关于PS抑制的研究主要集中在泛酰腺苷酸反应中间体类似物的合成[10，11]或高通量筛选中命中的鉴定。[12在此，我们描述了片段生长和片段连接方法的组合，导致发现了一系列新的M.结核病PS进一步验证PS作为结核病的潜在药物靶点。我们设计了一种用于片段筛选、验证和表征的系统策略[14]，需要一系列生物物理技术，包括热位移测定、基于配体的NMR光谱学、等温滴定量热法（ITC）和X射线晶体学（参见支持信息中的图1）。一旦关键片段的结合模式已经阐明，化合物的设计和阐述合成，使额外的相互作用，以确定在酶活性位点的帮助下，计算对接实验与黄金（遗传优化配体对接）。[15]然后对精心制作的片段重复ITC和X射线晶体学实验，并重复该过程以增加配体效力。化合物5-甲氧基吲哚（1）最初通过WaterLOGSY NMR光谱被鉴定为ATP竞争性命中。[16]ITC证实，该片段结合的KD值为1.1 mm，配体效率（LE）[17]为0.36（所讨论化合物的滴定见支持信息中的图6和图7）。通过将碎片浸泡在PS晶体中并通过X射线晶体学确定结构（分辨率为1.6），揭示了精确的结合模式（图1a）。发现该片段结合在ATP腺嘌呤基序的识别位点。结合到PS的1的晶体结构的分析揭示了两个关键的氢键相互作用，一个在吲哚的OMe基团和Val 187的主链氮原子之间，另一个在吲哚NH基团和硫酸根分子之间（存在于结晶缓冲液中的150 mm处），其本身与Ser 197骨架氮原子和Lys 160残基相互作用（参见支持信息中的图3a）。筛选的几个片段，在结构上密切相关的1揭示了一个狭窄的结构-活性关系，该片段。例如，用羟基或甲基取代甲氧基，或用苯并吡啶或苯并咪唑杂环取代吲哚核心，导致结合亲和力降低至少10倍。这些结果证明了形状互补性和特异性在片段结合中的重要性（参见支持信息中的图2）。因此，最初的碎片命中1被选择来启动...
Despite the fact that nearly 2 million people a year are killed by Mycobacterium tuberculosis (TB),[1] we still lack a robust pipeline of potential therapies to combat this burden.[2] Fragment-based approaches [3–5] have provided a new paradigm in the development of high-quality chemical ligands and have great potential to contribute as starting points in the development of drugs against TB.[6] Pantothenate synthetase (PS), the product of the panC gene, catalyzes the magnesium adenosine triphosphate (ATP) dependent condensation of pantoate with β-alanine to form pantothenate (vitamin B5).[7, 8] A pantothenate auxotroph of M. tuberculosis defective in the panC and panD genes was found to be highly attenuated in a mouse model of the disease.[9] This observation provides a level of genetic validation of the use of PS as a target for the development of new TB therapeutics. To date, studies on PS inhibition have focused on the synthesis of analogues of the pantoyl adenylate reaction intermediate [10, 11] or the identification of hits from high-throughput screening.[12, 13] Herein, we describe a combination of fragment-growing and fragment-linking approaches that led to the discovery of a new series of inhibitors of M. tuberculosis PS for the further validation of PS as a potential drug target for TB. We devised a systematic strategy for fragment screening, validation, and characterization [14] for which a series of biophysical techniques were required, including a thermalshift assay, ligand-based NMR spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography (see Figure 1 in the Supporting Information). Once the binding modes of key fragments had been elucidated, compounds were designed and elaborated synthetically to enable additional interactions to be identified at the enzyme active site with the aid of computational docking experiments with GOLD (genetic optimization for ligand docking).[15] ITC and X-ray crystallographic experiments were then repeated on the elaborated fragments, and the process was iterated to increase ligand potency.The compound 5-methoxyindole (1) was initially identified as an ATP-competitive hit by WaterLOGSY NMR spectroscopy.[16] ITC confirmed that the fragment bound with a KD value of 1.1 mm and a ligand efficiency (LE)[17] of 0.36 (titrations for the compounds discussed are shown in Figures6 and 7 in the Supporting Information). The precise binding mode was revealed by soaking the fragment into a crystal of PS and determining the structure by X-ray crystallography to a resolution of 1.6 (Figure1a). The fragment was found to bind at the recognition site of the ATP adenine motif. Analysis of the crystal structure of 1 bound to PS revealed two key hydrogen-bonding interactions, one between the OMe group of the indole and the backbone nitrogen atom of Val187 and another between the indole NH group and a molecule of sulfate (present at 150 mm in the crystallization buffer), which itself interacts with the Ser197 backbone nitrogen atom and the Lys160 residue (see Figure 3a in the Supporting Information). The screening of several fragments that were structurally closely related to 1 revealed a narrow structure–activity relationship for this fragment. For example, the replacement of the methoxy group with either a hydroxy group or a methyl group, or the replacement of the indole core with a benzopyridine or benzoimidazole heterocycle led to an at least 10-fold decrease in the binding affinity. These results demonstrated the importance of shape complementary and specificity in fragment binding (see Figure 2 in the Supporting Information). The initial fragment hit 1 was therefore selected to initiate a …