以结核分枝杆菌丙氨酸消旋酶为靶点的抗结核药物筛选及其与D-环丝氨酸的共结晶

【目的】阐释结核分枝杆菌丙氨酸消旋酶原有抑制剂D-环丝氨酸的作用机制,建立丙氨酸消旋酶抑制剂的高通量筛选模型,并用此模型筛选到新的抑制剂分子。【方法】将结核分枝杆菌中丙氨酸消旋酶基因克隆到pET-28a表达载体中,在大肠杆菌BL21(DE3)菌株中得到可溶性的大量表达。表达后的蛋白质通过镍离子亲和层析和阴离子交换层析得到纯化,一方面将纯化后的蛋白和D-环丝氨酸共结晶以解析其抑制剂作用的分子机制。另一方面建立并优化丙氨酸消旋酶抑制剂的高通量筛选体系,用D-环丝氨酸验证体系的可行性并用该体系筛选本实验室药物库中的384种小分子片段、792种化合物及2 200种中药样品。【结果】得到的共晶晶体衍射能力2.50?,晶体空间群为P41212,晶胞参数a=b=163.92?,c=57.44?。结构分析表明,D-环丝氨酸进入活性位点之后与磷酸吡哆醛相互作用形成磷酸吡哆胺,使得磷酸吡哆醛的C4?原子与K42之间的相互作用被破坏,从而改变了结核分枝杆菌丙氨酸消旋酶的活性中心氢键网络。同时经D-环丝氨酸验证建立的抑制剂筛选体系可行,获得阳性化合物分子2个。【结论】依据我们所建立的高通量筛选体系可以有效地为结核分枝杆菌丙氨酸消旋酶筛选到可信的抑制剂分子。

Anti-tuberculosis drug screening of alanine racemase from Mycobacterium tuberculosis and its co-crystallized with D-cycloserine

Objective] The aim of this work was to investigate the inhibition mechanism of D-cycloserine (DCS) on alanine racemase from Mycobacterium tuberculosis (AlrMtb), and build up a high-throughput screening assay to screen for new inhibitors of AlrMtb. Methods] The AlrMtb gene was cloned into pET28a vector and over-expressed in soluble form in E. coli strain BL21(DE3). The protein was purified using Ni2+-chelating chromatography followed by anion exchange chromatography. The AlrMtb protein was co-crystallized with DCS to make clear of the inhibitory mechanism. In addition, a high-throughput screening assay for new inhibitors was set up in order to obtain new anti-tuberculosis drugs that inhibited AlrMtb activity. About 384 small-molecule fragments, 792 chemicals and 2 200 components of traditional Chinese Medicine were tested in this assay where DCS was used as the positive reference. Results] The protein crystals of AlrMtb-DCS complex diffract to 2.5 ?. The space group of the crystals is P41212 with cell parameters a=b=163.92 ?, c=57.44 ?. In the AlrMtb-DCS structure model, DCS was shown to react with PLP and formed a new molecule PMP. This process disrupted previous interaction between the C4? atom of PLP and Lysine 42 in AlrMtb and thereby changed the hydrogen bonding network in the active site. Via the high-throughput screening assay, we successfully identified two new inhibitors of AlrMtb. Conclusion] The high-throughput inhibitor screening assay we built is an effective and powerful methodfor theidentification of new inhibitors of AlrMtb.