Drug targeting Mycobacterium tuberculosis cell wall synthesis:: Genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose
Drug targeting Mycobacterium tuberculosis cell wall synthesis:: Genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose
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DOI：
10.1128/aac.45.5.1407-1416.2001
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发表时间：
2001-05-01
影响因子：
4.9
通讯作者：
McNeil, MR
中科院分区：
文献类型：
--
作者：
Ma, YF;Stern, RJ;McNeil, MR
L鼠李糖基残基在结核分枝杆菌细胞壁中起着重要的结构作用。因此，由dTTP和葡萄糖-L-磷酸形成dTDP-鼠李糖的四种酶(Rm14~RmlD)是开发新的结核病治疗药物的重要靶点。结核分枝杆菌编码Rm1a、RmlC和RmlD的基因已被鉴定并在大肠杆菌中表达，目前在结核分枝杆菌基因组中只存在Rm1a到RmlD各一个亚型的基因。RmlB基因为Rv3463。Rv3264c编码的是ManB，而不是RMLA的第二个亚型。利用重组RmlB、-C和-D酶建立了一种筛选dTDP-鼠李糖形成抑制剂的微板法，这三种酶与dTDP-葡萄糖和NADPH孵育形成dTDP-鼠李糖和NADP(+)，并伴随着340 nm处光密度的下降(OD340)，监测抑制剂候选者降低OD340变化率的能力。为了测试该分析的稳定性和实用性，我们筛选了一个包含8,000个化合物的化学库，鉴定出11个在晚上10点有效的抑制剂；其中4个化合物对整个结核杆菌细胞具有活性，MIC从128到16个微克/毫升。其中3个酶抑制剂中存在一个罗丹宁结构基序，其中两个化合物对整个结核杆菌细胞具有活性。用酶试验筛选了60种已知在培养中抑制结核分枝杆菌生长的秘鲁植物提取物，其中两种提取物在酶试验中的浓度低于2µg/ml时是活性抑制物。
An (L)-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (Rml4 to RmlD) that form dTDP-rhamnose from dTTP and glucose-l-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed in Escherichia coli, It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3463. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose, The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP(+) with a concomitant decrease in optical density at 340 nm (OD340), Inhibitor candidates were monitored for their ability to lower the rate of OD340 change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened, Eleven inhibitors active at 10 pM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 mug/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 mug/ml.