Identification of Desaturase Targets

去饱和酶靶标的鉴定

• 批准号: 7084480
• 负责人: Patrick Joseph Brennan
• 金额: $ 30.13万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084480
• 关键词: Mycobacterium bovis; Mycobacterium tuberculosis; antitubercular agents; bacterial genetics; bacterial proteins; cell free system; coenzyme A; drug discovery /isolation; enzyme activity; epoxide hydrolase; fatty acid metabolism; gene expression; high throughput technology; hydroxy fatty acid; membrane proteins; multidrug resistance; oxidoreductase; oxidoreductase inhibitor; protein quantitation /detection; protein structure function; recombinant proteins; stearates; thiourea; tuberculosis

DESCRIPTION (provided by the applicant): Thiocarlide (THC), a thiourea, inhibits the synthesis of mycolic acids and fatty acids in Mycobacterium tuberculosis. Our evidence indicates that the main effect of THC on fatty acid synthesis is in inhibiting the synthesis of oleic acid, directly attributable to the inhibitory effect of THC on the activity of the membrane-associated stearoyl-CoA (delta9) desaturase DesA3 (Rv3229c). Sterculic acid, a known inhibitor of delta9 desaturases, emulates the effect of THC on oleic acid synthesis but does not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. Therefore, THC has at least one other enzymatic target in the mycolic acid biosynthetic pathway. Since our preliminary data also indicate that THC has no inhibitory effect on the activity of the FAS-I and FAS-II systems from Mycobacterium aurum, the second target of the drug could be an enzyme introducing functional groups into the meromycolate chain of mycolic acids such as a membrane-associated desaturase. Bioinformatics has revealed four putative membrane desaturases with the characteristic His-rich motif, all potential targets of THC/thioureas, as well as two soluble-type desaturases. Moreover, the genome of M. tuberculosis potentially encodes eleven proteins structurally related to mammalian-type epoxide hydrolases which are known in eukaryotes to be sensitive to urea inhibitors. All of these enzymes will be over-expressed and minimal inhibitory concentration to the THC compared. In a broader genetic approach, mutations in the genome of mycobacteria responsible for conferring high level resistance to THC will be characterized. The essentiality, function and 3D structure of the M. tuberculosis desaturases and newly identified target(s) of THC will be established and cell free assays/screens developed to a low screening of several libraries of inhibitors including our existing library of THC derivatives, facilitate a medicinal chemistry approach in the form of chemical synthesis and extended testing of products. THC was once acceptable for treatment of tuberculosis but lost favor due to untoward absorption kinetics and low bioavailability. This approach may overcome these aspects.

描述（由申请人提供）：Thiocarlide (THC)，一种硫脲，抑制结核分枝杆菌中分枝菌酸和脂肪酸的合成。我们的证据表明，THC 对脂肪酸合成的主要作用是抑制油酸的合成，这直接归因于 THC 对膜相关硬脂酰辅酶 A (delta9) 去饱和酶 DesA3 (Rv3229c) 活性的抑制作用。 Sterculic Acid 是一种已知的 delta9 去饱和酶抑制剂，它模拟 THC 对油酸合成的影响，但不影响分枝菌酸合成，表明该药物的两种作用之间缺乏关系。因此，THC 在分枝菌酸生物合成途径中至少有一个其他酶靶点。由于我们的初步数据还表明 THC 对金分枝杆菌的 FAS-I 和 FAS-II 系统的活性没有抑制作用，因此该药物的第二个靶点可能是将功能基团引入分枝菌酸的分枝菌酸链中的酶，例如膜相关的去饱和酶。生物信息学揭示了四种假定的具有富含组氨酸基序的膜去饱和酶（THC/硫脲的所有潜在靶标）以及两种可溶型去饱和酶。此外，结核分枝杆菌的基因组可能编码十一种在结构上与哺乳动物型环氧化物水解酶相关的蛋白质，已知这些蛋白质在真核生物中对尿素抑制剂敏感。 所有这些酶都将与过表达的 THC 的最小抑制浓度进行比较。在更广泛的遗传方法中，将表征负责赋予高水平 THC 抗性的分枝杆菌基因组突变。将建立结核分枝杆菌去饱和酶和新确定的 THC 靶标的本质、功能和 3D 结构，并开发无细胞测定/筛选，以对多个抑制剂库（包括我们现有的 THC 衍生物库）进行低筛选，促进化学合成和产品扩展测试形式的药物化学方法。 THC 曾经被接受用于治疗结核病，但由于不良的吸收动力学和低生物利用度而失去了青睐。这种方法可以克服这些方面。