Identification of Desaturase Targets

描述（由申请方提供）：硫卡内酯（THC）是一种硫脲，可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对膜相关的硬脂酰-CoA（delta 9）去饱和酶DesA 3（Rv 3229 c）的活性的抑制作用。已知的δ 9去饱和酶抑制剂Sterculic acid模拟了THC对油酸合成的作用，但不影响分枝菌酸合成，表明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具有至少一个其它酶靶。由于我们的初步数据还表明，THC对来自金分枝杆菌的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官能团引入分枝菌酸的部分分枝菌酸链中的酶，例如膜相关去饱和酶。生物信息学已经揭示了四个假定的膜去饱和酶的特征富含组氨酸的基序，所有的THC/硫脲的潜在目标，以及两个可溶型去饱和酶。此外，M.结核杆菌潜在地编码11种结构上与已知在真核生物中对尿素抑制剂敏感的卡氏型环氧化物水解酶相关的蛋白质。 所有这些酶将过表达，并与THC的最小抑制浓度进行比较。在更广泛的遗传方法中，将表征负责赋予对THC高水平抗性的分枝杆菌基因组中的突变。介绍了M.将建立结核病去饱和酶和新鉴定的THC靶点的无细胞测定/筛选，并将无细胞测定/筛选开发为几种抑制剂文库的低筛选，包括我们现有的THC衍生物文库，促进化学合成形式的药物化学方法和产品的扩展测试。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。

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.