Identification of Desaturase Targets

描述（由申请人提供）：硫代硫化物（THC），一种硫脲，抑制结核分枝杆菌中霉菌酸和脂肪酸的合成。我们的证据表明，四氢大麻酚对脂肪酸合成的主要作用是抑制油酸的合成，直接归因于四氢大麻酚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，它可以模拟四氢大麻酚对油酸合成的影响，但不影响霉菌酸的合成，这表明该药物的两种作用之间缺乏关系。因此，四氢大麻酚在霉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，四氢大麻酚对来自金分枝杆菌的fas - 1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一种将官能团引入霉菌酸链的酶，如膜相关的去饱和酶。生物信息学已经揭示了四种可能的膜去饱和酶，它们具有富his基元的特征，都是THC/硫脲的潜在靶点，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11种与哺乳动物型环氧化物水解酶结构相关的蛋白质，这些蛋白质在真核生物中已知对尿素抑制剂敏感。所有这些酶都将过表达和最小抑制浓度对THC进行比较。在更广泛的遗传方法中，负责赋予四氢大麻酚高水平耐药性的分枝杆菌基因组突变将被表征。将建立结核分枝杆菌去饱和酶的必要性、功能和3D结构以及新发现的四氢大麻酚靶点，并开发无细胞测定/筛选，以低筛选几种抑制剂库，包括我们现有的四氢大麻酚衍生物库，促进以化学合成形式的药物化学方法和产品的扩展测试。四氢大麻酚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.