Identification of Desaturase Targets

描述(由申请人提供)：硫脲(THC)，一种硫脲，抑制结核分枝杆菌中真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。已知的Delta9去饱和酶抑制剂胸脂酸模拟了THC对油酸合成的影响，但不影响霉菌酸合成，表明该药物的这两种作用之间缺乏联系。因此，THC在霉菌酸的生物合成途径中至少还有一个其他的酶靶点。由于我们的初步数据也表明THC对金分枝杆菌Fas-I和Fas-II系统的活性没有抑制作用，该药物的第二个靶点可能是在分枝杆菌酸的分枝杆菌酸链中引入功能基团的酶，如膜相关去饱和酶。生物信息学已经揭示了四种可能的富含组氨酸的膜去饱和酶，所有的潜在靶点都是THC/硫脲，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种结构上与哺乳动物类型的环氧化物水解酶相关的蛋白质，这些蛋白质在真核生物中对尿素抑制剂敏感。所有这些酶都将过度表达，并比较其对THC的最低抑制浓度。在更广泛的遗传学方法中，将对导致对THC产生高水平耐药性的分枝杆菌基因组突变进行表征。结核分枝杆菌脱饱和酶和新确定的靶点(S)的重要性、功能和3D结构将得到确定，无细胞分析/筛选将发展到较低水平，包括我们现有的THC衍生物库在内的几个抑制剂库的筛选，促进以化学合成和产品扩展测试形式的药物化学方法。THC曾被接受用于治疗结核病，但由于吸收动力学不良和生物利用度低而失去了青睐。这种方法可能会克服这些方面。

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.