Gros Ludovic, Castillo-Acosta Victor Manuel, Jiménez Jiménez Carmen, Sealey-Cardona Marco, Vargas Sofia, Manuel Estévez Antonio, Yardley Vanessa, Rattray Lauren, Croft Simon L, Ruiz-Perez Luis M, Urbina Julio A, Gilbert Ian H, González-Pacanowska Dolores
Welsh School of Pharmacy, Cardiff University, UK.
Antimicrob Agents Chemother. 2006 Aug;50(8):2595-601. doi: 10.1128/AAC.01508-05.
A series of azasterol derivatives, designed as potential inhibitors of the Delta(24)-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.
设计了一系列氮杂甾醇衍生物作为δ(24)-甾醇甲基转移酶（24-SMT）的潜在抑制剂，并对其抗寄生原生动物的活性进行了评估。对体外培养的布氏罗得西亚锥虫血流型的半数有效剂量达到了纳摩尔范围的值。为了研究作用模式，克隆并过表达了布氏锥虫亚种布氏锥虫的24-SMT，并对化合物的抑制活性进行了测定。所测试的抑制剂均未显示出对该酶有活性。甾醇组成分析表明，血流型细胞膜中仅存在胆甾烷型甾醇，而麦角甾醇是前循环型甾醇提取物的主要成分。有趣的是，Northern印迹分析表明，寄生虫的前循环型和血流型中均存在24-SMT mRNA，尽管后者的mRNA水平低三倍。同样，蛋白质印迹分析和活性测定证明了寄生虫两种形式中均存在活性酶。我们得出结论，所设计的化合物作用于布氏锥虫中24-SMT以外的位点。
