Liendo A, Visbal G, Piras M M, Piras R, Urbina J A
Laboratorio de Quimica Biológica, Centro de Bioquímica y Biofisica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.
Mol Biochem Parasitol. 1999 Oct 25;104(1):81-91. doi: 10.1016/s0166-6851(99)00129-2.
A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.
本文对克氏锥虫临床相关细胞内（无鞭毛体）阶段存在的内源性固醇进行了详细分析。将寄生虫在培养的Vero细胞中培养，分别添加或不添加不同的固醇生物合成抑制剂，包括C14α脱甲基酶抑制剂酮康唑以及两种δ24(25)-固醇甲基转移酶抑制剂，即20-哌啶-2-基-5α-孕甾-3β-20-R-二醇（22,26-氮杂固醇）和24-(R,S),25-表氨基羊毛甾醇。从宿主细胞中分离并纯化无鞭毛体，提取中性脂质，通过色谱和质谱方法进行分离和分析。对照（未处理）无鞭毛体所含的主要内源性固醇为24-甲基胆甾-7-烯-3β-醇（麦角甾-7-烯-3β-醇）及其24-乙基类似物，以及少量其前体麦角甾-7,24(28)二烯-3β-醇；这些细胞还含有胆固醇（占总固醇重量的80%），可能来源于宿主细胞。在10 nM酮康唑（最低抑菌浓度，MIC）存在下增殖24小时的无鞭毛体，其内源4-去甲基固醇含量急剧降低，同时24-甲基二氢羊毛甾醇和24-亚甲基二氢羊毛甾醇积累。另一方面，在相同时间段内用两种24(25)-SMT抑制剂（各自的MIC为100 - 300 nM）处理的无鞭毛体积累了大量C27固醇，就22,26-氮杂固醇而言，其结构表明δ14固醇还原酶也受到了抑制。酮康唑使[2-(14)C]-乙酸掺入寄生虫内源C4-去甲基固醇的量呈剂量依赖性降低，IC50为50 nM，与先前报道的细胞外前鞭毛体形式的值无差异。综上所述，结果表明无鞭毛体的固醇生物合成途径比先前描述的前鞭毛体更简单，缺乏δ5和δ22还原酶。结果还表明，与前鞭毛体相比，抗真菌唑类作为抗增殖剂对无鞭毛体的效力高100倍，很可能是由于细胞内寄生虫的内源性固醇池较小。
