22-26-azasterol and 24-25-iminolanosterol

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 μM) and 24(R,S),25-epiminolanosterol (10 μM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Topics: Animals; Antitrichomonal Agents; Cell Line; Cell Proliferation; Cholestanol; Dogs; Enzyme Inhibitors; Lanosterol; Methyltransferases; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Parasitic Sensitivity Tests; Toxicity Tests; Trichomonas vaginalis

The antiproliferative effects of two azasterols, 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R,S)-diol, AZA) and 24,25(R,S)-epiminolanosterol (EIL), in combination with sulphadiazine (SDZ) and pyrimethamine (PYR) were studied against Toxoplasma gondii tachyzoites growing in cultured mammalian cells. We had previously shown that AZA and EIL, two known inhibitors of Delta24(25)sterol methyl transferase in fungi and protozoa, have a potent and selective anti-T. gondii activity, although no 24-alkyl sterols have been detected in this parasite. We now report that when these sterol analogues were used in combination with the conventional SDZ/PYR treatment, potent synergistic effects were observed, ranging from 10- to 100-fold reductions of the IC50 values in the presence of sub-optimal doses of azasterols. When exposed to these drug combinations, intracellular T. gondii parasites displayed diverse subcellular alterations, including mitochondrial swelling, the arrest of the endodiogeny process with fragmented nuclei and subsequent cell lysis. These results suggest a potential new approach for the treatment of toxoplasmosis, which could significantly lower the required levels of antifolates and thus their adverse side effects.

Topics: Animals; Cell Line; Cholestanol; Coccidiostats; Drug Synergism; Drug Therapy, Combination; Folic Acid Antagonists; Host-Parasite Interactions; Lanosterol; Macaca mulatta; Parasitic Sensitivity Tests; Pyrimethamine; Sulfadiazine; Toxoplasma

We report potent and selective inhibitory effects of 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, known inhibitors of Delta24(25)-sterol methyltranferase (SMT) in fungi and protozoa, on the proliferation of Toxoplasma gondii in LLCMK2 cells. These compounds produced a dose-dependent reduction in parasite proliferation. 22,26-azasterol had an IC50 of 5.3 microM after 24 h and 4.5 microM after 48 h, while for 24,25-(R,S)-epiminolanosterol the IC50 values were 1 microM after 24 h and 0.12 microM after 48 h. The rapid reduction of parasite load suggested these compounds have selective cytotoxic effects against T. gondii. However, we were unable to detect 24-alkyl sterols in purified T. gondii tachyzoites using highly sensitive gas-liquid chromatography/mass spectrometry methods, a fact which indicated that the anti-proliferative effects of these azasterols were not mediated by inhibition of SMT. Transmission electron microscopy showed that the mitochondrion was the major target of drugs. Ultrastructural effects on plasma membrane, apicoplast and the formation of autophagosomal structures were also observed.

Topics: Animals; Cell Line; Cell Membrane; Cholestanol; Coccidiostats; Gas Chromatography-Mass Spectrometry; Lanosterol; Macaca mulatta; Methyltransferases; Mitochondria; Parasitic Sensitivity Tests; Phagosomes; Sterols; Toxoplasma

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.

Topics: Animals; Chlorocebus aethiops; Cholestanol; Cytochrome P-450 Enzyme Inhibitors; Ketoconazole; Lanosterol; Methyltransferases; Oxidoreductases; Sterol 14-Demethylase; Sterols; Trypanosoma cruzi; Vero Cells

The accepted mechanism for the antiproliferative effects of sterol biosynthesis inhibitors (SBI) against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, is the depletion of specific parasite sterols that are essential growth factors and cannot be replaced by cholesterol, the main sterol present in the vertebrate host. However, the precise metabolic roles of these specific parasite sterols are unknown. We approached this problem by subjecting T. cruzi epimastigotes to two types of SBI, inhibitors of sterol C-14 demethylase and delta 24(25) methyl transferase, and investigating the modification of lipid composition and enzyme activities in the plasma membranes of the parasite. We found in purified plasma membrane from SBI-treated cells that, together with the expected changes in the sterol composition, there was also an inversion of the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio and a large increase in the content of saturated fatty acids esterified to phospholipids. The modification of the phospholipid headgroup composition correlated with a 70% reduction in the specific activity of the membrane-bound PC-PE-N-methyl transferase SBI-treated cells; it was shown that this inhibition was not due to a direct effect of the drug on the enzyme. Finally, the specific activity of the Mg(2+)-dependent, vanadate-sensitive ATPase present in the membranes was also inhibited by ca. 50% in SBI-treated cells. The results suggest that one of the primary effects of the depletion of endogenous sterols induced by SBI in T. cruzi is a modification of the cellular phospholipid composition as a consequence of a reduced activity of PE-PC-N-methyl transferase and probably of the acyl delta 9 and delta 6 desaturases; this, in turn, could affect the activity of other enzymatic and transport proteins.

Topics: Animals; Ca(2+) Mg(2+)-ATPase; Cholestanol; Ketoconazole; Lanosterol; Membrane Lipids; Phospholipids; Sterols; Trypanosoma cruzi