Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents.

The antifungal drug miconazole and the cholesterol-lowering agent lovastatin (mevinolin) were used in combination to assess their potency as anti-leishmanial agents. The drug combination was synergistic, being more potent in terms of inhibition of promastigote proliferation, macrophage infection and amastigote numbers. In promastigote cultures the effect was more marked in Leishmania amazonensis than L. donovani. Analysis of the sterol compositions of both promastigote and amastigote cultures revealed the inhibition of sterol 14 alpha-demethylation by miconazole and showed some apparent evidence of inhibition of sterol biosynthesis by lovastatin.

Paclobutrazol inhibition of sterol biosynthesis in a cell suspension culture and evidence of an essential role for 24-ethylsterol in plant cell division.

Growth of a celery (Apium gravidens) cell suspension culture was inhibited by the synthetic plant growth regulator paclobutrazol. Paclobutrazol caused a reduction in the incorporation of [2-14C]acetate into the 4-demethyl sterols (campesterol, sitosterol, stigmasterol) but radioactivity accumulated in the 4 alpha-methylsterols. The accumulating 4 alpha-methylsterols were identified as obtusifoliol and cycloeucalenol indicating that paclobutrazol was inhibiting sterol biosynthesis by blocking 14 alpha-demethylation. The inhibition of celery cell growth by paclobutrazol could be partially overcome by addition of cholesterol to the culture medium. However, addition of stigmasterol restored growth to the control value suggesting an essential role for a 24-ethylsterol to support plant cell division.

Effects of sinefungin on growth and sterol composition of Leishmania promastigotes.

The S-adenosylmethionine analogue sinefungin was tested in vitro against promastigotes of various strains of Leishmania. The IC50 values for the Leishmania mexicana, Leishmania major, and Leishmania donovani strains used were of the order of 10 ng/ml but the Leishmania amazonensis strain tested was more resistant to the drug, the IC50 value being 6 micrograms/ml. Sterol profiles, in which 24-alkyl (C28) sterols predominated, were relatively unaffected by sinefungin. Incorporation of label derived from either [methyl-2H3]methionine or [methyl-14C]methionine into sterols was not appreciably affected by treatment at a growth-inhibiting concentration of sinefungin. It was concluded that sinefungin had only a limited effect on sterol production at the 24-transmethylation step and it is unlikely that this is the primary cause of cell death.

Effects of an azasterol inhibitor of sterol 24-transmethylation on sterol biosynthesis and growth of Leishmania donovani promastigotes.

Leishmania donovani promastigotes were cultured in the presence of an azasterol (20-piperidin-2-yl-5 alpha-pregnane-3 beta,20-diol) to determine the effects on sterol biosynthesis and cell proliferation. Inhibition of growth increased gradually with azasterol concentrations up to 5 micrograms/ml; concentrations of azasterol exceeding 5 micrograms/ml were lethal. Sterol biosynthesis was affected by the azasterol when administered at concentrations as low as 100 pg/ml. The primary site of action was the alkylation at C-24 of a delta 24-sterol precursor. The 24-alkylated sterols [ergosta-5,7,24(24(1))-trien-3 beta-ol and ergosta-5,7,22-trien-3 beta-ol] of the protozoan were replaced by delta 24-cholesta-type sterols which then accumulated in the cells. Administration of the azasterol together with a bis-triazole inhibitor of the 14 alpha-methylsterol 14-demethylase reaction, which operates in sterol biosynthesis, resulted in depletion of 24-alkylsterols and their replacement with predominantly 14 alpha-methylsterols lacking a 24-alkyl group. Continuous subculture of promastigotes in the presence of the azasterol resulted in gradual depletion of 24-alkylsterols and their complete replacement by delta 24-cholesta-type sterols. Transfer of the azasterol-treated cells to medium lacking azasterol resulted in a gradual restoration, after several subcultures, of the normal 24-alkylsterol pattern. The results indicate that, although 24-alkylsterols are normally produced by the protozoan, it can nevertheless survive with sterols possessing only the cholestane skeleton. Thus there is no absolute requirement for 24-alkylsterols to fulfil some essential 'sparking' role associated with cell growth in promastigotes.