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1.
Mol Biochem Parasitol ; 160(1): 52-9, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18485498

ABSTRACT

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.


Subject(s)
Cholestanol/analogs & derivatives , Drug Resistance , Leishmania major/drug effects , Leishmania major/enzymology , Methyltransferases/isolation & purification , Animals , Cholestanol/pharmacology , Endoplasmic Reticulum/enzymology , Escherichia coli/enzymology , Leishmania major/ultrastructure , Microscopy, Electron , Microscopy, Fluorescence , Recombinant Proteins/isolation & purification , Transfection , Transport Vesicles/enzymology , Trypanocidal Agents/pharmacology
2.
Antimicrob Agents Chemother ; 50(8): 2595-601, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16870747

ABSTRACT

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.


Subject(s)
Antiprotozoal Agents/pharmacology , Aza Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Sterols/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Animals , Aza Compounds/chemical synthesis , Aza Compounds/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Methyltransferases/chemistry , Methyltransferases/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Sterols/chemical synthesis , Sterols/chemistry , Structure-Activity Relationship , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & development
3.
Antimicrob Agents Chemother ; 48(8): 2937-50, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15273104

ABSTRACT

This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites. Azasterols are a known class of (S)-adenosyl-L-methionine: Delta24-sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa. The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp. and Trypanosoma spp. The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT. The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed. When an acetate was attached to the 3beta-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism. Thus, an acetate group on the 3beta-OH may have application as a prodrug. However, there may be an additional mode(s) of action for these acetate derivatives. These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum. The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis.


Subject(s)
Antiprotozoal Agents/pharmacology , Aza Compounds/pharmacology , Leishmaniasis/drug therapy , Trypanocidal Agents/pharmacology , Trypanosomiasis/drug therapy , Animals , Humans , KB Cells , Leishmania donovani/drug effects , Leishmania donovani/enzymology , Leishmania donovani/growth & development , Leishmania major/drug effects , Leishmania mexicana/drug effects , Leishmania mexicana/enzymology , Leishmania mexicana/growth & development , Leishmaniasis/parasitology , Lipids/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Microscopy, Electron , Sterols/metabolism , Structure-Activity Relationship , Trypanosoma brucei rhodesiense/drug effects , Trypanosoma brucei rhodesiense/enzymology , Trypanosoma brucei rhodesiense/growth & development , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/growth & development , Trypanosomiasis/parasitology
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