Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei.
Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.
Zhou
Wenxu
W
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Cross
George A M
GA
Nes
W David
WD
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001692:Biological Transport; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004875:Ergosterol / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D020128:Inhibitory Concentration 50; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015394:Molecular Structure; D021261:Parasitic Sensitivity Tests; D051381:Rats; D013261:Sterols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000981:Antiprotozoal Agents; D013261:Sterols; D002784:Cholesterol; D008780:Methyltransferases; D004875:Ergosterol