Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Wenxu Zhou, George A. M. Cross, and W. David Nes

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409

Corresponding Author: wdavid.nes{at}ttu.edu

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 three orders of magnitude different with cholesterol 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 structure of these sterols reveal a non-conventional 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 [K_i 39 nM], was found to inhibit the growth of the procyclic and bloodstream forms at an IC₅₀ of ca. 1 µM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using 25-azalanosterol-treated procyclic form which, compared to control cultures, causes a change in cellular sterol content from ergostenols to cholesterol. However, growth of the blood stream form disrupted by 25-azalanosterol is 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.

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