Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei

Abstract

SUMMARY Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Herein, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while it has no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min-1/0.24 min-1; partition ratio of 1.08) whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ24-sterol is a promising strategy for developing new treatment for trypanosomiasis.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design

“…To examine the properties of the two Ac SMTs, a soluble enzyme extract was prepared by methods previously employed in the study of protozoa SMT cloned and expressed in bacterial cells that do not synthesize sterols. Although we observed increasing enrichment of Ac SMT during purification by SDS-PAGE (data not shown), differential loss of SMT activity as noted in previous studies in SMT purification can occur, yielding homogenous SMT with a low turnover number of ∼0.6 min −1 ( 15 , 16 , 19 ). Recently, we determined the reason for major loss of activity during protein purification is attributable to the allosteric nature of SMT (homotetramer showing cooperativity among its subunits), which contains a Walker motif for ATP binding in the SMT sequence.…”

“…9C ), gave for the intercept a maximum rate of inactivation of ∼0.4 min −1 and T 1/2 of 17 min. The partition ratio ( k cat / k inact ), a measure of formation of C26 methyl monol product per inactivation event, generated by 24-SMT incubated with DHL is ∼1, which is close to the partition ratio of 3 for 26-flourocholesta-5,7,24-trieniol against Tb SMT ( 16 ), suggesting DHL is a potent inactivator of Ac SMT activity. Selectivity of the time-dependent nature of covalent inhibitors such as DHL with their target proteins is often measured as k inact / K i values in preference to IC 50 values ( 12 ).…”

“…However, no relevant C4-methyl sterol intermediate or DHL metabolite was evident by GC/MS analysis. The lack of accumulation of cycloartenol following in vivo treatments by DHL is probably a result of inactivation of the 24-SMT affecting the enzyme turnover which, like the tight binding EL, blocks 24-SMT action ( 16 ). In contrast to our observations of 26-fluorolanosterol and DHL incubations in T. brucei where the lanosterol-based inhibitor was not a suicide substrate but rather was converted to an acceptable zymosterol analog capable of SMT binding (and thus acted as prodrugs, 16, 17), 15 µM DHL-treated cells at 48 h harvest showed DHL (RRTc, 1.24, M + 424) and 26-methyl DHL (RRTc, 1.26, M + 438) at 1% total sterol in an ∼10:1 ratio; no C26-methyl diol (M + 456) was present within our limits of detection in GC/MS analysis.…”

Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model

“…rhodesiense bloodstream forms in vitro [60]. Different enzyme inhibitors targeting CYP51 and C24-methyltransferase have also been identified [18,61–63]. Posaconazole is a potent inhibitor of the Cyp450-dependent lanosterol 14α-demethylase in yeasts and molds [64,65].…”

Evaluation of Secondary Metabolites from the Red Sea Tunicate Polyclinum Constellatum

“…A recent study has reported 24-Methylenecyclopropane as new class of steroidal inhibitors-mechanism-based suicide substrates with promising activity against T. brucei [26]. It would be interesting to investigate 24-methylene cholesterol as inhibitor of ergosterol biosynthesis as potential drug target [27]. …”