Steroidal Antimetabolites Protect Mice against Trypanosoma brucei

Abstract

Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.

Druggable Sterol Metabolizing Enzymes in Infectious Diseases: Cell Targets to Therapeutic Leads

“…In cell-based T. brucei and T. cruzi , MCP was shown to inhibit growth generating IC 50 of 6 µM and against the cell-free enzyme to exhibit time-dependent inhibition, results that are consistent with its irreversible inhibitor properties [ 103 ]. These observations partially satisfied the Bait and Switch approach for k cat inhibitors specific to blocking ergosterol production in parasites, Section 3.2 [ 72 , 100 , 101 , 113 ].…”

“…In other routine screening of steroidal analogs common to yeast used as negative controls for growth inhibition, we observed quite unexpectedly that cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), generated rapid cell death against the parasitic A. castellanii and T. brucei cells [ 112 , 113 ]. Since there was no precedent for the induced toxicity in protozoa or any other eukaryotic system resulting from feeding yeast sterols to the growth media, the growth responses were anomalous.…”

“…In similar fashion, CHT and ERGT independently were shown to inhibit growth of A. castellanii trophozoites generating IC 50 values of 51 nM with total cell lysis occurring within 48 h of treatment. Further, testing CHT in a mouse model of T. brucei infection led to significant increases in survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg [ 113 ], showing promise for these compounds as therapeutic leads. Incubation of CHT and ERGT up to 40 μM with HEK cells failed to affect growth, even though the compounds were accumulated by the cells, and minimally metabolized in the ring system of Δ 5,7 to Δ 5 (as in cholesterol biosynthesis)—not side chain, and they did not have any effect on cholesterol production.…”

“…Consequently, we next focused our investigations on incubations of CHT and ERGT with cloned C24-SMTs from T. brucei and A. castellani . Kinetic study of bound yeast sterols to TbSMT1, AcSMT1 and AcSMT2, chemical analysis of the analog covalently bound to C24-SMT by Tandem MS, mutagenesis in Region I of the C24-SMT, and GCMS analysis of extracts of enzymes reacted with the yeast sterols showed that the active site of this enzyme from different protozoa recognized the yeast sterols [ 72 , 112 , 113 ]. What made the natural product yeast sterols even more potent than the synthetic analogs we had designed, based on their structural attributes, to generate HEIs at C24 and C25, was the apparent misplacement of the sterol side chain in a region of the active site not normally occupied by the intermediate ion.…”

Editorial: A Feasible Approach for Natural Products to Treatment of Diseases

“…As a flagellated parasitic protozoan, Trypanosoma brucei causes African trypanosomiasis, a neglected tropical disease. According to Chaudhuri et al [7], two natural antimetabolites, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), possess antiprotozoal properties as a trypanocidal agent against T. brucei. In addition, CHT/ERGT protected mice infected with T. brucei by increasing their survival time.…”