*Article* **Steroidal Antimetabolites Protect Mice against** *Trypanosoma brucei*

**Minu Chaudhuri 1,\*, Ujjal K. Singha 1,†, Boden H. Vanderloop <sup>2</sup> , Anuj Tripathi <sup>1</sup> and W. David Nes 2,\***


**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 EC<sup>50</sup> 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.

**Keywords:** ergosta-5,7,22,24(28)-tetraenol (ERGT); cholesta-5,7,22,24-tetraenol (CHT); ergosterol biosynthesis; antimetabolite; suicide substrate; *Trypanosoma brucei*
