Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications

Wenxu Zhou, Emilio Ramos, Xunlu Zhu, Paxtyn M. Fisher, Medhanie E. Kidane, Boden H. Vanderloop, Crista D. Thomas, Juqiang Yan, Ujjal Singha, Minu Chaudhuri, Michael T. Nagel, W. David Nes

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Abstract

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via 24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via 25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation- interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Original languageEnglish
Pages (from-to)981-994
Number of pages14
JournalJournal of lipid research
Volume60
Issue number5
DOIs
StatePublished - 2019

Keywords

  • Anti-amoeba drugs
  • Sterol biosynthesis
  • Suicide substrate

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    Zhou, W., Ramos, E., Zhu, X., Fisher, P. M., Kidane, M. E., Vanderloop, B. H., Thomas, C. D., Yan, J., Singha, U., Chaudhuri, M., Nagel, M. T., & David Nes, W. (2019). Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications. Journal of lipid research, 60(5), 981-994. https://doi.org/10.1194/jlr.M091587