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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 Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(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-π 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.

A set of sterol methyl transferases (SMT) from amoeba, 2 synthesized in Acanthamoeba castellanii (Ac), and 1 in Naegleria gruberi (Ng), were cloned, functionally expressed in Escherichia coli and their substrate specificities determined. Bioinformatic analysis of the amino acid sequences for the protozoan-distinct SMT’s revealed that they possessed relatedness more to plants than to fungi. Enzyme-generated substrate and product specificities were determined by a combination of GC/MS, NMR, HPLC, and isotopic labelling with D3-SAM. The favored substrate for Ac24SMT was cycloartenol, Ac-28SMT was 24-methylenelophenol, and Ng24SMT was zymosterol, which corresponds to their natural distribution in the respective amoebas. Ac24SMT and Ng24SMT catalyzed sterol methylation through the Δ24(28) pathway affording a single product. Alternatively, Ac28SMT catalyzed sterol methylation through the Δ25(27) pathway, affording 4α-methyl-24βethyl-stigmasta-7,25(27)-dienol and other minor products. Inhibition of the three SMTs was demonstrated using high-affinity transition state analogs (TSA) of the sterol methylation reaction; the TSA were equally potent against the Ac and Ng SMTs and exhibited Ki’s in the low nanomolar range, consistent with their mechanism of action. These results show that a heretofore unrecognized substrate preference and product outcome of SMT catalyzed reactions that could influence ergosterol biosynthesis and growth of these parasitic organisms. SMT is a clear target enzyme that warrants further study in rational drug design targeting pathogenic amoeba.