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 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.
Highlights
Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT)
Our previous studies of A. castellanii cells revealed that: i) two functional classes of SMT can operate in tandem, such that the 24(28)-olefin pathway catalyzed by SMT1 precedes the 25(27)-olefin pathway catalyzed by SMT2; ii) C29-sterol biosynthesis proceeds as a branch pathway of the canonical C28-sterol biosynthesis pathway to ergosterol (Fig. 2); and iii) the C28- and C29-sterol composition can undergo marked changes as trophozoites differentiate into resting cysts or lyse/ die (16, 17)
We further discovered that the proportion of C28- and C29sterols at growth arrest can change by supplementing the medium with either a tight binding inhibitor, such as 25-azacycloartanol, or a suicide substrate inhibitor, such as 26,27-dehydrolanosterol, that selectively target the AcSMTs that, thereby, kill trophozoites (Fig. 2, supplemental Fig. S1C)
Summary
Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Substrate analogs prepared with a toxic functional group to inactivate enzymes catalyzing the sterol C14-demethylation (CYP51) and sterol C24-methylation (SMT) reactions have proven harmful to protozoa viability by their ability to covalently bind CYP51 or SMT (14–17) These analogs may be considered a synthetic chemotype of antimetabolite for blocking ergosterol biosynthesis via protein alkylation. Foremost, 22,24-sterols, recognized as a new class of antibiotic, could affect metabolic challenges as variables in sterol genealogy/biosynthesis driven by SMT gene gain Likewise, these antimetabolites could replace intermediates or serve as product to compromise an evolving cholesterol biosynthesis pathway in animals capable of 22-introduction but constrained by reduced SMT gene expression or loss (19–21). We found the newly identified fungal antibiotics capable of protein alkylation in amoeba sterol biosynthesis provide a mechanism to limit the C28-/ C29-sterol assemblage across phylogeny
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