TY - JOUR
T1 - Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications
AU - Zhou, Wenxu
AU - Ramos, Emilio
AU - Zhu, Xunlu
AU - Fisher, Paxtyn M.
AU - Kidane, Medhanie E.
AU - Vanderloop, Boden H.
AU - Thomas, Crista D.
AU - Yan, Juqiang
AU - Singha, Ujjal
AU - Chaudhuri, Minu
AU - Nagel, Michael T.
AU - David Nes, W.
N1 - Publisher Copyright:
© 2019 Zhou et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Anti-amoeba drugs
KW - Sterol biosynthesis
KW - Suicide substrate
UR - http://www.scopus.com/inward/record.url?scp=85065540841&partnerID=8YFLogxK
U2 - 10.1194/jlr.M091587
DO - 10.1194/jlr.M091587
M3 - Article
C2 - 30709898
AN - SCOPUS:85065540841
VL - 60
SP - 981
EP - 994
JO - Journal of Lipid Research
JF - Journal of Lipid Research
SN - 0022-2275
IS - 5
ER -