title: 24-Methylenecyclopropane steroidal inhibitors: A Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei.
abstract: A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15±5μM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24β-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (kcat/kinact=0.13min-1/0.08min-1; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC-MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases.
authors.affiliation: Department of Chemistry and Biochemistry and Center for Chemical Biology, Texas Tech University, Lubbock, TX 79409, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Miller
authors.affiliation: Department of Chemistry and Biochemistry and Center for Chemical Biology, Texas Tech University, Lubbock, TX 79409, USA.
authors.forename: Presheet
authors.initials: P
authors.lastname: Patkar
authors.affiliation: Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37208, USA.
authors.forename: Ujjal K
authors.initials: UK
authors.lastname: Singha
authors.affiliation: Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37208, USA.
authors.forename: Minu
authors.initials: M
authors.lastname: Chaudhuri
authors.affiliation: Department of Chemistry and Biochemistry and Center for Chemical Biology, Texas Tech University, Lubbock, TX 79409, USA. Electronic address: wdavid.nes@ttu.edu.
authors.forename: W
authors.initials: W
authors.lastname: David Nes
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D003521:Cyclopropanes / Q000494:pharmacology*; D004875:Ergosterol / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008745:Methylation / Q000187:drug effects; D008780:Methyltransferases / Q000378:metabolism; D010802:Phylogeny; D015800:Protozoan Proteins / Q000378:metabolism; D013256:Steroids / Q000037:antagonists & inhibitors*; D013261:Sterols / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000378:metabolism; D014353:Trypanosomiasis, African
chemical_list: D003521:Cyclopropanes; D015800:Protozoan Proteins; D013256:Steroids; D013261:Sterols; C508672:methylenecyclopropane; D002784:Cholesterol; D008780:Methyltransferases; D004875:Ergosterol
keywords: Ergosterol biosynthesis inhibitor; Human African trypanosomiasis; Sleeping sickness; Sterol C24-methyltransferase; Suicide substrate