title: Crystal structures of Trypanosoma brucei sterol 14alpha-demethylase and implications for selective treatment of human infections.
abstract: Sterol 14alpha-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 A resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA. galina.i.lepesheva@vanderbilt.edu
authors.forename: Galina I
authors.initials: GI
authors.lastname: Lepesheva
authors.forename: Hee-Won
authors.initials: HW
authors.lastname: Park
authors.forename: Tatiana Y
authors.initials: TY
authors.lastname: Hargrove
authors.forename: Benoit
authors.initials: B
authors.lastname: Vanhollebeke
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Wawrzak
authors.forename: Joel M
authors.initials: JM
authors.lastname: Harp
authors.forename: Munirathinam
authors.initials: M
authors.lastname: Sundaramoorthy
authors.forename: W David
authors.initials: WD
authors.lastname: Nes
authors.forename: Etienne
authors.initials: E
authors.lastname: Pays
authors.forename: Minu
authors.initials: M
authors.lastname: Chaudhuri
authors.forename: Fernando
authors.initials: F
authors.lastname: Villalta
authors.forename: Michael R
authors.initials: MR
authors.lastname: Waterman
mesh_terms: D000595:Amino Acid Sequence; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D008024:Ligands; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation; D058886:Sterol 14-Demethylase; D013261:Sterols / Q000096:biosynthesis; D013379:Substrate Specificity; D014344:Trypanocidal Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000188:drug therapy*
chemical_list: D001549:Benzamides; C548418:CYP51A1 protein, human; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D008024:Ligands; D013261:Sterols; D014344:Trypanocidal Agents; C037689:benzamide; D003577:Cytochrome P-450 Enzyme System; D058886:Sterol 14-Demethylase