title: CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds.
abstract: CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogensTrypanosoma brucei,Trypanosoma cruziandLeishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killingT. cruziamastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity.TrypanosomatidaeCYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction.
authors.affiliation: Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA.
authors.forename: Tatiana Y
authors.initials: TY
authors.lastname: Hargrove
authors.forename: Kwangho
authors.initials: K
authors.lastname: Kim
authors.forename: Maria
authors.initials: M
authors.lastname: de Nazaré Correia Soeiro
authors.forename: Cristiane França
authors.initials: CF
authors.lastname: da Silva
authors.forename: Denise da Gama Jaen
authors.initials: DD
authors.lastname: Batista
authors.forename: Marcos Meuser
authors.initials: MM
authors.lastname: Batista
authors.forename: Eugenia M
authors.initials: EM
authors.lastname: Yazlovitskaya
authors.forename: Michael R
authors.initials: MR
authors.lastname: Waterman
authors.forename: Gary A
authors.initials: GA
authors.lastname: Sulikowski
authors.forename: Galina I
authors.initials: GI
authors.lastname: Lepesheva
keywords: CYP51; Crystal structure; Inhibition; Sterol 14α-demethylase