Sterol 14?-demethylases are the cytochrome P450 enzymes found in all biological kingdoms and, regardless of their low (22-35%) sequence identity across phylogeny, grouped into one family (CYP51) because of their strict functional conservation. From bacteria to humans, they all catalyze the same unusual three-step reaction of the oxidative removal of the 14?-methyl group from one or more of five cyclized sterol precursors (14?- methyl ?14?-alcohol?14?-aldehyde?14?-demethylated product plus formic acid). Eukaryotic microsomal membrane-bound CYP51s use NADPH-cytochrome P450 reductase (CPR) as their redox partner, while water-soluble bacterial orthologs accept electrons from ferredoxins and/or flavodoxins. The CYP51 reaction is required for biosynthesis of sterols, which are essential for eukaryotic membrane biogenesis and also serve as precursors for a variety of regulatory molecules that are involved in cellular growth, development, and division processes (hormones, vitamins, nuclear receptors, etc.). For more than 50 years, the CYP51 reaction has served as the target for clinical antifungal drugs and agricultural fungicides (imidazoles, triazoles, or sometimes pyridines), yet the enzyme per se has not been included in the drug discovery paradigm because of the difficulties of its handling. Our long-term goal is to understand what makes/keeps a CYP51 a CYP51 and what structural features of this P450 can be used to make rationally designed, potent, and functionally irreversible species-selective inhibitors. We have found that while upon binding of exogenous ligands (azoles, pyridines, and even a substrate analog) CYP51s remain in their resting, ligand-free-like state, accommodation of the physiological substrate causes a large-scale conformational switch that involves the backbone of the active site and the surface of interaction with the electron donor partner, preparing the enzyme for catalysis.
The aims of the current renewal application are 1) to determine, by combining cryo-electron microscopy and X-ray crystallography, the structures of the complex of the substrate-bound CYP51/CPR and the substrate- bound Methylococcus capsulatus CYP51/ferredoxin fusion; 2) to use computational structural biology to better understand CYP51 molecular dynamics; 3) to evaluate the efficacy of our two VNI derivatives with optimized pharmacokinetics in the mouse models of Chagas disease (caused by three naturally drug resistant strains of Trypanosoma cruzi) and in the mouse model of sleeping sickness (Trypanosoma brucei), to analyze our in-house library of CYP51 inhibitors against a fungus Cryptococcus neoformans (cryptococcal meningitis) and two free-living pathogenic amoebas, Acanthamoeba castellanii (blinding keratitis) and Nagleria fowleri (primary amebic meningoencephalitis), and to test our two potent functionally irreversible inhibitors of human CYP51 in cancer cell lines and in cytomegalovirus infected human cells.

Public Health Relevance

Structural characterization of CYP51 complex with its redox partner may lead to a novel type of inhibitors that block the CYP51 electron transfer channel, which would help with the problem of azole resistance. Understanding CYP51 structural dynamics will rationalize the development of potent, functionally irreversible species-oriented CYP51 inhibitors. Testing the VNI derivatives with optimized pharmacokinetics in animal models of Chagas disease and sleeping sickness will advance the best candidate to clinical trials; inhibition of CYP51 from pathogenic amoebas and Cryptococcus neoformans may provide a cure for primary amebic meningoencephalitis, blinding keratitis, and more efficient treatment of cryptococcal meningitis; potent inhibitors of human CYP51 may add to the arsenal of drugs for cholesterol-related diseases and post-cancer chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067871-18
Application #
10077559
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Garcia, Martha
Project Start
2004-01-01
Project End
2023-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
18
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
965717143
City
Nashville
State
TN
Country
United States
Zip Code
37203
Hargrove, Tatiana Y; Wawrzak, Zdzislaw; Fisher, Paxtyn M et al. (2018) Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51. J Biol Chem 293:19344-19353
Friggeri, Laura; Hargrove, Tatiana Y; Wawrzak, Zdzislaw et al. (2018) Sterol 14?-Demethylase Structure-Based Design of VNI (( R)- N-(1-(2,4-Dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)) Derivatives To Target Fungal Infections: Synthesis, Biological Evaluation, and Crystallographic J Med Chem 61:5679-5691
Friggeri, Laura; Hargrove, Tatiana Y; Rachakonda, Girish et al. (2018) Sterol 14?-Demethylase Structure-Based Optimization of Drug Candidates for Human Infections with the Protozoan Trypanosomatidae. J Med Chem :
Lepesheva, Galina I; Friggeri, Laura; Waterman, Michael R (2018) CYP51 as drug targets for fungi and protozoan parasites: past, present and future. Parasitology 145:1820-1836
Hargrove, Tatiana Y; Friggeri, Laura; Wawrzak, Zdzislaw et al. (2017) Structural analyses of Candida albicans sterol 14?-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis. J Biol Chem 292:6728-6743
Mwenechanya, Roy; Ková?ová, Julie; Dickens, Nicholas J et al. (2017) Sterol 14?-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana. PLoS Negl Trop Dis 11:e0005649
Hargrove, Tatiana Y; Garvey, Edward P; Hoekstra, William J et al. (2017) Crystal Structure of the New Investigational Drug Candidate VT-1598 in Complex with Aspergillus fumigatus Sterol 14?-Demethylase Provides Insights into Its Broad-Spectrum Antifungal Activity. Antimicrob Agents Chemother 61:
Guedes-da-Silva, F H; Batista, D G J; Da Silva, C F et al. (2017) Antitrypanosomal Activity of Sterol 14?-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain. Antimicrob Agents Chemother 61:
Porta, Exequiel O J; Jäger, Sebastián N; Nocito, Isabel et al. (2017) Antitrypanosomal and antileishmanial activity of prenyl-1,2,3-triazoles. Medchemcomm 8:1015-1021
Lepesheva, Galina; Christov, Plamen; Sulikowski, Gary A et al. (2017) A convergent, scalable and stereoselective synthesis of azole CYP51 inhibitors. Tetrahedron Lett 58:4248-4250

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