Sterol 14?-demethylase (CYP51) is the monooxygenase that catalyzes a unique three step reaction (14?- methyl ?14?-alcohol?14?-aldehyde?14?-demethylated product plus formic acid), removing the 14?-methyl group from the cyclized sterol precursors. CYP51 is regarded as a possible evolutionary ancestor to all members of the currently existing P450 superfamily. The CYP51 reaction is required for biosynthesis of cholesterol in humans, ergosterol in eukaryotic microbial human pathogens (such as fungi and protozoa), and various phytosterols in plants. The vast majority of the sterols are utilized for the formation of viable eukaryotic membranes (fluidity, permeability, modulation of multiple functions of integrated membrane proteins), while some sterols serve as precursors for a number of regulatory molecules essential for cell growth, development, and multiplication. Blocking sterol biosynthesis in unicellular organisms is lethal. CYP51 inhibitors (azoles) are used as the major clinical antifungal drugs and agricultural fungicides. Two of these drugs (posaconazole and ravuconazole) are presently in clinical trials for the deadly human infection caused by the protozoan pathogen Trypanosoma cruzi (Chagas disease). All of the azoles currently in use were discovered empirically, i.e. by screening their effects on fungal cell growth, and are far from ideal in terms of efficiency, safet, side effects, and sensitivity to resistance. The long-term goal of our research is to understand the molecular basis for the CYP51 functional conservation, drug action, and drug resistance. Here we propose to apply the information on the CYP51 structure/function that was gained during the previous cycles of funding to rationalize the CYP51-targeting drug discovery paradigm. We will perform protozoan/fungal/human CYP51 structure-guided derivatization of the highly potent, non-toxic experimental VNI scaffold to make pharmacologically-optimized drug candidates suitable for clinical trials for Chagas disease (including the infections caused by naturally resistant strains of the parasite) and visceral leishmaniasis (Aim 1a), as well as to develop new custom-designed inhibitors of fungal CYP51s (Aim 1b).
Aim 2 will evaluate human CYP51 as a potential drug target for cholesterol- related human diseases, including cancer and Alzheimer's disease.
Aim 3 of this proposal is to determine the structure of CYP51 in complex with its electron donor partner, NADPH-cytochrome P450 reductase, which will reveal the key residues involved in the protein-protein interaction, and may lead to the development of a novel type of CYP51 inhibitors. The results of this research will direct future approaches in drug discovery, enabling the creation of rationally designed species-oriented drug candidates directed at saving millions of human lives.

Public Health Relevance

The proposed research is directly relevant to public health because it is aimed at optimization of a new drug for Chagas disease and visceral leishmaniasis, and development of new structure-based inhibitors of fungal CYP51 enzymes. This research will also provide new insights into the potential applicability of human CYP51 as a target for treating cholesterol-related human diseases. The structural information on the interaction between CYP51 and its protein partner will open new avenues for the discovery of a novel type of CYP51 inhibitors that block the enzyme catalysis by preventing electron delivery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067871-16
Application #
9614297
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2004-01-01
Project End
2019-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
16
Fiscal Year
2019
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
37240
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
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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
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:

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