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 #
5R01GM067871-15
Application #
9394012
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
2018-01-01
Budget End
2018-12-31
Support Year
15
Fiscal Year
2018
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
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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
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Hoekstra, William J; Hargrove, Tatiana Y; Wawrzak, Zdzislaw et al. (2016) Clinical Candidate VT-1161's Antiparasitic Effect In Vitro, Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi. Antimicrob Agents Chemother 60:1058-66
Hargrove, Tatiana Y; Friggeri, Laura; Wawrzak, Zdzislaw et al. (2016) Human sterol 14?-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design. J Lipid Res 57:1552-63
Yu, Xiaofeng; Nandekar, Prajwal; Mustafa, Ghulam et al. (2016) Ligand tunnels in T. brucei and human CYP51: Insights for parasite-specific drug design. Biochim Biophys Acta 1860:67-78
Yu, Xiaofeng; Cojocaru, Vlad; Mustafa, Ghulam et al. (2015) Dynamics of CYP51: implications for function and inhibitor design. J Mol Recognit 28:59-73
Hargrove, Tatiana Y; Wawrzak, Zdzislaw; Lamb, David C et al. (2015) Structure-Functional Characterization of Cytochrome P450 Sterol 14?-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs. J Biol Chem 290:23916-34

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