The cytochrome P450 superfamily contains more than 6,500 genes constituting at least 711 gene families. Only one of these gene families, CYP51, is found in all biological kingdoms, bacteria to humans. CYP51 catalyzes an essential step in sterol biosynthesis, the sterol 141-demethylation reaction which is the first step in the postsqualene portion of the pathway. This competing renewal will continue studies of CYP51 with a long term goal of developing detailed understanding of inhibition of this important drug target. We will determine the high resolution X-ray structure of this enzyme from different eukaryotes, including rat, Candida albicans and trypanosomes (T. brucei and T. cruzi). The more than 100 CYP51 sequences known throughout biology contain 29 conserved amino acids, the CYP51 signature. When certain of these amino acids are mutated in the soluble form of CYP51 from bacteria (M. tuberculosis) and compared to the same mutation in membrane bound eukaryotic forms (human, trypanosomes), significant functional differences are observed. Therefore we believe that 3D structure including functional conformational dynamics in the prokaryotic CYP51 may be quite different from those in eukaryotic forms emphasizing the need to determine eukaryotic CYP51 structure to compare with the bacterial structure, leading to better understanding of the general principles of CYP51 conservation. CYP51 is a well known drug target for pathogenic yeast/fungal infections and azole CYP51 inhibitors are used to treat such infections. However, it is challenging to develop drugs which are specific for the pathogen and do not target host (human) P450s. We will investigate two types of inhibitors for their effectiveness in inhibition of CYP51 from different organisms, particularly those from trypanosomes. Both substrate-based and nitrogen-based inhibitors will be examined because they bind differently in the P450 active site region. Also we will carry out high throughput screening of a 160,000 chemical library to search for tight binding molecules which might also be inhibitors of CYP51. Having identified potent inhibitors in vitro we will co-crystallize them with CYP51 orthologs (emphasis on trypanosomal forms) in order to understand in detail the structural basis of inhibition. This study will provide insight into how the CYP51 protein structure influences tight binding of both types of inhibitors and will suggest what structural features of catalytic inhibitors of CYP51 are most important for this inhibition process. We will also study the effect of the most potent inhibitors in cellular forms of trypanosomes and subsequently in well established mouse models for T. cruzi infection. These studies will lead to a detailed understanding of the structure/function relationship of this essential P450, and also will establish a detailed paradigm for discovery of specific drugs for the infectious protozoa, T. brucei and T. cruzi, and perhaps information on potential lead compounds for treatment of such infections. It can be expected that a paradigm for development of specific inhibitors of sterol 141-demethylase will arise from these studies. These inhibitors will be specific for the enzymes in pathogenic protozoa such as trypansomes and have little or no effect on the host (human) enzyme. As a result of this inhibition, the protozoa will not be able to synthesize sterols and can not survive.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067871-08
Application #
8022865
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2003-04-01
Project End
2011-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
8
Fiscal Year
2011
Total Cost
$327,502
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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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
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:
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
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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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