CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds

Hargrove, Tatiana Y.; Kim, Kwang‐Ho; Soeiro, Maria de Nazaré Correia; Silva, Cristiane França da; Batista, Denise da Gama Jaén; Batista, Marcos Meuser; Yazlovitskaya, Eugenia M.; Waterman, Michael R.; Sulikowski, Gary A.; Lepesheva, Galina I.

doi:10.1016/j.ijpddr.2012.06.001

Cited by 46 publications

(78 citation statements)

References 42 publications

(50 reference statements)

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“…The objective of this work was to examine structure/function relationships and drugability of human CYP51. (48,62) as inhibitors of protozoan (T. cruzi), fungal (A. fumigatus), and human CYP51 enzymes. In these experiments, we used a 60 min reaction because the longer incubation time affords higher sensitivity and, therefore, is helpful in screening out less effective inhibitors (48,51,57).…”

Section: Resultsmentioning

confidence: 99%

“…(48,62) as inhibitors of protozoan (T. cruzi), fungal (A. fumigatus), and human CYP51 enzymes. In these experiments, we used a 60 min reaction because the longer incubation time affords higher sensitivity and, therefore, is helpful in screening out less effective inhibitors (48,51,57). Interestingly, under the same experimental conditions, while only a 2-fold molar excess over the enzyme was needed for the most potent compounds to cause complete inhibition of T. cruzi CYP51 (VNI derivatives) or 80-95% inhibition of A. fumigatus CYP51 (voriconazole, posaconazole, and VNI), human CYP51 displayed resistance to inhibition even at a 50-fold molar excess of the compounds.…”

Section: Resultsmentioning

confidence: 99%

“…VNI, VNT, VNF, and VFV were synthesized by the Chemical Synthesis Core Facility (Vanderbilt Institute of Chemical Biology) (48).…”

Section: Methodsmentioning

confidence: 99%

“…Our experimental CYP51 structure-based VNI derivative, VFV, was designed to extend the antiprotozoal spectrum of activity (48,62). VFV has low cytotoxicity [e.g., NIH/3T3 mouse embryonic fibroblasts, EC 25 >50 M (41)], excellent cellular permeability, favorable pharmacokinetics, broad tissue distribution, weak inhibitory effects on human drug-metabolizing P450s, and no adverse side effects when used in vivo in mouse models of Chagas disease and visceral leishmaniasis (62).…”

Section: Inhibitory Effects Of Vfv On Proliferation Of Different Typementioning

confidence: 99%

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Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

Hargrove

Friggeri

Wawrzak

et al. 2016

Journal of Lipid Research

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…VNI, VNT, VNF, and VFV were synthesized by the Chemical Synthesis Core Facility (Vanderbilt Institute of Chemical Biology) (48).…”

Section: Methodsmentioning

confidence: 99%

Section: Inhibitory Effects Of Vfv On Proliferation Of Different Typementioning

confidence: 99%

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Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

Hargrove

Friggeri

Wawrzak

et al. 2016

Journal of Lipid Research

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“…For the eukaryotic cytochrome P450 enzymes that contain transmembrane segments, most X-ray structures have been obtained after removal of the N-terminal transmembrane domain to facilitate expression and crystallization (13)(14)(15)(16)(17)(18), as is the case with many other monospanning membrane protein structures. Resolved CYP51 structures include the soluble enzyme from Mycobacterium tuberculosis (19) and N-terminal truncated enzymes from Homo sapiens (20), Trypanosoma cruzi (21), Trypanosoma brucei (22), and Leishmania infantum (23). The available eukaryotic cytochrome P450 structures show the catalytic subunit has a conserved fold surrounding a central prosthetic heme group.…”

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confidence: 99%

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

283

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Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.M embrane proteins that span the lipid bilayer once constitute around 50% of all integral membrane proteins (1). Although monospanning membrane proteins carry out numerous key biological functions, including environmental sensing, organellespecific catalysis, and the regulation of cell morphology, only individual domains or subdomains are currently represented in the Protein Data Bank, and structural information about interactions between their transmembrane domains and extramembranous components is lacking. Cytochrome P450 proteins are prominent enzymes with orthologs found in all kingdoms of life. In eukaryotes, microsomal members of this major family of mixed-function mono-oxygenases contain a single transmembrane helix and can be grouped in two broad functional categories: biodefense, such as the first phase of xenobiotic detoxification, and core metabolism including reactions in sterol biosynthesis and fatty acid oxidation (2).The lanosterol 14α-demethylases or CYP51 enzymes, probably the most genetically ancient of the cytochrome P450 families, play a central role in cholesterol or ergosterol biosynthesis (3). CYP51s carry out three consecutive mono-oxygenase reaction cycles to remove the 14α-methyl group from lanosterol to yield 4,4-dimethyl-cholesta-8,14,24-trienol, a key precursor in cholesterol and ergosterol biosynthesis, releasing water and formic acid (3). Because of the key roles that CYP51s play in yeast, filamentous fungi, and some parasitic protozoa, these enzymes are therapeutic targets for antimicrobial agents, including fluconazole (FLC), voriconazole (VCZ), and itraconazole (ITC) (4). Fungal infections play an increasingly significant role in disease, impacting agriculture ecosystems and human health, especially in immunocompromised individuals (5-7) for whom antifungal resistance continually poses a threat (8). In humans CYP51 is being tested as a target for cholesterol-lowering drugs (9) and in antiangiogenic cancer therapies (10). A limited set of cytochrome P450 isoforms (1A2, 2C8, 2C...

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Dynamics of CYP51: implications for function and inhibitor design

Cojocaru

Mustafa

et al. 2015

J of Molecular Recognition

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Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes.

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CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds

Cited by 46 publications

References 42 publications

Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Dynamics of CYP51: implications for function and inhibitor design

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