Chapter 13
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Three-Dimensional Modeling of Cytochrome P450 14α-Demethylase (CYP51) and Interaction of Azole Fungicide Metconazole with CYP51 1
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Atsushi Ito , Keiichi Sudo , Satoru Kumazawa, Mami Kikuchi , and Hiroshi Chuman 3
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Nishiki Research Laboratories, Kureha Chemical Industry Company, Ltd., Fukushima, Japan Bio-Medical Research Laboratories, Kureha Chemical Industry Company, Ltd., Tokyo, Japan Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan
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We have reported the quantitative structure-activity relationships (QSAR) of metconazole, a triazole fungicide and its related compounds, and proposed the interaction mode between CYP51 and metconazole. To confirm our model, we constructed three-dimensional model of phytopathogen's CYP51 by homology modeling on the basis of the recently reported crystal structure of CYP51 of Mycobacterium tuberculosis (MTCYP51). Plant pathogen's CYP51s exhibit 25-28% amino acid sequence identities with MTCYP51. The residues located within 8 Å from fluconazole in the crystallized complex of MTCYP51 were identified. The selected amino acids were replaced with those of CYP51 of Botrytis cinerea (BCCYP51), and a model of interaction between metconazole and BCCYP51 was constructed. The modeled three-dimensional structure corresponded to our putative interaction model proposed with the QSAR analyses.
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© 2005 American Chemical Society Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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1 Introduction During the past two decades, many azole fungicides have been investigated all over the world (7). Azoles are a large and an important group of fungicides used for the control of fungal diseases in agriculture and medicine. The mode of action of azole fungicide is the inhibition of biosynthesis of ergosterol, which is an important component of fungal membranes (2-4). In the ergosterol biosynthesis, lanosterol 14a-demethylase (P450i m» CYP51) catalyzes the transformation of lanosterol to ergosterol by elimination of 14a-methyl group of lanosterol to give the A ' unsaturated sterol. Azole fungicide binds to this CYP51 and inhibits this reaction. Metconazole and ipconazole shown in Figure 1 are Kureha's azole fungicides (5). Metconazole is a foliar fungicide for controlling a wide range of cereal diseases. It is particularly effective against Fusarium, Septoria and rust diseases on cereals. Ipconazole is a seed treatment fungicide for controlling seed borne rice diseases such as 'bakanae' disease, Helminthosporium leaf spot and blast. 4D
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metconazole (CARAMBA®)
ipconazole (TECHLEAD®)
Figure 1. Structures of metconazole and ipconazole
The two fungicides are similar in chemical structure. The difference between them is the alkyl group on the cyclopentane ring. Metconazole has gem-dimethyl group and ipconazole has isopropyl group on cyclopentane ring, respectively. We have studied the structure-activity relationship of these fungicides and proposed the interaction mode between our azole fungicides and the target receptor, CYP51 (6). The interaction mode is schematically illustrated in Figure 2. The following interactions at the molecular level are proposed: A. The nitrogen atom at the 4-position of the triazole ring coordinates to the heme iron of CYP51. B. A hydrophobic pocket may be existed in the active site. The pocket accommodates the /wa-chlorophenyl group. C. The increase of the activity with the introduction of gem-dimethyl and
Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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isopropyl groups to the cyclopentane ring indicates that these alkyl groups play an essential role in binding to CYP51. These structures are possibly assigned as another hydrophobic interaction site. D. The presence of the hydroxyl group at 1-position of the cyclopentane ring was experimentally confirmed to be important for the activity. This group is considered to be involved in either hydrogen bond or electrostatic interaction with an amino acid residue in the active site of CYP51. hydrophobic interaction (I)
Figure 2. A proposed interaction mode for metconazole to the target enzyme, CYP51
2 Molecular Modeling of CYP51 To confirm our model, we attempted to construct three-dimensional model of plant pathogen's CYP51 by homology modeling. Some homology models of CYP51 have been reported. Most of them are models of Candida species studied mainly in pharmaceutical research. Because the target enzyme Candida CYP51 is a membrane-bound enzyme, it is difficult to crystallize for X-ray analysis. So these models have been constructed based on the crystal structure of prokaryotic cytochrome P450 family, such as P450cam, P450terp, P450eryF and P450BM3. Amino acid sequence identities between these four P45ÛS and Candida CYP51 are low (18-20%). But their topologies have been reported to be quite similar, so their crystal structure data have been used in homology modeling (7-/2). And recently crystal structure of CYP51fromMycobacterium tuberculosis (MTCYP51) complexed with azole fongicide fluconazole was determined and interaction mode was reported in 2001 (13). So we tried to construct the model of plant pathogen's CYP51 on the basis of the crystal structure of MTCYP51.
Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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145 2.1 Amino Acid Sequence Alignment Amino acid sequences of plant pathogen's CYPSls were taken from GenBank / EMBL / DDBJ databases. There are twelve plant pathogens' CYP51s: Botrytis cinerea (AAF85983), Erysiphe graminis (ACC97606), Septoria tritici (AAF74756), Tapesia yallundae (AAG44832), Tapesia acuformis (AAF18468), Uncinula necator (014442), Venturia inaequalis (AAF71293), Venturia nashicola (CAC85409A Monilinia fructicola (AAL79180), Ustilago maydis (CAA88176), Pénicillium digitatum (BAB03658) and Pénicillium italicum (CAA89824). Table I lists the fungicidal activities of metconazole against these plant pathogens. Although there are not all fungicidal data to those pathogens, metconazole shows high fungicidal activity.
Table I. Fungicidal activity of metconazole against plant pathogen Plant pathogen
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Botrytis cinerea Erysiphe graminis Septoria triticii Tapesia yallundae Tapesia acufomis Uncinula necator Venturia inaequalis Venturia nashicola Moniliniafructicola Ustilago maydis Pénicillium digitatum Pénicillium italicum
MIC (mg/1) in vitro
ED
50
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0.8
15.4 5.2
0.1 0.8
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