P450 14DM Research Articles

Synthesis, antifungal and antimycobacterial activities of new bis-imidazole derivatives, and prediction of their binding to P450 14DM by molecular docking and MM/PBSA method

New bis-imidazole derivatives have been synthesized and their antifungal and antimycobacterial activity was determined. Almost all compounds exhibited a moderate to good activity against two clinical isolates of Candida albicans 3038 and Candida glabrata 123. The same compounds showed an interesting killing activity against Mycobacterium tuberculosis H 37Rv reference strain. Docking procedures combined with molecular dynamics simulations in the MM/PBSA framework of theory were applied to predict the binding mode of all compounds in the binding pocket of the cytochrome P450 14α-sterol demethylase (14DM) of C. albicans, for which no ligand–protein crystal structure is currently available. The results obtained in silico showed that the active compounds may interact at the active site of protein, and that their binding free energy values are in agreement with the corresponding experimental activity values.

Electronic and structural features of lanosterol in the 14α-demethylation

14α-demethylation is the reaction which leads directly to norlanosterol from lanosterol, and is carried out exclusively by lanosterol structure. To discover the features, which make lanosterol a unique molecule able to undergo this demethylation, the electronic and energetic parameters of lanosterol and other structurally related steroids, were calculated. Local and global parameters were analyzed, in order to insight into the reactivity and selectivity of every molecule studied. Electrostatic potential maps were used to find differences of selectivity in each molecule, along with total energy and hardness, discovering the differences in reactivity. Lanosterol shows specific orientation and unique shape of electrostatic potential map, which does not appear in other structures, except epilanosterol, because it differs only in the orientation of a hydroxyl group, therefore they present many similarities but many differences also. For this reason, epilanosterol has a similar shape of electrostatic potential map, but not its orientation. Aoyama et al. have found, three essential structural features in lanosterol to be demethylated, which generate a specific electrostatic potential map, the hydroxyl group on C-3, the position of the double bound between C8 and C9 on cycle B, and the side chain double bond. Our study agrees with some biochemical studies, which reveal that there are three key features essential for substrate recognition by the enzyme P-450 14DM. We think the present study is an alternative methodology to find features which are related with some parameters obtained via theoretical calculations.

The pharmacokinetics of voriconazole.

Voriconazole (Vfend® Pfizer Global Research and Development, Sandwich, Kent, UK) is a broad-spectrum antifungal triazole that is structurally related to fluconazole. As with all triazole antifungal agents, voriconazole works principally by inhibition of cytochrome P450 14-α-demethylase (P450 14DM) [1, 2]. This enzyme is in the sterol biosynthesis pathway that leads from lanosterol to ergosterol. This causes a build up of 14-α-methyl sterols and is thought to impair the function of membrane-bound enzyme systems by disrupting the close packing of the acyl chains of phospholipids. This results in reduced fungal growth. Compared with fluconazole, voriconazole inhibits P450 14DM to a greater extent [3–5]. Absorption of voriconazole is essentially complete but the elimination of voriconazole is characterized by nonlinear pharmacokinetics [6]. Consequently, pharmacokinetic parameters are dependent upon dose. In vivo and in vitro studies have demonstrated that voriconazole is extensively metabolized, with the major circulating metabolite resulting from N-oxidation [7]. Voriconazole is a substrate for CYP2C9, CYP2C19, and CYP3A4. Therefore, CYP2C19 genotype and/or coadministration of drugs that modulate CYP2C19 or CYP3A4 activities could affect voriconazole plasma concentrations [7]. The purpose of this Supplement is to present the oral and intravenous pharmacokinetics of voriconazole in humans; to demonstrate the effect of food on the pharmacokinetics of the drug during multiple dosing; to show that there are no clinically significant effects of a histamine H2 receptor antagonist (cimetidine), macrolide antibiotics (erythromycin and azithromycin), a CYP2C19 substrate (omeprazole) and a protease inhibitor (indinavir) on voriconazole pharmacokinetics; to confirm that voriconazole does not alter the steady-state disposition of digoxin; to recommend dose adjustments of voriconazole when given with phenytoin; to warn of the potential for prolonged bleeding times when the drug is given with warfarin. The papers presented in this Supplement gather together what is known about the pharmacokinetics of voriconazole in man. By documenting the clinically unimportant and important drug–drug interactions of voriconazole in one place the Supplement will assist physicians in the safe and effective use of this drug.

Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis

The genetic locus encoding cytochrome P450 51 (CYP51; P450 14DM) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14α-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.

Antifungal agents. 10. New derivatives of 1-[(aryl)[4-aryl-1H-pyrrol-3-yl]methyl]-1H-imidazole, synthesis, anti-candida activity, and quantitative structure-analysis relationship studies.

The synthesis, anti-Candida activity, and quantitative structure-activity relationship (QSAR) studies of a series of 2,4-dichlorobenzylimidazole derivatives having a phenylpyrrole moiety (related to the antibiotic pyrrolnitrin) in the alpha-position are reported. A number of substituents on the phenyl ring, ranging from hydrophobic (tert-butyl, phenyl, or 1-pyrrolyl moiety) to basic (NH(2)), polar (CF(3), CN, SCH(3), NO(2)), or hydrogen bond donors and acceptor (OH) groups, were chosen to better understand the interaction of these compounds with cytochrome P450 14-alpha-lanosterol demethylase (P450(14DM)). Finally, the triazole counterpart of one of the imidazole compounds was synthesized and tested to investigate influence of the heterocyclic ring on biological activity. The in vitro antifungal activities of the newly synthesized azoles 10p-v,x-c' were tested against Candida albicans and Candida spp. at pH 7.2 and pH 5.6. A CoMFA model, previously derived for a series of antifungal agents belonging to chemically diverse families related to bifonazole, was applied to the new products. Because the results produced by this approach were not encouraging, Catalyst software was chosen to perform a new 3D-QSAR study. Catalyst was preferred this time because of the possibility of considering each compound as a collection of energetically reasonable conformations and of considering alternative stereoisomers. The pharmacophore model developed by Catalyst, named HYPO1, showed good performances in predicting the biological activity data, although it did not exhibit an unequivocal preference for one enantiomeric series of inhibitors relative to the other. One aromatic nitrogen with a lone pair in the ring plane (mapped by all of the considered compounds) and three aromatic ring features were recognized to have pharmacophoric relevance, whereas neither hydrogen bond acceptor nor hydrophobic features were found. These findings confirmed that the key interaction of azole antifungals with the demethylase enzyme is the coordination bond to the iron ion of the porphyrin system, while interactions with amino acids localized in proximity of heme could modulate the biological activity of diverse antifungal agents. In conclusion, HYPO1 conveys important information in an intuitive manner and can provide predictive capability for evaluating new compounds.

A three-dimensional model of lanosterol 14alpha-demethylase of Candida albicans and its interaction with azole antifungals.

The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF. The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures. The model was then refined using molecular mechanics and molecular dynamics. The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model. These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments. The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals. The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans. All 14 azole antifungals are shown to have a similar docking mode in the active site. The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate. The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132. The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.

Expression, purification, reconstitution and inhibition of Ustilago maydis sterol 14α-demethylase (CYP51; P450 14DM)

Triadimenol and tebuconazole are potent inhibitors of the sterol 14α-demethylation reaction in fungi which is catalysed by CYP51, a haem-thiolate containing enzyme belonging to the cytochrome P450 monooxygenase superfamily. Using CYP51 from the phytopathogen Ustilago maydis, a comparison of the sensitivity of the fungal enzyme to triadimenol and tebuconazole has been carried out. U. maydis CYP51 was purified to homogeneity as determined by SDS-PAGE and specific haem content. Catalytic activity was investigated following reconstitution with its respective NADPH cytochrome P450 reductase and proposed endogenous substrate, 24-methylenedihydrolanosterol. Addition of the triadimenol and tebuconazole induced type II spectral changes in the enzyme, with saturation occurring at equimolar azole concentrations. Inhibition of reconstituted activities showed a one-to-one sensitivity of the fungal CYP51 as judged by IC 50 values. The implications for fungicide mode of action and treatment are discussed.

Molecular diversity of sterol 14α-demethylase substrates in plants, fungi and humans

Metabolism of lanosterol (LAN), 24-methylene-24,25-dihydrolanosterol (24-methyleneDHL), dihydrolanosterol (DHL) and obtusifoliol (OBT) by purified human, plant ( Sorghum bicolor) and fungal ( Candida albicans) sterol 14α-demethylase (CYP51; P450 14DM) reconstituted with NADPH cytochrome P450 reductases was studied in order to elucidate the substrate specificity and sterol stereo- and regio-structural requirements for optimal CYP51 activity. Both human and C. albicans CYP51 could catalyse 14α-demethylation of each substrate with varying levels of activity, but having slightly higher activity for their respective endogenous substrates in vivo, dihydrolanosterol for human CYP51 ( V max=0.5 nmol/min/nmol CYP51) and 24-methylene-24,25-dihydrolanosterol for C. albicans CYP51 ( V max=0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol ( V max=5.5 nmol/min/nmol CYP51) and was inactive towards 14α-demethylation of lanosterol, 24-methylene-24,25-dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4β-methyl group in the sterol molecule renders the plant CYP51 incapable of 14α-demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.

Isolation of an Ustilago maydis ERG11 gene and its expression in a mutant deficient in sterol 14α-demethylase activity

A gene ( ERG11) encoding cytochrome P450 sterol 14α-demethylase (P450 14DM) was isolated from the maize pathogen, Ustilago maydis, by amplifying part of the coding region of the gene using PCR and by employing the amplified DNA fragment as a hybridization probe to recover the complete gene from an U. maydis λEMBL3 genomic library. The deduced amino acid sequence of the U. maydis gene showed homology to P450 14DMs from other organisms and contained specific motifs which were hallmarks of P450s. Expression of the gene in an U. maydis mutant (A20) deficient in P450 14DM led to only a partial restoration of P450 14DM activity. Accumulation of ergosta-7,22-dienol and ergost-7-enol in A20 transformants containing the ERG11 gene implied that an additional mutation affecting sterol Δ 5,6-desaturase activity accompanied the P450 14DM lesion.

Isolation of an Ustilago maydis ERG11 gene and its expression in a mutant deficient in sterol 14 alpha-demethylase activity.

A gene (ERG11) encoding cytochrome P450 sterol 14 alpha-demethylase (P450(14DM)) was isolated from the maize pathogen, Ustilago maydis, by amplifying part of the coding region of the gene using PCR and by employing the amplified DNA fragment as a hybridization probe to recover the complete gene from an U. maydis lambda EMBL3 genomic library. The deduced amino acid sequence of the U. maydis gene showed homology to P450(14DM)s from other organisms and contained specific motifs which were hallmarks of P450s. Expression of the gene in an U. maydis mutant (A20) deficient in P450(14DM) led to only a partial restoration of P450(14DM) activity. Accumulation of ergosta-7,22-dienol and ergost-7-enol in A20 transformants containing the ERG11 gene implied that an additional mutation affecting sterol delta 5,6-desaturase activity accompanied the P450(14DM) lesion.

Sterol 14-Demethylase P450 Activity Expressed in Rat Gonads: Contribution to the Formation of Mammalian Meiosis-Activating Sterol

Sterol 14-demethylase P450 (P45014DM)-dependent formation of 4,4-dimethylcholesta-8,14,24-trienol, that has been reported for a mammalian meiosis-activating sterol, in rat ovaries was confirmed. Transient elevation of ovarian P45014DM activity, that might be related to the female sexual cycle of rat, was observed. The P45014DM activity of immature ovaries was induced by pregnant mare's serum gonadotropin. These facts suggest the contribution of ovarian P45014DM to the gonadotropin-dependent formation of the meiosis-activating sterol, and provide a clue to understanding the mechanism how gonadotropins initiate the resumption of meiosis of mammalian oocytes. It was also found that rat testicular P45014DM participated in the production of 4,4-dimethylcholesta-8,24-dienol that was reported for another meiosis-activating sterol isolated from bull testes.

Isolation and molecular characterisation of the gene encoding eburicol 14 alpha-demethylase (cYP51) from Penicillium italicum.

The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14 alpha-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of Pl italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14 alpha-demethylase.

Effects of 19-Oxygenated Lanosterol Derivatives on 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Activity and Lanosterol Demethylase Activity.

The effects of 19-oxygenated lanosterol derivatives on lanosterol 14 alpha-demethylase (P-450(14DM)) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were studied. 3 beta-Hydroxylanost-9(11)-en-19-oic acid (6) was found to be an effective inhibitor (IC50:0.5 microM) of P-450(14DM) in the reconstituted system using purified pig liver P-450(14DM) and was most active in inhibiting HMG-CoA reductase activity (IC50:1.0 microM) in mouse L cells. In [2-14C]acetate incorporation studies using mouse L cells, 6 was found to reduce the incorporation of acetate into C27-sterol (desmosterol) with a concomitant increase in radiolabeled C30-sterols. These results demonstrate that 6 is a dual-action inhibitor of cholesterol biosynthesis.

Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.

Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450(14DM) (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BENr was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Effects of elevated expression of the CYP51 (P450 14DM) gene on the sterol contents of Saccharomyces cerevisiae

Lanosterol metabolism was eleveted in yeast cells cultured both aerobically and semi-anaerobically by increased expression of lanosterol 14α-demethylase P450 (P450 14DM or CYP51). However, despite increases in the diene-intermediates of ergosterol biosynthesis, the ergosterol content was not affected. These results suggested that the levels of cellular sterols are controlled in at least tow stages, and that the concomitant elevation of P450 14DM and ergostadienols desaturase (P450 22DS) is required to increase the cellular ergosterol content.

Studies on the Mechanism of Expression of a Major Gene Mutation for Resistance to Triadimenol in the Filamentous Phytopathogenic Ascomycete Nectria haematococca var. cucurbitae

The chromosomal major gene mutation at tri-1 locus gives high resistance to triadimenol and low resistance to other triazoles. The same mutation has no effect on sensitivity to nontriazole sterol biosynthesis inhibitors or even increases the sensitivity to certain imidazole derivatives. The tri-1 gene reduces mainly the fungitoxicity of diastereoisomer A of triadimenol (res. factor 58) and has little effect on sensitivity to diastereoisomer B (res. factor 5.5), indicating stereospecificity of the mutation to triadimenol A. There was a low reduction of the ergosterol content in total nonsaponifiable lipids with triadimenol concentrations highly inhibitory to growth of the mutant strain. The higher sensitivity of tri-1 strains to certain imidazoles was correlated with higher inhibition of the 14α-demethylation step of the ergosterol biosynthetic pathway. Electrophoretic profiles of microsomal fractions from a wild-type and a tri-1 strain showed the presence of one hemoprotein band with a molecular weight in the region of 125 kDa and an isoelectric point of 4.4. The connection of the heme with the apoprotein was found more unstable in the case of the enzyme from the mutant strain. Moreover, the peroxidase activity of electrophoresed mutant microsomes was greatly reduced after long storing at a low temperature. The presence of triadimefon in the growth medium increased the intensity of the hemoprotein band to the same extent in both wild-type and mutant strain. From the above results it may be concluded that a structural change in the cytochrome P450 14DM is probably responsible for the resistance of tri-1 strains to triazoles with no effect on 14α-demethylation ability of the enzyme.

Modeling cytochrome P450 14α demethylase ( Candida albicans) from P450cam

The tertiary structure of cytochrome P450 14α demethyl-ase—Candida albicans (P450 CA) is modeled on the basis of sequence alignment with two closely related proteins and the cristallographic structure of Pseudomonas putida P450cam. The secondary structure prediction system used combines the information from several algorithms and trains the data to offer an optimized prediction of the known P450cam. The trained algorithm was then used to predict the secondary structure of the other P450 sequences. The prediction of the surface coil regions was aided by an alignment between P450 CA and the homologous sequences P450 I4a demethylase—Saccharomyes cerevisiae (66 SD) and P450 14α demethylase—Candida tropicalis (72 SD). The prediction and alignment information was combined to establish an alignment between P450 CA and P450cam, and to assign full secondary structure to the target protein. This secondary structure was folded from the template of P450cam and the predicted structure was relaxed by molecular dynamics. Model checking highlighted minor adjustments in the alignment, correctly orienting hydrophobic and hydrophilic side chains. The model offers explanations for several known experimental results and suggests further investigations that may prove fruitful in understanding the structure and mechanisms of the P450 family (Porter, T.D. and Coon, M.J. Minireview cytochrome P450. J. Biol. Chem. 1991, 266, 13469-13472. Waterman, M.R. Cytochrome P450 cellular distribution and structural considerations. Current Opinion in Structural Biology 1992, 2, 384-387. Aoyama, Y., Yoshida, Y., Sonondo, Y. and Sato, Y. Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14α demethylase (P450 14DM) of yeast. Biochim. Biophys. Acta 7992, 1122, 251-255.).

Occurrence of a P450 Showing High Homology to Yeast Lanosterol 14-Demethylase (P450 14DM) in the Rat Liver

The rat liver cDNA library in λgt11 was screened with the antisera against rat liver lanosterol 14α-demethylase (P450 14DM), and one clone (pRT-9) was isolated. The open reading frame of pRT-9 cDNA showed high homology to yeast P450 14DM (48.9 % in nucleotides and 37.7 % in amino acids). Homology of the heme-binding region of pRT-9 protein to that of yeast P450 14DM was 80 %. Three additional regions showing more than 60 % homology were identified and one of them was assignable as the distal helix. These findings suggest that pRT-9 protein is P450 14DM and P450 14DMs of rat and yeast may be orthologous. This is the first evidence suggesting the occurrence of orthologous P450 in widely diverged species.

Resistance to Imidazoles and Triazoles in Saccharomyces cerevisiae as a New Dominant Marker

The imidazole and triazole fungicides inhibit cytochrome P450 14α-lanosterol demethylase (P450 14DM) implicated in the ergosterol biosynthesis pathway, which is specific to fungi and yeasts. Two plasmids were obtained which allow triazole and imidazole resistance in Saccharomyces cerevisiae . The low copy number plasmid (pFD 383) encodes cytochrome P450 C14α lanosterol demethylase under the control of phospho-glycerate-kinase promoter. S. cerevisiae transformed by the pFD 383 plasmid are resistant to imidazoles and triazoles. Moreover, this transformed strain shows increased levels of P450 14DM mRNA and of cytochrome P450. A second low copy number plasmid (pFD 384) carries a mutant cytochrome P450 14α lanosterol demethylase gene, which increases imidazole and triazole resistance. These constructions can be used on a dominant selection marker to transform wild-type yeasts and to confer imidazole and triazole resistance in industrial fermentation.

Inhibition by a novel azole antifungal agent with a geranyl group on lanosterol 14α-demethylase of yeast

AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2 E)-3,7-dimethylocta-2,6-dienyloxy)ethyl]-1 H-imidazole) is a new imidazole derivative characterized by a geranyl substituent showing strong antifungal activity. Azole antifungal agents are known to be potent inhibitors of lanosterol 14α-demethylase (P450 14DM) of fungi. The role of the geranyl group of AFK-108 on interaction of AFK-108 with the target was studied by using Saccharomyces cerevisiae P450 14DM as the model enzyme. AFK-108 and some of its derivatives bound to oxidized P450 14DM with one-to-one stoichiometry and inhibited the demethylase activity. AFK-108 derivatives having the longer farnesyl or the shorter prenyl group showed lower affinity than AFK-108 for the enzyme. AFK-108 caused 100% inhibition at the equivalent concentration to P450 14DM in the reaction mixture (0.07 μM), while the farnesyl derivative inhibited the activity by 60% at the same concentration. AFK-108 interfered with the binding of CO to the ferrous P450 14DM. However, the interfering effect of the prenyl derivative was lower than that of AFK-108. Another AFK-108 derivative having the saturated 3,7-dimethyloctyl group was also a weaker inhibitor than AFK-108. These experiments suggest that the geranyl group of AFK-108 interacts with the substrate binding site of P450 14DM that recognises the side chain of the substrate. AFK-108 is the first example of an azole derivative interacting with the side chain recognising region of the substrate binding site of P450 14DM.