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Expression and homology modeling of sterol 14α-demethylase from Penicillium digitatum
Abstract
Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.
Supplementary resource (1)
Nucleotide Sequence
January 2006
... Homology modeling has been widely applied in three-dimensional model building of CYP51s to understand molecular interactions between inhibitors and target enzymes [17,21]. The crystal structure of M. tuberculosis CYP51 (MtCYP51) has been used in many CYP51 modeling studies [4,22,23]. However, MtCYP51 has only 27.2% sequence identity with the PiCYP51A. ...
... In the current study, we have constructed a PiCYP51A model for the first time with the core region of PiCYP51 proteins (Figure 2), containing the most conserved fold structure reported in the other P450 proteins [16][17][18][21][22][23], including helices E, I, J and L. Each of these helices has at least two aromatic-ring-containing residues (Phe and/or Tyr) involved in the hydrophobic interacting with some specific receptor(s). On the other hand, the present PiCYP51A model also suggested the critical role of nitrogen (N) atoms in the azole and aromatic rings in the coordination bond formation, as experimentally reported for the ligand-heme Fe(II) interaction in PiCYP51A active site [4]. ...
... The homology model of PiCYP51 was built based on the crystal structure of the human CYP51 (PDB: 3LD6) by SWISS-MODEL, as previously described [22,27]. The model was further relaxed by molecular dynamic (MD) simulations using GROMACS 4.5.5 program package [23,28]. The 5 ns MD simulation was processed under GROMOS96 43a1 force field, within SPC3 water model, and at 300 K/1 atm conditions. ...
Sterol 14α-demethylases from Cytochrome P450 family (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. The 3D structure of CYP51A from Penicillium italicum (PiCYP51A) was constructed through homology modeling based on the crystal structure of human CYP51A (PDB: 3LD6). Molecular dynamics (MD) simulation was operated to relax the initial model and followed by quality assessment using PROCHECK program. On the basis of the docking information on the currently available CYP51s with the patent demethylase inhibitors (DMIs), pharmacophore-based virtual screening combined with docking analysis was performed to pick out twelve new compounds from ZINC database. Six hits revealed in the ligand database suggested potential ability to inhibit PiCYP51A. Compared to patent fungicide triazolone, the top three lead compounds had similar or higher affinity with the target enzyme, and accordingly, exhibited comparable or lower EC50 values to P. italicum isolates. The results could provide references for de novo antifungal drug design.
... The pharmacophoric conformations of azole antifungal agents were docked into the active site of the enzyme and azolebinding residues in the active site were identified. The crystal structure of MtCYP51 was used in many CYP51 modeling studies (Sheng et al. 2004; 2009 , Zhao et al. 2007); however, MtCYP51 has only 27% sequence identity with the PdCYP51 enzyme and is unusually exposed to the bulk solvent at the substrate binding site. The model based on MtCYP51 is not accurate enough to elucidate the structure of PdCYP51 and the binding site of fungicides and other potential inhibitors. ...
... Expression of PdCYP51 and spectral analysis of the binding of inhibitors Penicillium digitatum was obtained from the China General Microbiological Culture Collection Centre (CGMCC 550). The PdCYP51 was expressed in bacteria and purified as previously described (Zhao et al. 2007). In brief, cloning and expression of PdCYP51 was carried out by using reverse transcription PCR (RT-PCR) with the purified mRNA isolated from P. digitatum. ...
... N-4 of the triazole ring was bound to the heme iron with a distance less than 3.0 A ˚ ´ . The diflurophenyl or monochlorophenyl groupTable 1 The molecular docking interaction energy (kcal mol -1 ) of PdCYP51 with inhibitors and K d values obtained from spectra assays the determination of the binding of inhibitors to P450s (Zhao et al. 2007). Based on their abilities to inhibit 14a-demethylase activity, the binding capabilities of diniconazole, tebuconazole, triadimenol and triadimefon with the purified PdCYP51 were examined. ...
Sterol 14α-demethylase from Penicillium digitatum (PdCYP51) is a prime target of antifungal drugs for citrus disease in plants. To design novel antifungal compounds, a homology model of PdCYP51 was constructed using the recently reported crystal structure of human CYP51 as the template. Molecular docking was performed to investigate the interaction of four commercial fungicides with the modeled enzyme. The side chain of these compounds interplayed with PdCYP51 mainly through hydrophobic and van der Waals interactions. Biochemical spectra analysis of inhibitors combined with PdCYP51 are also compatible with the docking results. This is the first molecular modeling for PdCYP51 based on the eukaryotic crystal structure of CYP51. The structural information and binding site mapping of PdCYP51 for different inhibitors obtained from this study could aid in screening and designing new antifungal compounds targeting this enzyme.
... The ERG11 gene sequences of a wide range of fungal species that are harmful to agricultural crops and human health have been determined and characterized. These species include Antrodia cinnamomea (Lee et al., 2010), Aspergillus fumigatus (Mellado et al., 2001;Warrilow et al., 2010), Botrytis cinerea (Albertini et al., 2002), Candida albicans (Lai and Kirsch, 1989;Park et al., 2011), Candida glabrata (Kairuz et al., 1994), Cryptococcus neoformans (Revankar et al., 2004;Sheng et al., 2009), Penicillium digitatum (Zhao et al., 2007), Malassezia globosa , Monilinia fructicola, previ-ously known as Monilia fructicola (Luo and Schnabel, 2008), Pneumocystis carinii (Morales et al., 2003), Saccharomyces cerevisiae (Kalb et al., 1987), Oculimacula yallundae, previously known as Tapesia yallundae (Wood et al., 2001), Uncinula necator (Délye et al., 1997) and Ustilago maydis (Lamb et al., 1998). The ERG11 gene also has an important role in the steroid biosynthetic pathways of bacteria, plants and mammals in which its gene product has the same metabolic role as its fungal counterpart (Rozman et al., 1996;Bak et al., 1997;Bellamine et al., 1999;Jackson et al., 2003;Pietila et al., 2006). ...
... The SRS1 (YxxF/L(I)xxPxFGxxVxF/YD/a) and SRS4 regions (GQ/hHT/sS) of M. perniciosa CYP51 were very similar to those of other organisms (Figure 3). The SRS1 region (B'helix/B'/loopC) forms the upper surface of the substrate binding cavity of P450 and the replacement of some conserved residues in this region can lead to partial or complete loss of CYP51 activity in Homo sapiens and Mycobacterium tuberculosis (Zhao et al., 2007). The SRS4 domain is located in the C-terminal region of the P450 I-helix, which forms the right wall of the distal surface of the substrate binding cavity (Lepesheva and Waterman, 2007). ...
The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.
... Due to the importance of CYP51A, many homology models have been built in previous studies. [7][8][19][20][21][22] In comparison, our study has several significant improvements. First, the templates we used have higher identity (up to 50.7%) with the target sequences. ...
... Many fungal CYP51 homology models have been constructed, for example, Candida albicans, Cryptococcus neoformans, Penicillium digitatum, S. cerevisiae, and A. fumigatus, by using the crystal structure of Mycobacterium tuberculosis CYP51 as a template. 8,[19][20][21][22] Although functionally important regions are conserved among the members of this fungal CYP51 family, these structural studies have suffered from a nonorthologous, low-homology template for homology modeling. As the structure of the human CYP51A protein has been determined, which is 41% identical to the A. fumigatus protein across its complete amino acid sequence, a new model based on the human CYP51A orthologue that provides a higher-quality model for A. fumigatus CYP51A is reported. ...
Mutations of CYP51A protein (Cytochrome P450 14-α Sterol demethylase) play a central role in the azole resistance of Aspergillus fumigatus. The available structural models of CYP51A protein of A. fumigatus are built based on that of Homo sapiens and that of Mycobacterium tuberculosis, of which the amino acid homology is only 38% and 29% compared with CYP51A protein of A. fumigatus, respectively. In the present study, we constructed a new 3D structural model of A. fumigatus CYP51A protein based on a recently resolved crystal structure of the homologous protein in the fungus S. cerevisiae, which shares 50% amino acid homology with A. fumigatus CYP51A protein. Three azole molecules, itraconazole, voriconazole, and posaconazole, were docked to the wild-type and the
mutant A. fumigatus CYP51A protein models, respectively, to illustrate the impact of cyp51A mutations to azole-resistance. We found the mutations that occurred at L98, M220, and Y431 positions would decrease the binding
affinity of azoles to the CYP51A protein and therefore would reduce their inhibitory effects. Additionally, the mutations
of L98 and G432 would reduce the stability of the protein, which might lead to conformational change of its binding pocket
and eventually the resistance to azoles.
... The ERG11 gene sequences of a wide range of fungal species that are harmful to agricultural crops and human health have been determined and characterized. These species include Antrodia cinnamomea (Lee et al., 2010), Aspergillus fumigatus (Mellado et al., 2001;Warrilow et al., 2010), Botrytis cinerea (Albertini et al., 2002), Candida albicans (Lai and Kirsch, 1989;Park et al., 2011), Candida glabrata (Kairuz et al., 1994), Cryptococcus neoformans (Revankar et al., 2004;Sheng et al., 2009), Penicillium digitatum (Zhao et al., 2007), Malassezia globosa , Monilinia fructicola, previ-ously known as Monilia fructicola (Luo and Schnabel, 2008), Pneumocystis carinii (Morales et al., 2003), Saccharomyces cerevisiae (Kalb et al., 1987), Oculimacula yallundae, previously known as Tapesia yallundae (Wood et al., 2001), Uncinula necator (Délye et al., 1997) and Ustilago maydis (Lamb et al., 1998). The ERG11 gene also has an important role in the steroid biosynthetic pathways of bacteria, plants and mammals in which its gene product has the same metabolic role as its fungal counterpart (Rozman et al., 1996;Bak et al., 1997;Bellamine et al., 1999;Jackson et al., 2003;Pietila et al., 2006). ...
... The SRS1 (YxxF/L(I)xxPxFGxxVxF/YD/a) and SRS4 regions (GQ/hHT/sS) of M. perniciosa CYP51 were very similar to those of other organisms (Figure 3). The SRS1 region (B'helix/B'/loopC) forms the upper surface of the substrate binding cavity of P450 and the replacement of some conserved residues in this region can lead to partial or complete loss of CYP51 activity in Homo sapiens and Mycobacterium tuberculosis (Zhao et al., 2007). The SRS4 domain is located in the C-terminal region of the P450 I-helix, which forms the right wall of the distal surface of the substrate binding cavity (Lepesheva and Waterman, 2007). ...
The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.
... Cloning and expression of PdCYP51 were carried out by using reverse transcription PCR (RT-PCR) with the purified mRNA as a template isolated from P. digitatum. 12 The amplified PdCYP51 cDNA fragment was cloned into pMD-T vector for sequencing and then subcloned into pET-28 for expression. The recombinant plasmid pET-PdCYP51 was transformed into Escherichia coli BL21 (DE3), and the positive colonies were selected. ...
... Docking Model of Compound 6e with PdCYP51. On the basis of our previous homology modeling of PdCYP51, 12 compound 6e was docked in the active site with the N3 of the imidazole ring forming a coordinate bond with the Fe of heme ( Figure 5). In the binding model, the distance between the N atom of compound 6e and the heme atom was 2.2 Å; there should therefore be interactions between them. ...
A series of new 2-azolyl-3,4-dihydroquinazolines 6 was synthesized by direct cyclization of imidazole or 1,2,4-triazole with carbodiimides 4, which were obtained from aza-Wittig reaction of iminophosphorane 3 with isocyanate. The preliminary bioassay results demonstrated that most of the 2-imidazolyl-3,4-dihydroquinazolines 6a-6i exhibited good to significant fungicidal activity against Penicillium digitatum whereas 2-triazolyl-3,4-dihydroquinazolines 6j-6t exhibited low fungicidal activity. Some of the 2-imidazolyl-3,4-dihydroquinazolines 6a-6i also exhibited strong binding interaction with the cytochrome P450-dependent sterol 14-demethylase (CYP51). For example, compound 6e showed the best fungicidal activity against P. digitatum with IC50 4.14 g/mL, and the best CYP51 binding activity with Kd 0.34 g/mL, both superior to the agricultural fungicide Triadimefon.
... Despite a lack of overall sequence identity, MtCyp51p shares conserved substrate recognition site motifs and structure with its mammalian, plant and fungal orthologs [48]. Homology models have been previously used to flag up possible protein-drug interactions by providing an insight into which residues in Cyp51 appear to be important for ligand binding [56]. In fact, Cyp51 homology models, based on prokaryotic P450s, have been reported for Candida albicans, Candida krusei and S. cerevisiae [57][58][59][60][61]. Currently, the M. tuberculosis Cyp51p is being used as a template in models for fungal Cyp51 enzymes such as Penicillium digitatum, C. albicans, A. fumigatus and Cryptococcus neoformans [28,55,56]. ...
... Homology models have been previously used to flag up possible protein-drug interactions by providing an insight into which residues in Cyp51 appear to be important for ligand binding [56]. In fact, Cyp51 homology models, based on prokaryotic P450s, have been reported for Candida albicans, Candida krusei and S. cerevisiae [57][58][59][60][61]. Currently, the M. tuberculosis Cyp51p is being used as a template in models for fungal Cyp51 enzymes such as Penicillium digitatum, C. albicans, A. fumigatus and Cryptococcus neoformans [28,55,56]. Moreover, several models have been previously inferred for A. fumigatus Cyp51A in combination with itraconazole, voriconazole and posaconazole, providing predictions about how the different azoles interact with AfCyp51A [28][29][30]. ...
Aspergillus lentulus, an Aspergillus fumigatus sibling species, is increasingly reported in corticosteroid-treated patients. Its clinical significance is unknown, but the fact that A. lentulus shows reduced antifungal susceptibility, mainly to voriconazole, is of serious concern. Heterologous expression of cyp51A from A. fumigatus and A. lentulus was performed in Saccharomyces cerevisiae to assess differences in the interaction of Cyp51A with the azole drugs. The absence of endogenous ERG11 was efficiently complemented in S. cerevisiae by the expression of either Aspergillus cyp51A allele. There was a marked difference between azole minimum inhibitory concentration (MIC) values of the clones expressing each Aspergillus spp. cyp51A. Saccharomyces cerevisiae clones expressing A. lentulus alleles showed higher MICs to all of the azoles tested, supporting the hypothesis that the intrinsic azole resistance of A. lentulus could be associated with Cyp51A. Homology models of A. fumigatus and A. lentulus Cyp51A protein based on the crystal structure of Cyp51p from Mycobacterium tuberculosis in complex with fluconazole were almost identical owing to their mutual high sequence identity. Molecular dynamics (MD) was applied to both three-dimensional protein models to refine the homology modelling and to explore possible differences in the Cyp51A-voriconazole interaction. After 20ns of MD modelling, some critical differences were observed in the putative closed form adopted by the protein upon voriconazole binding. A closer study of the A. fumigatus and A. lentulus voriconazole putative binding site in Cyp51A suggested that some major differences in the protein's BC loop could differentially affect the lock-up of voriconazole, which in turn could correlate with their different azole susceptibility profiles.
... Regarding the DMI group, there are so far no accounts of tebuconazole 398 resistance in isolates of P. oryzae derived from rice. Resistance to DMIs has been reported worldwide, as for Blumeria jaapii(Proffer et al., 2006), B. graminis 400(Wyand and Brown., 2005), Monilinia fructicola(Luo and Schnabel., 2008),401 Penicillium digitatum(Zhao et al., 2007), Venturia inaequalis(Schnabel & Jones, 402 2001), Fusarium asiaticum and F. graminearum (Yin et al., 2009), and 403 Zymoseptoria tritici (Leroux et al., 2011). Among these, the largest amount of 404 information is found on Z. tritici. ...
The long-term dynamics of fungicide resistance of the rice blast fungus Pyricularia oryzae was monitored by examining the reaction of the fungal field isolates, collected over a period of 26 years, to the active ingredients of commercially relevant fungicides. The in vitro sensitivity of all isolates was measured against Quinone outside inhibitors (QoI), Melanin biosynthesis inhibitors (MBI) and Sterol demethylation inhibitor (DMI) fungicides, namely azoxystrobin (QoI), tricyclazole (MBI), tebuconazole (DMI), and trifloxystrobin + tebuconazole (QoI + DMI). Over the 26-year collection period, a gradual rise in the EC50 estimates for mycelial growth sensitivity was observed for all fungicides, but most strikingly for azoxystrobin. A rise in conidial germination and appressorium formation was also noted, most markedly for azoxystrobin. Consistently, the earlier isolates were much more sensitive to the active ingredients than the more contemporary isolates. The sequencing of the amplified cyt b fragment distinguished two haplotypes, H1 and H2. Haplotype H1 (six isolates) contained the G to C transversion at codon 143 (resulting in change G143A), linked to the resistant phenotype QoI-R. Haplotype H2 (40 isolates), gathered the isolates sensitive to QoI. This work documents the gradual rise in the frequency of fungicide resistant isolates in Pyricularia oryzae rice populations on a long-term basis.
... Zhou et al. employed pET28a to successfully express the complete ORF of CYP51 from Penicillium digitatum in E. coli Rosetta (DE3) [36]. However this strategy did not work for LbCYP51. ...
Leishmaniasis, a neglected tropical disease, is a major cause of morbidity and mortality worldwide. Of the three main clinical forms, cutaneous leishmaniasis (CL) is the most common and 40 million people are at risk in the endemic areas. Currently, the available drugs to fight leishmaniasis have high toxicity and poor efficiency. Then, it is very important to search for effective and safe drugs that would target essential enzymes from the parasite, such as lanosterol 14-alpha demethylase (CYP51, EC 1.14.13.70) from Leishmania braziliensis. Because most drug design efforts have been directed for Leishmania non-braziliensis species, there is no structural or kinetic data regarding L. braziliensis CYP51. Herein, we present for the first time molecular biology efforts and purification protocol to obtain the enzyme LbCYP51. These results lay the ground for future investigation of drugs against this target.
... Therefore, identifying new target genes and the designing of new drugs is of great significance to control the spread of resistant fungi. Previously, we reported the cloning, expression, and characterization of cyp51 from P. digitatum and Ustilago maydis [52][53][54][55]. The structural characteristics of the interaction between heterologous CYP51 and commercial azoles were also analyzed by binding assays. ...
Penicillium
digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium
tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.
... MtCYP51 homology models for various fungal CYP51 proteins have been calculated to predict amino acids involved in azole–protein interaction and the effects of their substitution on structural integrity. This concerned the human pathogens A. fumigatus (Gollapudy et al. 2004; Snelders et al. 2010; Xiao et al. 2004), Candida albicans (Rueda et al. 2011; Park et al. 2011; Rupp et al. 2005; Xiao et al. 2004), Candida krusei (Fukuoka et al. 2003), Cryptococcus neoformans (Sheng et al. 2009), and the plant pathogens Botrytis cinerea (Ito et al. 2005), M. oryzae (Yang et al. 2009), Mycosphaerella fijiensis (CanasGutierrez et al. 2009), P. digitatum (Zhao et al. 2007), and Ustilago maydis (Han et al. 2010). Although the crystal structure of MtCYP51 considerably advanced knowledge, it lacks some information for consistent homology modeling of eukaryotic CYP51s as it is shorter (451 amino acid (aa)) than the proteins in human (503 aa) and fungi (∼515 to 550 aa; Fraczek et al. 2011). ...
Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel azole structures.
... DMI fungicides have been widely used in medicine and agriculture and constitute some of the most successful fungicides. However, resistance to DMIs has now been reported in many fungal species (Hildebrand et al., 1988; Delye et al., 1998; Ma et al., 2006; Leroux et al., 2007; Gao et al., 2009; Sombardier et al., 2010), and the molecular mechanism leading to DMI resistance has been investigated in several plant pathogenic species, including Blumeriella jaapii (Ma et al., 2006; Proffer et al., 2006), Blumeria graminis (Delye et al., 1998; Wyand and Brown, 2005), Monilinia fructicola (Luo and Schnabel, 2008), Mycosphaerella graminicola (Leroux et al., 2007; Brunner et al., 2008; Bean et al., 2009), Mycosphaerella fijiensis (Canas-Gutierrez et al., 2009), Penicillium digitatum (Nakaune et al., 1998; Hamamoto et al., 2001; Zhao et al., 2007), Tapesia yallundae (Wood et al., 2001), Venturia inaequalis (Schnabel and Jones, 2001), Fusarium asiaticum and Fusarium graminearum (Yin et al., 2009). Currently, the DMI fungicides, such as propiconazole, are widely used for the control of rice blast disease (Skamnioti and Gurr, 2009 ). ...
The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.
... Due to the importance of CYP51 in antifungal drug studies, many CYP51 homology models have been made, e.g., C. albicans, Cryptococcus neoformans, Penicillium digitatum, and Saccharomyces cerevisiae, by using the crystal structure of M. tuberculosis CYP51 as a template (10,15,26,27). Although this approach has several limitations, functionally important regions are conserved among the members of this fungal CYP51 family. ...
Molecular studies have shown that the majority of azole resistance in Aspergillus fumigatus is associated with amino acid substitutions in the cyp51A gene. To obtain insight into azole resistance mutations, the cyp51A gene of 130 resistant and 76 susceptible A. fumigatus isolates was sequenced. Out of 130 azole-resistant isolates, 105 contained a tandem repeat of 34 bp in the promoter region
and a leucine-to-histidine substitution in codon 98 (designated TR/L98H). Additionally, in 12 of these TR/L98H resistant isolates,
the mutations S297T and F495I were found, and in 1 isolate, the mutation F495I was found. In eight azole-resistant isolates,
known azole resistance mutations were detected in codon G54, G138, or M220. In three azole-susceptible isolates, the mutation
E130D, L252L, or S400I was found and in 13 azole-susceptible isolates but also in 1 azole-resistant isolate, the mutations
F46Y, G98G, M172V, N248T, D255E, L358L, E427K, and C454C were found. All of the nonsynonymous mutations, apart from the mutations
in codons G54, G138, and M220 and L98H, were located at the periphery of the protein, as determined by a structural model
of the A. fumigatus Cyp51A protein, and were predicted neither to interact with azole compounds nor to affect structural integrity. Therefore,
this wide diversity of mutations in the cyp51A gene in azole-susceptible A. fumigatus isolates is not correlated with azole resistance. Based on the Cyp51A protein homology model, the potential correlation of
a mutation to azole resistance can be predicted.
Background
1,2,4-Triazoles are important five-membered heterocyclic scaffolds due to their extensive biological activities. These products have been an area of growing interest to many researchers around the world as of their diverse biological activities including antifungal and antibacterial activity against broad spectrum of microbial pathogens.
Methods
A series of 2-((1H-1,2,4-triazol-1-yl)methyl) derivatives was designed, synthesized and evaluated as antifungal agents against five plant pathogenic fungi (Alternaria tennis, Botryodiplodia theobromae, Fusarium moniliforme, Fusarium oxysporum and Helminthosporium turcicum) to meet the urgent need of new antifungal agents with improved activity and broader spectrum. In silico studies including pharmacophore modeling, virtual screening, drug-likeness analysis and ADMET prediction were examined. In addition, the elucidation of the activity is based on the molecular docking to the active site of the Sterol 14α-Demethylase Cytochrome P450 (CYP51) was investigated.
Results
The results of antifungal activity indicated that the compounds containing tert or sec-butyl as hydrophobic substituents on a phenyl ring significantly increased the activity (compounds 4, 5 and 6) with EC50 in the range of 8-84 mg/L of all tested fungi. The pharmacophore modeling produced an accurate projecting model (Hypo 1) from these derivatives. The superlative Hypo1 consists of three features counting two hydrogen bond acceptors (HBA) and one hydrophobic (HYD). The docking results showed approximately a similar binding degree at the active sites of the fungal enzyme (CYP51) as a standard fungicide penconazole.
Conclusion
According to data obtained, some derivatives, especially those with tert or sec-butyl substituents on the phenyl ring, were more potent against phytopathogenic fungi. These compounds (e.g., 4, 5, and 6) should develop into new potential fungicides as a desirable activity.
CYP51 is an enzyme of sterol biosynthesis pathway present in animals, plants, protozoa and fungi. This enzyme is described as an important drug target that is still of interest. Therefore, in this work we reviewed the structure and function of CYP51 and explored the molecular modeling approaches for the development of new antifungal and antiprotozoans that target this enzyme. Crystallographic structures of CYP51 of some organisms have already been described in the literature, which enable the construction of homology models of other organisms enzymes and molecular docking studies of new ligands. Some new series of azoles with antifungal or antiprotozoan activities present their binding mode and interactions described showing important residues of the active site. Molecular modeling is an important tool to be explored for the discovery and optimization of CYP51 inhibitors with better activities, pharmacokinetics and toxicological profiles. This article is protected by copyright. All rights reserved.
Substituted styrene 1 was reacted with KHSO5 in the presence of the chiral catalyst 2 to give aryloxiranes 3, which were subsequently treated with 1H-1,2,4-triazole and potassium carbonate to generate enantioriched 1-aryl-2-(1H-1,2,4-triazol-1-yl)ethanols 4. Some of the triazoles 4 exhibited strong binding interactions with the cytochrome P450-dependent sterol 14α-demethylase (CYP51). triazole, chiral, 14α-demethylase; fungicidal activity.
Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals are the treatment of choice, however, resistance has arisen against these compounds, which could lead to devastating consequences. Therefore, it is important to understand how these fungicides are used and how the resistance arises to fully tackle the problem. Here, we give an overview of the problem and discuss the mechanisms that mediate azole resistance in agriculture (point mutations in the CYP51 amino acid sequence, overexpression of the CYP51 enzyme and overexpression of genes encoding efflux pumps proteins).
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To get a better optimization expression of the Ustilago maydis CYP51 (P450-14DM, UmCYP51) protein in E. coli BL21(DE3), the different lengths of UmCYP51 gene that lacked the coding region for the putative membrane-spanning segment of the N-terminus were truncated. The first one is the wild type, the second one with 20 amino acids (60 base pairs) in N-terminus was truncated and the third one with 35 amino acids (105 base pairs) was truncated. Then these genes were incorporated into different expression vectors (pET28, pET32 and pGEX-KG) to construct nine recombinant expression plasmids (pET28-Um, pET28-Um-20, pET28-Um-35, pET32-Um, pET32-Um-20, pET32-Um-35, pGEXKG-Um, pGEXKG-Um-20 and pGEXKG-Um-35). The expression of recombinant plasmids were performed using 0.5 mmol/L of isopropyl ?-D-thiogalactoside (IPTG) at 30?. The culture harvested every 2 h up to 8 h. It was found that only recombinant plasmid pET32-Um-35 was expressed in E. coli BL21(DE3). Codon usage database (http//:www.kazusa.or.jp/coden) was used for the analysis of rare codon and software RNAStructure 4.5 was employed to study the mRNA secondary structure of translation initiation region. The results showed that rare codons rate in UmCYP51 gene is only 4.63%, the Rosetta (DE3) strain expressing some rare codons is not suitable for the protein expression of UmCYP51. Only the lowest energy of mRNA structure for pET32-Um-35 can obtained protein expression. These results are compatible with the experiments. Moreover, to design novel antifungal compounds against UmCYP51, based on the recently determined X-ray crystal structure human CYP51, a three-dimensional structure model of UmCYP51 was built through homology modeling using MODELLER 9V7 program. After refinement of the energy minimization and MD simulation using GROMACS 4.0.3 package, the UmCYP51 model was evaluated by PROCHECK Ramachandran plot statistics that indicated the designed model was in good quality. Commercial fungicide tebuconazole was docked into the model protein using Autodock 4.2.3 program to form the binding pattern of inhibitor with UmCYP51. The docking conformation of tebuconazole in the active site of UmCYP51 showed that the N-4 of the triazole ring was bound to heme iron with a distance about 0.245 nm. The hydroxy group of tebuconazole formed hydrogen-bonding interaction with the oxygen atom of carbonyl group for Ala265 with a distance about 0.245 nm. The mechanism of inhibitory activity of tebuconazole against UmCYP51 obtained from this study could aid in designing newantifungal compounds targeting this enzyme.
Corn smut is one of the most serious diseases affecting corn crops caused by Ustilago maydis. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis and has been shown to be an effective target of antifungal drugs. To design novel antifungal compounds against UmCYP51, in this study, homology modeling and expression of CYP51 from U. maydis (UmCYP51) were carried out. A-three-dimensional structure model of UmCYP51 was built through homology modeling based on the crystal structure of Mycobacterium tuberculosis. Only pET32-Um-35 could be highly expressed in E. coli BL21 (DE3)successfully after many attempts, which indicated N terminal transmembrane regions and the lowest energy of mRNA structure are importment for the protein expression of UmCYP51, combining of bioinformatics analysis of transmembrane prediction and mRNA secondary structure of translation initiation region. These results will provide some information for predicting the expression of protein from the angle of bioinformatics, and will set the stage for insighting the feature of the UmCYP51.
Penicillium digitatum Sterol 14α-demethylase (PdCYP51), a prime enzyme in membrance sterol biosynthesis, is a key target of antifungal drugs for citrus disease. Based on the recently determined X-ray crystal structure human CYP51, a three-dimensional structure model of PdCYP51 was built through homology modeling. After molecular dynamics (MD) simulation, the refined model was assessed by Verify-3D and Procheck, which confirmed that the refined model was reliable. Further evaluation on the model quality was performed by investigating the interaction of some sterol 14α-demethylase inhibitors (DMIs) with the modeled enzyme. Molecular docking program was employed to determine such interactions. The binding pattern predicted by the docking revealed that DMI inhibitor interacted with PdCYP51 mainly through hydrogen-bonding and hydrophobic interactions. Moreover, the results are compatible with the spectra assay data in the laboratory. The docking complex provided further refinement of the DMI binding interaction that may be used as a basis for virtual screening and for novel design to discover more potent compounds.
A series of chiral β-arylalkyl-1H-1,2,4-triazole derivatives were prepared and both their in vitro antifungal activities against Penicillium digitatum and binding activity toward CYP51 protein of P. digitatum (PdCYP51) were tested. In general, the in vitro inhibitory activities of R- and S-enantiomers were in good agreement with the corresponding binding activities in cultured cells. Furthermore, the preliminary molecular docking modeling of representative compounds are described to provide more insight into their stereoselective interaction, as well as further rationalize the observations of the activities of chiral azoles as demethylase inhibitors for defense against postharvest pathogens.Graphical abstractMolecular docking results show that the S-isomer more favorably fits the active site of the cytochrome. The more tightly the compounds bind to the PdCYP51, the greater their antifungal activity.Research highlights► Excellent in vitro antifungal activities against P. digitatum. ► Potential demethylase inhibitors for defense against postharvest pathogens. ► Respective enantiomers showed significantly different inhibitory activities. ► Chiral triazoles present obvious stereoselective interaction with enzyme.
Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis aldehyde dehydrogenase (BlALDH) and identified two different residues, Glu255 and Cys289, that might be responsible for the catalytic function of the enzyme. The role of these residues was further investigated by site-directed mutagenesis and biophysical analysis. The expressed parental and mutant proteins were purified by nickel-chelate chromatography, and their molecular masses were determined to be approximately 53 kDa by SDS-PAGE. As compared with the parental BlALDH, a dramatic decrease or even complete loss of the dehydrogenase activity was observed for the mutant enzymes. Structural analysis showed that the intrinsic fluorescence and circular dichroism spectra of the mutant proteins were similar to the parental enzyme, but most of the variants exhibited a different sensitivity towards thermal- and guanidine hydrochloride-induced denaturation. These observations indicate that residues Glu255 and Cys289 play an important role in the dehydrogenase activity of BlALDH, and the rigidity of the enzyme has been changed as a consequence of the mutations.
Ustilago maydis infection is a serious disease affecting corn crops worldwide. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis and an effective target of antifungal drugs. To further study the interaction between CYP51 and drugs and exploit more specific 14α-demethylase inhibitor (DMI) fungicides for U. maydis, in this study homology modeling of CYP51 from U. maydis (UmCYP51) templated as the eukaryotic orthologues (the human CYP51) and screening of new DMI fungicides based on optimized expression were carried out for the first time. In addition, XF-113 and ZST-4 were screened by analyzing the spectral characteristics between the purified UmCYP51-35 and fungicides. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal drugs.
Sterol 14α-demethylase (CYP51), a key target of azole (DMI) fungicides, can be expressed in both prokaryotes and eukaryotes. Green mould of citrus, caused by Penicillium digitatum (Pers.) Sacc., is a serious post-harvest disease. To develop specific and more effective fungicides against this disease, the characteristics of the interaction between sterol 14α-demethylase from P. digitatum (PdCYP51) and possible new fungicides were analysed. The cyp51 gene of P. digitatum was cloned and expressed under different conditions in Escherichia coli (Mig.) Cast. & Chalm., and the binding spectra of PdCYP51 were explored by the addition of two commercial azoles and four new nitrogen compounds.
The yield of soluble protein (PdCYP51) was largest when expressed in Rosetta (DE3) induced by 0.5 mM IPTG for 8 h at 30 °C. Compound B (7-methoxy-2H-benzo[b][1,4]thiazine-3-amine) showed the strongest binding activity of the four new nitrogen compounds, with a K(d) value of 0.268 µM. The K(d) values of the six compounds were significantly correlated with their EC(50) values.
The spectral analysis and bioassay results could be used to screen the new chemical entities effectively. Compound B, selected by virtual screening from a commercial chemical library, is a candidate for a new DMI fungicide. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal agents.
Cytochrome P450 sterol 14alpha-demethylases (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. All fungal CYP51s are integral membrane proteins, making structural and biophysical characterization more challenging. The X-ray crystallographic structure of CYP51 isolated from Mycobacterium tuberculosis (MT-CYP51) is the unique reported one hitherto. In the present study, a homology modeling three-dimensional structure of CYP51 from Penicillium digitatum (PD-CYP51) was generated by CPHmodels, in which the accuracy of sequence alignment could be improved by taking into account further structural conservation information, using MT-CYP51 as the template. Interaction mechanism between the active site of PD-CYP51 and its inhibitors were further investigated by molecular dynamics simulating and molecular docking. With the effective docking process and interaction analysis information, structure-based virtual screening was performed to pick out the thirty new potential inhibiting compounds with structural diversity by using a new virtual screening strategy including Flex-Pharm/PMF/GOLD//FlexX/PMF/GOLD molecular docking procedures, and finally, seven new hit compounds out of SPECs database with potent inhibitory ability were validated by bioaffinity assays at enzyme level and on P. digitatum in vitro. The positive results indicated that all modeling strategies and screening processes presented in the current study most like to be an encouraging way in search of novel lead compounds with structural diversity for the specifically individual fungal CYP51s of both plants and human pathogens in the future.
A series of chiral gamma-aryl-1H-1,2,4-triazole derivatives has been synthesized and the respective analogues have been tested for their inhibitory activities against Penicillium digitatum (P. digitatum). In vitro experiments were indicative of a strong inhibitory effect of all of the compounds on P. digitatum, and seven of the compounds 5 exhibited better inhibition than the commercial fungicides triadimefon and triadimenol. The respective pairs of enantiomers showed significantly different inhibitory activities, most notably in the case of 5g-R and 5g-S, for which a 230-fold difference was observed. These observations suggest that P. digitatum discriminates the enantiomers and that the R enantiomer better fits the active site of cytochrome P450.
Cryptococcus neoformans is one of the most important causes of life-threatening fungal infections in immunocompromised patients. Lanosterol 14α-demethylase
(CYP51) is the target of azole antifungal agents. This study describes, for the first time, the 3-dimensional model of CYP51
from Cryptococcus neoformans (CnCYP51). The model was further refined by energy minimization and molecular-dynamics simulations. The active site of CnCYP51
was well characterized by multiple-copy simultaneous-search calculations, and four functional regions important for rational
drug design were identified. The mode of binding of the natural substrate and azole antifungal agents with CnCYP51 was identified
by flexible molecular docking. A G484S substitution mechanism for azole resistance in CnCYP51, which might be important for
the conformation of the heme environment, is suggested.
1H-1,2,4-Triazole reacted with 2-butenal in the presence of diaryl prolinol silyl ether 3 and benzonic acid to give 3-(1H-1,2,4-triazol-1-yl)butanal 4, which was subsequently reduced and then treated with various acyl chloride to generate enantioriched 3-(1H-1,2,4-triazol-1-yl)butyl benzoates 6. Some of triazoles 6 exhibited strong binding interactions with the cytochrome P450-dependent sterol 14alpha-demethylase (CYP51). For example, compound (R)-6f showed the best binding activity with K(d) 0.3381 microM.
Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants.
A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.
Rice blast, caused by the ascomycete fungus Magnaporthe grisea, is one of the most serious diseases for cultivated rice. Sterol 14 alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and an important drug target for microbial pathogenic infections. In the present study CYP51 with truncation of N-terminal residues from M. grisea (MGCYP51) was cloned and expressed in E. coli, drug binding spectrum of MGCYP51 induced by addition of diniconazole was determined. In order to exploit more selective and effective fungicides for M. grisea, homology model of MGCYP51 was established based on crystal structure of Mycobacterium tuberculosis and diniconazole was docked into the active site by FlexX. The spectral data showed that diniconazole exhibited a high affinity for MGCYP51, coincided with the implication of molecular docking. The results in a way elucidated the reasonability and reliability of the 3D model and docking model, which can be hopefully used to virtually screen the more potent azole fungicides for MGCYP51.
A model of the joint dynamics of change in population size N and evolution in a quantitative trait z, as a result of a general form of density dependence, local stabilizing selection, and immigration of individuals deviating from the local optimum, is analyzed. For weak selection and migration, a reduction in total equilibrium population size below the initial level without immigration, K, is shown to occur if the immigrants deviates more than square root of 8 = 2.83 genetic standard deviations from the optimum and if the rate of migration m is sufficiently large relative to the strength of stabilizing selection s. For the Lotka-Volterra form of density dependence, two additional equilibria are shown to exist below K, provided that the strength of selection is large relative to the strength of density dependence. Reintroduction of an initially extinct population is possible if the immigrants are not too maladapted and if the genetic variance is sufficiently large. For a simplified version of the model corresponding to competition between similar species or different haplotypes, the equilibrium population size is always exactly at K if m < Ksz1(2) and is above K otherwise, which shows the importance of including recombination in the model.
When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.
Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) is required for ergosterol biosynthesis and is the target for azole antifungal compounds. The amino acid substitution Y132H in CYP51 from clinical isolates of Candida albicans can cause fluconazole resistance by a novel change in the protein. Fluconazole binding to the mutant protein did not involve normal interaction with haem as shown by inducing a Type I spectral change. This contrasted to the wild-type protein where fluconazole inhibition was reflected in coordination to haem as a sixth ligand and where the typical Type II spectrum was obtained. The Y132H substitution occurred without drastic perturbation of the haem environment or activity allowing resistant mutants to produce ergosterol and retain fitness, an efficient strategy for resistance in nature.
Cytochrome P450 14alpha-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14alpha-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-A crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences-a bent I helix and an open conformation of BC loop-that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.
Differentially expressed genes between normal SPC-A1 cells and SPC-A1 cells infected by RSV were investigated using differential display. The novel zlg10 gene codes for a novel protein, ZLG10, which has previously been reported to be up-regulated in RSV-infected SPC-A1 cells. Its putative open reading frame was also identified. To better understand the structure, function, and possible role of ZLG10 as a potential candidate for diagnosis and vaccine studies, the intact region encoding ZLG10 was obtained by PCR and expressed in Escherichia coli as a GST-fusion protein. After purification, GST-ZLG10 fusion protein was used to immunize the adult rabbits following standard protocols. Consequently, we found that the produced antiserum of the novel fusion protein significantly suppressed the infection by RSV on SPC-A1 cells by using neutral red uptake assay and quantitative measurement. Together, our data demonstrate that ZLG10, a novel protein expressed and purified in this report, might be a potential effective therapeutic candidate for treating RSV infections.
Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were > or = 80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherichia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[alpha]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycyclic aromatic hydrocarbons.
The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.
Resistance to sterol 14α-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14α-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.
We report on a test of FLEXX, a fully automatic docking tool for flexible ligands, on a highly diverse data set of 200 protein–ligand complexes from the Protein Data Bank. In total 46.5% of the complexes of the data set can be reproduced by a FLEXX docking solution at rank 1 with an rms deviation (RMSD) from the observed structure of less than 2 Å. This rate rises to 70% if one looks at the entire generated solution set. FLEXX produces reliable results for ligands with up to 15 components which can be docked in 80% of the cases with acceptable accuracy. Ligands with more than 15 components tend to generate wrong solutions more often. The average runtime of FLEXX on this test set is 93 seconds per complex on a SUN Ultra-30 workstation. In addition, we report on “cross-docking” experiments, in which several receptor structures of complexes with identical proteins have been used for docking all cocrystallized ligands of these complexes. In most cases, these experiments show that FLEXX can acceptably dock a ligand into a foreign receptor structure. Finally we report on screening runs of ligands out of a library with 556 entries against ten different proteins. In eight cases FLEXX is able to find the original inhibitor within the top 7% of the total library. Proteins 1999;37:228–241. ©1999 Wiley-Liss, Inc.
The conjugate gradient method is particularly useful for minimizing functions of very many variables because it does not require the storage of any matrices. However the rate of convergence of the algorithm is only linear unless the iterative procedure is restarted occasionally. At present it is usual to restart everyn or (n + 1) iterations, wheren is the number of variables, but it is known that the frequency of restarts should depend on the objective function. Therefore the main purpose of this paper is to provide an algorithm with a restart procedure that takes account of the objective function automatically. Another purpose is to study a multiplying factor that occurs in the definition of the search direction of each iteration. Various expressions for this factor have been proposed and often it does not matter which one is used. However now some reasons are given in favour of one of these expressions. Several numerical examples are reported in support of the conclusions of this paper.
Food related allergic reactions following inadvertent ingestion are increasingly common, with nuts, including Brazil nut, placed firmly in the top 10 food groups whose presence within a product should be declared. The presence of hidden allergens as a result of adulteration or contamination of ingredients presents a problem for both the food industry and the consumer. A sensitive and specific immunoassay for Brazil nut is described with a limit of detection of 1 ppm. Based upon the detection of the abundant 2S protein the assay is suitable for detection of raw and roasted Brazil nut in a range of food matrices.
This study investigates the effects of coating with low molecular weight chitosan (LMWC, Mw = 15 kDa) and high molecular weight chitosan (HMWC, Mw = 357 kDa) on the decay of Murcott tangor and the maintenance of its quality. A 0.1% LMWC coating substantially slowed the decay of Murcott tangor stored at 15 °C in relation to a control sample and reduced decay by over 20% as compared to the fungicide TBZ. A concentration of 0.2% LMWC was more effective in controlling the growth of fungus on citrus fruits caused by Penicillium digitatum and Penicillium italicum, exhibiting effective antifungal activity. LMWC coating improved firmness, titratable acidity, ascorbic acidity and the water content for Murcott tangor stored at 15 °C for 56 days. Consequently, Murcott tangor coated with LMWC exhibited greater antifungal resistance than TBZ and its quality was maintained for longer.
The crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068–3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh’s analysis [J. Biol. Chem. 267 (1992) 83–90]. While Gotoh’s SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.
The marketing of organic citrus fruit has markedly increased during the last few years. However, these fruits are not treated with chemical fungicides and suffer from relatively high rates of decay. In this study, we examined the possible use of a new hot water brushing (HWB) treatment, to disinfect the fruits and reduce decay development during postharvest storage. Preliminary observations have shown that a minimum exposure period of 20 s at 56°C was required to inhibit Penicillium digitatum (Pers.: Fr.) Sacc spore germination in vitro. In vivo studies, carried out by rinsing and brushing the fruit 24 h after artificial inoculation with a P. digitatum spore suspension, indicated that HWB at 56, 59 and 62°C for 20 s, reduced decay development in the infected wounds to only 20, 5 and less than 1%, respectively, of that in untreated control fruits or fruits treated with tap water. The effects of HWB at 56, 59 and 62°C for 20 s on epiphytic microflora, was further confirmed by the reduction of the microbial counts (CFU) on the fruit surface to only 24, 12, and less than 1%, respectively, of those on fruit that had been rinsed and brushed with tap water. Scanning electron microscopy showed that HWB at 56°C for 20 s had smoothed the fruit epicuticular waxes and thus covered and sealed stomata and cracks on the fruit surface, which could have served as potential pathogen invasion sites. Postharvest storage experiments using various organically grown citrus cultivars such as ‘Minneola’ tangerines, ‘Shamouti’ oranges and ‘Star Ruby’ red grapefruit, showed that HWB at 56°C for 20 s reduced decay development by 45–55%. The HWB treatment at 56°C did not cause surface damage, and did not influence fruit weight loss or internal quality parameters.
FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc.cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Mechanism of action of azole antifungal agents was studied by analyzing interaction of ketoconazole, itraconazole, triadimefon and triadimenol with a purified yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (P-45014DM). These antifungal agents formed low-spin complexes with P-45014DM, indicating the interaction of their azole nitrogens with the heme iron. Affinity of these antifungal agents for the cytochrome was extremely high compared with usual nitrogenous ligands. Upon reduction with sodium dithionite, the azole complexes of ferric P-45014DM were converted to the corresponding ferrous derivatives. Spectral analysis of these complexes suggested that geometric orientation of the azole moiety of an antifungal agent to the ferrous heme iron was regulated by the interaction between the N-1 substituent and the heme environment. CO could not readily replace ketoconazole or itraconazole co-ordinating to the heme iron of ferrous P-45014DM while triadimefon and triadimenol complexes of the cytochrome were promptly converted to the CO complexes. The inhibitory effects of ketoconazole and itraconazole on the P-45014DM-dependent lanosterol 14 alpha-demethylation were higher than that of triadimenfon. The substituents at N-1 of the azole moieties of ketoconazole and itraconazole are extremely large while those of triadimefon and triadimenol are relatively small. Accordingly, observations described above suggest that the N-1 substituent of an azole antifungal agent regulates the mobility of the molecule in the heme crevice of ferrous P-45014DM and determines the inhibitory effect of the compound.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
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.
We report on a test of FLEXX, a fully automatic docking tool for flexible ligands, on a highly diverse data set of 200 protein-ligand complexes from the Protein Data Bank. In total 46.5% of the complexes of the data set can be reproduced by a FLEXX docking solution at rank 1 with an rms deviation (RMSD) from the observed structure of less than 2 A. This rate rises to 70% if one looks at the entire generated solution set. FLEXX produces reliable results for ligands with up to 15 components which can be docked in 80% of the cases with acceptable accuracy. Ligands with more than 15 components tend to generate wrong solutions more often. The average runtime of FLEXX on this test set is 93 seconds per complex on a SUN Ultra-30 workstation. In addition, we report on "cross-docking" experiments, in which several receptor structures of complexes with identical proteins have been used for docking all cocrystallized ligands of these complexes. In most cases, these experiments show that FLEXX can acceptably dock a ligand into a foreign receptor structure. Finally we report on screening runs of ligands out of a library with 556 entries against ten different proteins. In eight cases FLEXX is able to find the original inhibitor within the top 7% of the total library.
A trypsin-streptavidin (TRYPSA) fusion protein was designed and its expression in Escherichia coli was evaluated. The streptavidin gene was PCR modified and cloned into the pET expression vector. The trypsin gene was subsequently inserted into this plasmid, thus generating a colinear fusion of trypsin and streptavidin genes (pTRYPSA). This engineering strategy was verified, and TRYPSA was expressed after IPTG induction using the E. coli strains, BL21(DE3) and BL21(DE3)pLysS. Standard protein fractions of the cell lysate were prepared and trypsin activity was primarily detected in the periplasmic and inclusion body fractions. Immunoblotting showed a single Western-positive band exhibiting a molecular weight of 39,000 Da. A biotinylated porous glass affinity matrix was prepared and selective adsorption resulted in a one-step purification and immobilization of TRYPSA from crude cell lysate. Trypsin activity was verified using a synthetic substrate. This enzyme bioreactor should serve as an excellent prototype for future studies that will examine the effect of limited proteolysis on functional characteristics of milk proteins, including gelling, emulsifying and foaming properties.
CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.
Chronic infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Latin America. Drug treatments for the associated illness, Chagas disease, are toxic and frequently unsuccessful. In a screening effort against the drug target protein farnesyltransferase, we identified a series of disubstituted imidazoles with highly potent anti-T. cruzi activity that apparently acted through a mechanism independent of protein farnesylation. Metabolic labeling studies of T. cruzi suggested that sterol biosynthesis was inhibited. Combined GC/MS analysis confirmed depletion of cellular sterols and suggested that the site of action was sterol 14-demethylase, a cytochrome P450 enzyme. Spectral studies with recombinant T. cruzi sterol 14-demethylase demonstrated that the compounds bind directly to this enzyme. Two of the compounds were well absorbed when given orally to mice, gave sustained plasma levels, and were well tolerated. The compounds were administered orally to mice with acute T. cruzi infection and caused dramatic decrease in parasitemia and led to 100% survival. These disubstituted imidazole compounds can be prepared by a relatively short synthetic route and represent a structural class with potent anti-T. cruzi activity.
New isoforms of CYP51 (sterol 14alpha-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei(TB), T. vivax, T. cruzi, and Leishmania major. The sequences share approximately 80% amino acid identity and are approximately 25% identical to sterol 14alpha-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT)) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min(-1)). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14alpha-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.
To investigate the properties of wheat cytochrome P450 and the characteristics of herbicide metabolism by cytochrome P450 in vitro, deeply understand the mechanisms of herbicide selectivity, recombinant wheat cytochrome P450 monooxygenase (CYP71Cv1) heterologously expressed in yeast was purified by DE-52 cellulose chromatography and fast protein liquid chromatography (FPLC) with Mono-Q column. The degree of purification was 1366-fold. The specific activity of purified cytochrome P450 reached to 512 nmol min-1 mg-1 protein with herbicide chlorsulfuron as substrate. The purified cytochrome P450 exhibited one band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular mass was 52.5 kDa. Kinetic parameter was determined in vitro. The Km values for chlorsulfuron and triasulfuron were 57 (+/-15) and 38 (+/-16) microM, respectively; and Vmax for chlorsulfuron and triasulfuron were 4.1 (+/-0.7) and 2.7 (+/-0.5) nmol min-1 mg-1protein in vitro, respectively.
Sterol 14alpha-demethylase (CYP51) is the most widely distributed of all members of the cytochrome P450 gene superfamily and the only CYP family found in both prokaryotes and eukaryotes. It is well known as a drug target for microbial pathogenic infections. Studies of CYP51 gene regulation have been carried out primarily in animals because its regulation is similar to those of other genes involved in the cholesterol biosynthetic pathway. The function of CYP51 has been studied widely throughout biology including in animals, plants, yeast/fungi, protozoa, and bacteria. The structure has been determined by X-ray crystallography for the soluble prokaryotic form of CYP51 from Mycobacterium tuberculosis. Together these studies provide the most detailed understanding of any single cytochrome P450 and this minireview summarizes this information.
In a continuing effort to develop highly potent azole antifungal agents, the three-dimensional quantitative structure-activity relationship methods, CoMFA and CoMSIA, were applied using a set of novel azole antifungal compounds. The binding mode of the compounds at the active site of lanosterol 14alpha-demethylase was further explored using the flexible docking method. Various hydrophobic, van der Waals, pi-pi stacking, and hydrogen bonding interactions were observed between the azoles and the enzyme. Based on results from the molecular modeling, a receptor-based pharmacophore model was established to guide the rational optimization of the azole antifungal agents. Thus, a total of 57 novel azoles were designed and synthesized by a three-step optimization process. In vitro antifungal assay revealed that the antifungal activities of these novel azoles were greatly improved, which confirmed the reliability of the model from molecular modeling.
Many 3D ligand-based and structure-based computational approaches have been used to predict, and thus help explain, the metabolism catalyzed by the enzymes of the cytochrome P450 superfamily (P450s). P450s are responsible for >90% of the metabolism of all drugs, so the computational prediction of metabolism can help to design out drug-drug interactions in the early phases of the drug discovery process. Computational methodologies have focused on a few P450s that are directly involved in drug metabolism. The recently derived crystal structures for human P450s enable better 3D modelling of these important metabolizing enzymes. Models derived for P450s have evolved from simple comparisons of known substrates to more-elaborate experiments that require considerable computer power involving 3D overlaps and docking experiments. These models help to explain and, more importantly, predict the involvement of P450s in the metabolism of specific compounds and guide the drug-design process.
Novel tetrahydroisoquinoline compounds were designed by coupling structure-based de novo design based on the structure of lanosterol 14alpha-demethylase (CYP51). The chemical synthesis and the antifungal activities in vitro of them were reported. The results exhibited that all of the lead compounds showed potent antifungal activities, in which compounds 6 and 7 had equal or stronger antifungal activities against five test fungi than that of fluconazole. The studies presented here provided the antifungal lead compounds. The affinity of the lead molecules for CYP51 was mainly attributed to their non-bonding interaction with the apoprotein, which was different from the azole antifungal agents.
Chagas disease is caused by infection with the protozoan pathogen, Trypanosoma cruzi. The only approved therapeutics for treating Chagas disease are two nitroheterocyclic compounds (benznidazole and nifurtimox) that are suboptimal due to poor curative activity for chronic Chagas disease and high rates of adverse drug reactions. Sterol 14-demethylase inhibitors include azole antifungal drugs such as ketoconazole, fluconazole, itraconazole, and others. The first reports of potent activity of azole antifungal drugs against Trypanosoma cruzi came out about 25 years ago. Since then, a sizeable literature has accumulated on this topic. Newer triazole compounds such as posaconazole and D0870 have been shown to be effective at curing mice with chronic Trypanosoma cruzi infection. Small clinical studies with-ketoconazole or itraconazole in humans with chronic Chagas disease have not demonstrated significant curative activity. However, there is good reason for optimism that newer compounds with greater potency and improved pharmacokinetic properties might be more efficacious. Data have been published demonstrating synergistic activity of azole drugs with various other compounds, indicating that combination chemotherapy may be an effective strategy as this field moves ahead. In light of the near absence of adequate therapeutics for curing patients with chronic Chagas disease, additional effort to develop better drugs needs to be a priority.
Designing better drugs
- De Groot MJ
- De Groot MJ
Extraction and purification of filamentous fungi DNA
- M P Moore
- A J Carrera
- D M Facile
Moore MP, Carrera AJ & Facile DM (1995) Extraction and
purification of filamentous fungi DNA. Biotechniques 18:
975-976.