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1.
Artigo em Inglês | MEDLINE | ID: mdl-30783005

RESUMO

Recombinant Candida albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50 compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations. In vitro testing of select CaCYP51 mutations in C. albicans showed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50 values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Esterol 14-Desmetilase/metabolismo , Sequência de Aminoácidos , Candida albicans/genética , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Itraconazol/farmacologia , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Esterol 14-Desmetilase/genética , Triazóis/farmacologia , Voriconazol/farmacologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-29439966

RESUMO

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 µg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 µg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 µg/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 µg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 µg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.


Assuntos
Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Triazóis/farmacologia , Administração Intranasal , Animais , Aspergillus fumigatus/isolamento & purificação , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Citocinas/sangue , Farmacorresistência Fúngica , Células Epiteliais/metabolismo , Ergosterol/biossíntese , Proteínas Fúngicas/antagonistas & inibidores , Galactose/análogos & derivados , Humanos , Hifas/metabolismo , Mananas/sangue , Camundongos , Testes de Sensibilidade Microbiana , Rhizopus/efeitos dos fármacos , Trichophyton/efeitos dos fármacos , Voriconazol/farmacologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-28483956

RESUMO

Prior to characterization of antifungal inhibitors that target CYP51, Trichophyton rubrum CYP51 was expressed in Escherichia coli, purified, and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [Kd ] values, 22.7, 20.3, and 20.9 µM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min-1; Km value, 2 µM). The investigational agent VT-1161 bound tightly to T. rubrum CYP51 (Kd = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole (Kd values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole (Kd = 53 nM). Determinations of 50% inhibitory concentrations (IC50s) using 0.5 µM CYP51 showed that VT-1161 was a tight-binding inhibitor of T. rubrum CYP51 activity, yielding an IC50 of 0.14 µM, whereas itraconazole, fluconazole, and ketoconazole had IC50s of 0.26, 0.4, and 0.6 µM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of T. rubrum growth, with MIC50, MIC90, and geometric mean MIC values of ≤0.03, 0.06, and 0.033 µg ml-1, respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating T. rubrum infections in patients.


Assuntos
Antifúngicos/farmacologia , Piridinas/farmacologia , Tetrazóis/farmacologia , Trichophyton/efeitos dos fármacos , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Clotrimazol/farmacologia , Farmacorresistência Fúngica , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Itraconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana , Esterol 14-Desmetilase/metabolismo , Especificidade por Substrato , Voriconazol/farmacologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-28223388

RESUMO

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 µM and 0.22 µM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 µg/ml, while those of voriconazole ranged from 0.064 to 4 µg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 µg/ml, whereas voriconazole (0.019 to >1 µg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 µg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.


Assuntos
Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Benzamidas/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Triazóis/farmacologia , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/isolamento & purificação , Células Cultivadas , Sistema Enzimático do Citocromo P-450 , Humanos , Itraconazol/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Voriconazol/farmacologia
5.
Antimicrob Agents Chemother ; 60(8): 4530-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27161631

RESUMO

Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 µM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 µM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 µM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes.


Assuntos
Antifúngicos/farmacologia , Cryptococcus/efeitos dos fármacos , Piridinas/efeitos adversos , Piridinas/farmacologia , Esterol 14-Desmetilase/metabolismo , Tetrazóis/efeitos adversos , Tetrazóis/farmacologia , Antifúngicos/efeitos adversos , Clotrimazol/efeitos adversos , Clotrimazol/farmacologia , Cryptococcus/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ergosterol/metabolismo , Fluconazol/efeitos adversos , Fluconazol/farmacologia , Humanos , Itraconazol/efeitos adversos , Itraconazol/farmacologia , Cetoconazol/efeitos adversos , Cetoconazol/farmacologia , Lanosterol/análogos & derivados , Lanosterol/metabolismo , Voriconazol/efeitos adversos , Voriconazol/farmacologia
6.
Mol Biol Evol ; 31(7): 1793-802, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24732957

RESUMO

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.


Assuntos
Ascomicetos/genética , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Ascomicetos/classificação , Ascomicetos/efeitos dos fármacos , Azóis/farmacologia , Evolução Molecular , Fungicidas Industriais/farmacologia , Hordeum/microbiologia , Filogenia , Seleção Genética , Análise de Sequência de DNA
7.
Antimicrob Agents Chemother ; 59(12): 7771-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26459890

RESUMO

The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 µM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 µM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 µM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 µM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Aspergillus fumigatus/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana , Mutação Puntual , Proteínas Recombinantes/química
8.
Antimicrob Agents Chemother ; 59(8): 4707-13, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26014948

RESUMO

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 µM), whereas inhibition by fluconazole was weak (IC50, 30 µM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Antifúngicos/farmacologia , Azóis/farmacologia , Esterol 14-Desmetilase/metabolismo , Acanthamoeba castellanii/metabolismo , Amebíase/microbiologia , Proliferação de Células/efeitos dos fármacos , Fluconazol/farmacologia , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Triazóis/farmacologia , Voriconazol/metabolismo
9.
Appl Environ Microbiol ; 81(10): 3379-86, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25746994

RESUMO

Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.


Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Esterol 14-Desmetilase/química , Sequência de Aminoácidos , Ascomicetos/química , Ascomicetos/genética , Candida albicans/enzimologia , Candida albicans/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Lanosterol/análogos & derivados , Lanosterol/química , Lanosterol/metabolismo , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredução , Alinhamento de Sequência , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Especificidade por Substrato , Temperatura
10.
Appl Environ Microbiol ; 80(19): 6154-66, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25085484

RESUMO

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 µM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 µM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 µg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 µg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 µg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 µM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 µg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Antifúngicos/farmacologia , Clotrimazol/farmacologia , Doenças dos Peixes/tratamento farmacológico , Saprolegnia/efeitos dos fármacos , Animais , Antifúngicos/química , Azóis/química , Azóis/farmacologia , Vias Biossintéticas , Clotrimazol/química , Doenças dos Peixes/microbiologia , Peixes , Testes de Sensibilidade Microbiana/veterinária , Filogenia , Saprolegnia/enzimologia , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Esteróis/análise
11.
Antimicrob Agents Chemother ; 57(3): 1352-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23274672

RESUMO

Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K(s), 14 to 18 µM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K(d), ~30,500 nM) and voriconazole (K(d), ~2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (K(d), ~40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC(50)) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC(50), ~150 µM) and did not significantly inhibit Δ60HsCYP51.


Assuntos
Antifúngicos/química , Candida albicans/enzimologia , Proteínas Fúngicas/química , Esterol 14-Desmetilase/química , Candida albicans/química , Clotrimazol/química , Escherichia coli/genética , Fluconazol/química , Proteínas Fúngicas/genética , Humanos , Itraconazol/química , Cetoconazol/química , Cinética , Lanosterol/química , NADP/química , Ligação Proteica , Pirimidinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade da Espécie , Esterol 14-Desmetilase/genética , Triazóis/química , Voriconazol
12.
Appl Environ Microbiol ; 79(5): 1639-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23275516

RESUMO

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Inibidores Enzimáticos/farmacologia , Esterol 14-Desmetilase/metabolismo , Triazóis/farmacologia , Antifúngicos/metabolismo , Inibidores Enzimáticos/metabolismo , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Triazóis/metabolismo , Voriconazol
13.
Biotechnol Appl Biochem ; 60(1): 52-64, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23586992

RESUMO

Mycosphaerella graminicola is a key fungal pathogen of wheat and a major target for azole fungicides, many of whose central mode of action is through inhibition of cytochrome P450 51 (lanosterol 14α-demethylase) in the ergosterol biosynthetic pathway. The range of activities of other fungal CYPs is thought to be a reflection of the differences between different organisms and their range of secondary metabolic pathways as a response to their niche environments, for example, in the production of mycotoxins. The present study collates information from a range of databases, to classify the CYPs found in M. graminicola and assign them an internationally recognized nomenclature, which, when referenced to the recent publication of the JGI version 2.0 genome model, creates a current, robust model for the CYP complement (CYPome) of M. graminicola. These CYPome data, which examined 82 CYPs and one pseudo-gene, may be utilized not only to further characterize and describe the physiology of the organism but also to enhance our understanding of CYP function and diversity.


Assuntos
Ascomicetos/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética
14.
Antimicrob Agents Chemother ; 56(4): 2099-107, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22252802

RESUMO

The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (K(s), 7.1 and 8.0 µM, respectively) compared to the wild-type CaCYP51 protein (K(s), 13.5 µM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher K(d) values observed.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Antifúngicos/farmacologia , Candida albicans/enzimologia , Candida albicans/genética , Fluconazol/farmacologia , Mutação Puntual/genética , Esterol 14-Desmetilase/genética , Sequência de Aminoácidos , Azóis/metabolismo , Ligação Competitiva/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , DNA Fúngico/genética , Cinética , Lanosterol/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/biossíntese
15.
Antimicrob Agents Chemother ; 56(12): 6417-21, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23027188

RESUMO

Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 µg ml(-1)) were found to be ERG2 mutants, wherein Δ(8)-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr(121) in ERG2; the corresponding residue (Thr(119)) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candidíase/microbiologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Esteroide Isomerases/genética , Sequência de Aminoácidos , Azóis/farmacologia , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Pirimidinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Esteróis/química , Triazóis/farmacologia , Voriconazol
16.
Antimicrob Agents Chemother ; 56(8): 4223-32, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22615281

RESUMO

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 µg ml(-1), respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ((glc)YM). However, when grown on sterol-supplemented (glc)YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ(7)-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented (glc)YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using (glc)YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using (glc)YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 µg AMB ml(-1), respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Azóis/farmacologia , Candida glabrata/metabolismo , Farmacorresistência Fúngica Múltipla/genética , Mutação de Sentido Incorreto , Esterol 14-Desmetilase/genética , Sequência de Bases , Transporte Biológico , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candida glabrata/isolamento & purificação , Ergosterol/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Humanos , Testes de Sensibilidade Microbiana , Pirimidinas/farmacologia , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Esteróis/metabolismo , Triazóis/farmacologia , Voriconazol
17.
PLoS One ; 17(3): e0265227, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35312722

RESUMO

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min-1), palmitic (1.61 min-1) and stearic acids (1.24 min-1), at both the ω-1- and ω-2-positions. However, CYP168A1 only hydroxylates unsaturated fatty acids, including palmitoleic (0.38 min-1), oleic (1.28 min-1) and linoleic acids (0.35 min-1), at the ω-1-position. CYP168A1 exhibited a catalytic preference for palmitic, oleic and stearic acids as substrates in keeping with the phosphatidylcholine-rich environment deep in the lung that is colonized by P. aeruginosa.


Assuntos
Ácidos Graxos , Pseudomonas aeruginosa , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Esteáricos
18.
Appl Environ Microbiol ; 77(11): 3830-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21478305

RESUMO

The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.


Assuntos
Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Azóis/metabolismo , Farmacorresistência Fúngica , Fungicidas Industriais/metabolismo , Esterol 14-Desmetilase/metabolismo , Substituição de Aminoácidos/genética , Ascomicetos/genética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Triticum/microbiologia
19.
Appl Environ Microbiol ; 77(4): 1460-5, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21169436

RESUMO

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 µg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.


Assuntos
Ascomicetos/efeitos dos fármacos , Ascomicetos/metabolismo , Farmacorresistência Fúngica , Esterol 14-Desmetilase/metabolismo , Triazóis/metabolismo , Eletroforese em Gel de Poliacrilamida , Ergosterol/metabolismo , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Fungicidas Industriais/farmacologia , Lanosterol/análogos & derivados , Lanosterol/metabolismo , Doenças das Plantas , Ligação Proteica/efeitos dos fármacos
20.
Antimicrob Agents Chemother ; 54(11): 4920-3, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20733045

RESUMO

Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 µg ml⁻¹, respectively).


Assuntos
Antibacterianos/farmacologia , Aspergillus fumigatus/enzimologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Doxiciclina/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Clotrimazol/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Ergosterol/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Teste de Complementação Genética , Itraconazol/farmacologia , Pirimidinas/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Triazóis/farmacologia , Voriconazol
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