RESUMO
Tetrazole antifungals designed to target fungal lanosterol 14α-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain of Candida albicans LDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-length Saccharomyces cerevisiae LDM with a C-terminal hexahistidine tag (ScLDM6×His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates of C. albicans and Candida glabrata VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.
Assuntos
Antifúngicos/metabolismo , Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Proteínas Fúngicas/química , Piridinas/metabolismo , Esterol 14-Desmetilase/química , Tetrazóis/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida glabrata/enzimologia , Candida glabrata/genética , Candida glabrata/crescimento & desenvolvimento , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Piridinas/química , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Especificidade por Substrato , Tetrazóis/química , Tetrazóis/farmacologia , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Lanosterol/metabolismo , Esterol 14-Desmetilase/metabolismo , Azóis/farmacologia , Candida albicans/metabolismo , Candida glabrata/metabolismo , Domínio Catalítico/efeitos dos fármacos , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Triazóis/farmacologiaRESUMO
Infections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 Å. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Lanosterol/química , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Esterol 14-Desmetilase/química , Aspergillus fumigatus/efeitos dos fármacos , Azóis/química , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Cristalografia por Raios X/métodos , Farmacorresistência Fúngica Múltipla , Fluconazol , Ligação de Hidrogênio , Triazóis/química , Triazóis/farmacologiaRESUMO
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.
RESUMO
The cytochrome P450 enzyme lanosterol 14α-demethylase (LDM) is the target of the azole antifungals used widely in medicine and agriculture as prophylaxis or treatments of infections or diseases caused by fungal pathogens. These drugs and agrochemicals contain an imidazole, triazole or tetrazole substituent, with one of the nitrogens in the azole ring coordinating as the sixth axial ligand to the LDM heme iron. Structural studies show that this membrane bound enzyme contains a relatively rigid ligand binding pocket comprised of a deeply buried heme-containing active site together with a substrate entry channel and putative product exit channel that reach to the membrane. Within the ligand binding pocket the azole antifungals have additional affinity determining interactions with hydrophobic side-chains, the polypeptide backbone and via water-mediated hydrogen bond networks. This review will describe the tools that can be used to identify and characterise the next generation of antifungals targeting LDM, with the goal of obtaining highly potent broad-spectrum fungicides that will be able to avoid target and drug efflux mediated antifungal resistance.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Antifúngicos/farmacologia , Esterol 14-Desmetilase/química , Inibidores de 14-alfa Desmetilase/química , Inibidores de 14-alfa Desmetilase/economia , Inibidores de 14-alfa Desmetilase/uso terapêutico , Agroquímicos/química , Animais , Antifúngicos/química , Antifúngicos/economia , Antifúngicos/uso terapêutico , Azóis/química , Azóis/economia , Azóis/farmacologia , Azóis/uso terapêutico , Descoberta de Drogas , Ecossistema , Abastecimento de Alimentos , Humanos , Camundongos , Micoses/tratamento farmacológico , Esterol 14-Desmetilase/metabolismoRESUMO
The very different multi-step routes to the closely related pair of diformyl-carbazole head units, 1,8-diformyl-3,6-di-tert-butyl-9H-carbazole (1tBu) and 1,8-diformyl-9H-carbazole (1H), are detailed and compared. The first examples of Schiff base macrocycles derived from diformyl-carbazole head units are reported. Specifically, the direct cyclisation of 1tBu or 1H with diethylenetriamine gives the two metal-free [1 + 1] Schiff base macrocycles HLH and HLtBu in high yields. Four carbazole-based macrocyclic complexes, [CuIIL(OH2)]OAc and [NiIIL]OAc, where L = LH or LtBu, were accessed either by metallation of these macrocycles, or by metal templated reaction of the macrocycle components. [CuIILtBu(OH2)]OAc·0.5(Ether) and [NiLH]OAc·EtOH, were structurally characterised, confirming the nickel(ii) complexes are square planar (both show diamagnetic NMR spectra) and that the copper(ii) complexes are square pyramidal with a water molecule bound in the axial site. Like porphyrins, both of these N4-donor macrocycles, which differ only in the R group present at the 3 and 6 positions (H or tBu), impose a strong ligand field.
RESUMO
Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance.
Assuntos
Antifúngicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacorresistência Fúngica , Mutação de Sentido Incorreto , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Triazóis/farmacologia , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Testes de Sensibilidade Microbiana , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Tirosina/genéticaRESUMO
Azole antifungals, known as demethylase inhibitors (DMIs), target sterol 14α-demethylase (CYP51) in the ergosterol biosynthetic pathway of fungal pathogens of both plants and humans. DMIs remain the treatment of choice in crop protection against a wide range of fungal phytopathogens that have the potential to reduce crop yields and threaten food security. We used a yeast membrane protein expression system to overexpress recombinant hexahistidine-tagged S. cerevisiae lanosterol 14α-demethylase and the Y140F or Y140H mutants of this enzyme as surrogates in order characterize interactions with DMIs. The whole-cell antifungal activity (MIC50 values) of both the R- and S-enantiomers of tebuconazole, prothioconazole (PTZ), prothioconazole-desthio, and oxo-prothioconazole (oxo-PTZ) as well as for fluquinconazole, prochloraz and a racemic mixture of difenoconazole were determined. In vitro binding studies with the affinity purified enzyme were used to show tight type II binding to the yeast enzyme for all compounds tested except PTZ and oxo-PTZ. High resolution X-ray crystal structures of ScErg11p6×His in complex with seven DMIs, including four enantiomers, reveal triazole-mediated coordination of all compounds and the specific orientation of compounds within the relatively hydrophobic binding site. Comparison with CYP51 structures from fungal pathogens including Candida albicans, Candida glabrata and Aspergillus fumigatus provides strong evidence for a highly conserved CYP51 structure including the drug binding site. The structures obtained using S. cerevisiae lanosterol 14α-demethylase in complex with these agrochemicals provide the basis for understanding the impact of mutations on azole susceptibility and a platform for the structure-directed design of the next-generation of DMIs.
Assuntos
Inibidores de 14-alfa Desmetilase/química , Agroquímicos/química , Antifúngicos/química , Esterol 14-Desmetilase/química , Leveduras/enzimologia , Inibidores de 14-alfa Desmetilase/farmacologia , Agroquímicos/farmacologia , Antifúngicos/farmacologia , Azóis/química , Azóis/farmacologia , Sítios de Ligação , Domínio Catalítico , Testes de Sensibilidade Microbiana , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Esterol 14-Desmetilase/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Leveduras/efeitos dos fármacosRESUMO
The nickel(II) spin state in a series of N4-donor macrocycles differing in the number of imine bonds is tuned from low spin (2 imines) to low or high spin (1 imine; non- or coordinating co-ligand) to high spin (no imines).
RESUMO
A metal-free 16-membered N4-donor [1 + 1] Schiff-base macrocycle was isolated as HL(Pr)·2acid, by 1 : 1 condensation of diphenylamine-2,2'-dicarboxaldehyde (1), dipropylene triamine and an excess of either acetic or formic acid, or as HL(Pr)·(tosylic acid) when just one equivalent of tosylic acid was employed. Interestingly, the acid-free synthesis employed for the 14-membered analogue HL(Et) failed to generate pure HL(Pr) macrocycle, but nevertheless the crude product obtained was able to be used in subsequent complexation reactions to form five mononuclear complexes: Zn(II)L(Pr)(BF4)·H2O·0.5IPA (where IPA is isopropylalcohol), [Cu(II)L(Pr)](BF4), [Ni(II)L(Pr)](BF4), [Co(II)L(Pr)](BF4)·0.5H2O, [Fe(III)L(Pr)(NCS)2]·1.5H2O. Crystal structure determinations show that, like the HL(Et) analogues, [Ni(II)L(Pr)](BF4) features a square planar N4 coordinated Ni(II) centre and [Fe(III)L(Pr)(NCS)2]·0.15MeOH·0.2H2O features an octahedral N6 coordinated Fe(III) centre (two NCS anions bound axially). In both cases the N4-donor macrocycle is bound equatorially to the metal ion. Cyclic voltammograms of the BF4 complexes were carried out in MeCN vs. 0.01 mol L(-1) AgNO3/Ag and revealed multiple redox processes. The Zn(II) complex exhibits multiple ligand-centered redox processes. Interestingly, [Ni(II)L(Pr)](BF4) has two reversible redox processes, at E(m) = +0.38 (ΔE = 0.06 V) and -1.7 V (ΔE = 0.06 V), whereas the previously reported analogue [Ni(II)L(Et)](BF4) had a process at E(pc) = +0.59 V with only a weak return wave. Likewise, [Cu(II)L(Pr)](BF4) has a reversible process, at E(m) = -1.17 V (ΔE = 0.06 V) plus a process at E(pc) = +0.45 V, whereas previously reported [Cu(II)L(Et)](BF4) only featured irreversible processes, with the oxidation occurring at E(pc) = +0.50 V.