RESUMEN
BACKGROUND: Cystic fibrosis (CF), an inherited autosomal recessive disorder, is linked with high morbidity and mortality rates due to bacteria, filamentous, yeast and black yeast-like fungi colonisation in the upper respiratory tract. Although Candida species are the most common fungi isolated from CF patients, azole-resistant Aspergillus fumigatus (ARAf) is a big concern for invasive aspergillosis. Notably, the exact prevalences of Aspergillus species and the prevalence of ARAf isolates among Iranian CF patients have yet to be previously reported and are unknown. We aimed to investigate the prevalence of ARAf isolates in CF patients among Iranian populations by focusing on molecular mechanisms of the mutations in the target gene. METHODS: The 1 year prospective study recovered 120 sputum samples from 103 CF patients. Of these, 55.1% (86/156) yielded Aspergillus species, screened for ARAf using plates containing itraconazole (4 mg/L) and voriconazole (1 mg/L). According to the CLSI-M38 guidelines, antifungal susceptibility testing was performed using the broth microdilution method. In all phenotypically resistant isolates, the target of azole agents, the cyp51A gene, was sequenced to detect any possible single nucleotide polymorphisms (SNP) mediating resistance. RESULTS: Of 120 samples, 101 (84.2%) were positive for filamentous fungi and yeast-like relatives, with 156 fungal isolates. The most common colonising fungi were Aspergillus species (55.1%, 86/156), followed by Candida species (39.8%, 62/156), Exophiala species (3.8%, 6/156) and Scedosporium species (1.3%, 2/156). Forty out of 86 (46.5%) were identified for section Fumigati, 36 (41.9%) for section Flavi, 6 (7%) for section Nigri and 4 (4.6%) for section Terrei. Fourteen out of 40 A. fumigatus isolates were phenotypically resistant. The overall proportion of ARAf in total fungal isolates was 9% (14/156). cyp51A gene analysis in resistant isolates revealed that 13 isolates harboured G448S, G432C, T289F, D255E, M220I, M172V, G138C, G54E and F46Y mutations and one isolate carried G448S, G432C, T289F, D255E, M220I, G138C, G54E and F46Y mutations. Additionally, this study detects two novel cyp51A single-nucleotide polymorphisms (I242V and D490E). CONCLUSIONS: This study first investigated ARAf isolates in Iranian CF patients. Due to a resistance rate of up to 9%, it is recommended that susceptibility testing of Aspergillus isolates from CF patients receiving antifungal treatment be a part of the routine diagnostic workup. However, extensive multicentre studies with a high volume of CF patients are highly warranted to determine the impact of ARAf on CF patients.
Asunto(s)
Antifúngicos , Aspergillus fumigatus , Azoles , Fibrosis Quística , Sistema Enzimático del Citocromo P-450 , Farmacorresistencia Fúngica , Proteínas Fúngicas , Pruebas de Sensibilidad Microbiana , Humanos , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Irán/epidemiología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Fúngica/genética , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Estudios Prospectivos , Prevalencia , Sistema Enzimático del Citocromo P-450/genética , Azoles/farmacología , Azoles/uso terapéutico , Proteínas Fúngicas/genética , Masculino , Femenino , Aspergilosis/microbiología , Aspergilosis/epidemiología , Aspergilosis/tratamiento farmacológico , Adulto , Niño , Adolescente , Polimorfismo de Nucleótido Simple , Adulto Joven , Esputo/microbiología , Itraconazol/farmacología , Voriconazol/farmacología , Voriconazol/uso terapéutico , Preescolar , MutaciónRESUMEN
We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. This pathogen exhibited reduced azole susceptibility, similar to its close relatives within the Trichomonascus ciferrii species complex.
Asunto(s)
Candida , Genoma Fúngico , Otitis Media , Secuenciación Completa del Genoma , Humanos , Candida/genética , Candida/aislamiento & purificación , Candida/clasificación , Otitis Media/microbiología , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Azoles/farmacología , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de ADNRESUMEN
Purpose: Tuberculosis (TB) remains a major health threat worldwide, and the spread of drug-resistant (DR) TB impedes the reduction of the global disease burden. Ebselen (EbSe) targets bacterial thioredoxin reductase (bTrxR) and causes an imbalance in the redox status of bacteria. Previous work has shown that the synergistic action of bTrxR and sensitization to common antibiotics by EbSe is a promising strategy for the treatment of DR pathogens. Thus, we aimed to evaluate whether EbSe could enhance anti-TB drugs against Mycobacterium marinum (M. marinum) which is genetically related to Mycobacterium tuberculosis (Mtb) and resistant to many antituberculosis drugs. Methods: Minimum inhibitory concentrations (MIC) of isoniazid (INH), rifampicin (RFP), and streptomycin (SM) against M. marinum were determined by microdilution. The Bliss Independence Model was used to determine the adjuvant effects of EbSe over the anti-TB drugs. Thioredoxin reductase activity was measured using the DTNB assay, and its effects on bacterial redox homeostasis were verified by the elevation of intracellular ROS levels and intracellular GSH levels. The adjuvant efficacy of EbSe as an anti-TB drug was further evaluated in a mouse model of M. marinum infection. Cytotoxicity was observed in the macrophage cells Raw264.7 and mice model. Results: The results reveal that EbSe acts as an antibiotic adjuvant over SM on M. marinum. EbSe + SM disrupted the intracellular redox microenvironment of M. marinum by inhibiting bTrxR activity, which could rescue mice from the high bacterial load, and accelerated recovery from tail injury with low mammalian toxicity. Conclusion: The above studies suggest that EbSe significantly enhanced the anti-Mtb effect of SM, and its synergistic combination showed low mammalian toxicity in vitro and in vivo. Further efforts are required to study the underlying mechanisms of EbSe as an antibiotic adjuvant in combination with anti-TB drug MS.
Asunto(s)
Homeostasis , Isoindoles , Pruebas de Sensibilidad Microbiana , Compuestos de Organoselenio , Oxidación-Reducción , Estreptomicina , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/química , Isoindoles/farmacología , Animales , Ratones , Homeostasis/efectos de los fármacos , Estreptomicina/farmacología , Antituberculosos/farmacología , Antituberculosos/química , Mycobacterium marinum/efectos de los fármacos , Azoles/farmacología , Azoles/química , Relación Dosis-Respuesta a Droga , Antibacterianos/farmacología , Antibacterianos/química , Relación Estructura-Actividad , Estructura Molecular , Ratones Endogámicos BALB CRESUMEN
In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1Apo), fluconazole-bound state (Cdr1Flu), and milbemycin oxime-inhibited state (Cdr1Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.
Asunto(s)
Antifúngicos , Azoles , Candida albicans , Microscopía por Crioelectrón , Proteínas Fúngicas , Proteínas de Transporte de Membrana , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/antagonistas & inhibidores , Antifúngicos/farmacología , Antifúngicos/química , Azoles/farmacología , Azoles/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Farmacorresistencia Fúngica , Fluconazol/farmacología , Sitios de UniónAsunto(s)
Antifúngicos , Aspergillus fumigatus , Azoles , Farmacorresistencia Fúngica , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/aislamiento & purificación , Humanos , Dinamarca/epidemiología , Azoles/farmacología , Azoles/uso terapéutico , Antifúngicos/uso terapéutico , Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergilosis/epidemiología , Aspergilosis/tratamiento farmacológicoRESUMEN
The widespread and irrational use of azole antifungal agents has led to an increase of azole-resistant Candida albicans strains with an urgent need for combination drug therapy, enhancing the treatment efficacy. Here, we report the discovery of a first-in-class pyrazole-isoxazole, namely, 5b, that showed remarkable growth inhibition against the C. albicans ATCC 10231 strain in combination with voriconazole, acting as a downregulator of ERG 11 (Cyp51) gene expression with a significant reduction of the yeast-to-hypha morphological transition. Furthermore, C. albicans CYP51 enzyme assay and in-depth molecular docking studies unveiled the unique ability of the combination of 5b and voriconazole to completely fill the CYP51 binding sites. In vivo studies using a Galleria mellonella model confirmed the previously in vitro observed synergistic effect of 5b with voriconazole. Also considering its biocompatibility in a cellular model of human keratinocytes, these results indicate that 5b represents a promising compound for a further optimization campaign.
Asunto(s)
Antifúngicos , Candida albicans , Farmacorresistencia Fúngica , Isoxazoles , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Pirazoles , Voriconazol , Antifúngicos/farmacología , Antifúngicos/química , Voriconazol/farmacología , Candida albicans/efectos de los fármacos , Pirazoles/farmacología , Pirazoles/química , Animales , Humanos , Isoxazoles/farmacología , Isoxazoles/química , Sinergismo Farmacológico , Mariposas Nocturnas/microbiología , Mariposas Nocturnas/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Modelos Animales de Enfermedad , Relación Estructura-Actividad , Azoles/farmacología , Azoles/química , Azoles/uso terapéuticoRESUMEN
Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects. Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in a C. albicans isolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involves KSR1. Similar LOH events involving KSR1, which encodes a reductase in the sphingolipid biosynthesis pathway, were also detected in independently evolved fluconazole resistant isolates. We dissect the specific KSR1 codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV and KSR1 LOH results in a >500-fold decrease in azole susceptibility relative to the progenitor, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.
Asunto(s)
Antifúngicos , Candida albicans , Variaciones en el Número de Copia de ADN , Farmacorresistencia Fúngica , Proteínas Fúngicas , Pérdida de Heterocigocidad , Candida albicans/genética , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Antifúngicos/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fluconazol/farmacología , Azoles/farmacología , Pruebas de Sensibilidad Microbiana , Evolución Molecular , Candidiasis/microbiología , Candidiasis/tratamiento farmacológico , Candidiasis/genéticaRESUMEN
Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-É-methylergosta-8,24(28)-dien-3ß,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.
Asunto(s)
Antifúngicos , Candida albicans , Candidiasis , Farmacorresistencia Fúngica , Ergosterol , Proteínas Fúngicas , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Antifúngicos/farmacología , Ratones , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Animales , Candidiasis/microbiología , Candidiasis/metabolismo , Candidiasis/tratamiento farmacológico , Ergosterol/metabolismo , Azoles/farmacología , Esteroles/metabolismo , Fenotipo , Estrés Fisiológico , Pruebas de Sensibilidad Microbiana , Fluconazol/farmacologíaRESUMEN
Renal ischemia/reperfusion injury (RIRI) is an inevitable complication following kidney transplantation surgery, accompanied by the generation of a large amount of free radicals. A cascade of events including oxidative stress, extreme inflammation, cellular apoptosis, and thrombosis disrupts the microenvironment of renal cells and the hematological system, ultimately leading to the development of acute kidney injury (AKI). The current research primarily focuses on reducing inflammation and mitigating damage to renal cells through antioxidative approaches. However, studies on simultaneously modulating the renal hematologic system remain unreported. Herein, potent and novel drug-loaded nanomicelles can be efficiently self-assembled with magnolol (MG) and ebselen (EBS) by π-π conjugation, hydrophobic action and the surfactant properties of Tween-80. The ultrasmall MG/EBS nanomicelles (average particle size: 10-25 nm) not only fully preserve the activity of both drugs, but also greatly enhance drug utilization (encapsulation rates: MG: 90.1%; EBS: 49.3%) and reduce drug toxicity. Furthermore, EBS, as a glutathione peroxidase mimic and NO catalyst, combines with the multifunctional MG to scavenge free radicals and hydroperoxides, significantly inhibiting inflammation and thrombosis while effectively preventing apoptosis of vascular endothelial cells and renal tubular epithelial cells. This study provides a new strategy and theoretical foundation for the simultaneous regulation of kidney cells and blood microenvironment stability.
Asunto(s)
Compuestos de Bifenilo , Lignanos , Micelas , Compuestos de Organoselenio , Daño por Reperfusión , Daño por Reperfusión/prevención & control , Daño por Reperfusión/tratamiento farmacológico , Compuestos de Bifenilo/química , Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Lignanos/química , Lignanos/administración & dosificación , Humanos , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/administración & dosificación , Isoindoles/farmacología , Isoindoles/administración & dosificación , Isoindoles/química , Apoptosis/efectos de los fármacos , Nanopartículas/química , Nanopartículas/administración & dosificación , Azoles/química , Azoles/farmacología , Azoles/administración & dosificación , Animales , Riñón/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/química , Antioxidantes/administración & dosificación , Tamaño de la Partícula , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Portadores de Fármacos/química , Estrés Oxidativo/efectos de los fármacosRESUMEN
OBJECTIVES: This study evaluated the role of Upc2 in the development of azole resistance in Candida albicans isolates from Lebanese hospitalized patients and determined a correlation between resistance and virulence. METHODS: The UPC2 gene which codes for an ergosterol biosynthesis regulator was sequenced and analysed in two azole-resistant and one azole-susceptible C. albicans isolates. An amino acid substitution screening was carried out on Upc2 with a focus on its ligand binding domain (LBD) known to interact with ergosterol. Then, Upc2 protein secondary structure prediction and homology modelling were conducted, followed by total plasma membrane ergosterol and cell wall chitin quantifications. For virulence, mouse models of systemic infection were generated and an agar adhesion and invasion test was performed. RESULTS: Azole-resistant isolates harboured novel amino acid substitutions in the LBD of Upc2 and changes in protein secondary structures were observed. In addition, these isolates exhibited a significant increase in plasma membrane ergosterol content. Resistance and virulence were inversely correlated while increased cell wall chitin concentration does not seem to be linked to resistance since even though we observed an increase in chitin concentration, it was not statistically significant. CONCLUSIONS: The azole-resistant C. albicans isolates harboured novel amino acid substitutions in the LBD of Upc2 which are speculated to induce an increase in plasma membrane ergosterol content, preventing the binding of azoles to their target, resulting in resistance.
Asunto(s)
Antifúngicos , Azoles , Candida albicans , Candidiasis , Farmacorresistencia Fúngica , Ergosterol , Proteínas Fúngicas , Pruebas de Sensibilidad Microbiana , Mutación , Candida albicans/genética , Candida albicans/efectos de los fármacos , Candida albicans/aislamiento & purificación , Candida albicans/patogenicidad , Líbano , Humanos , Azoles/farmacología , Antifúngicos/farmacología , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Virulencia , Ratones , Candidiasis/microbiología , Sustitución de Aminoácidos , Quitina/metabolismo , Femenino , Pared Celular , Modelos Animales de EnfermedadRESUMEN
Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. IMPORTANCE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.
Asunto(s)
Antifúngicos , Aspergillus fumigatus , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Activación Transcripcional , Aspergillus fumigatus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Antifúngicos/farmacología , Azoles/farmacología , Sistema Enzimático del Citocromo P-450/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Farmacorresistencia Fúngica/genética , Dominios ProteicosRESUMEN
Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis, is currently the most common and damaging disease in apple orchards. Two strains of V. inaequalis (S755 and Rs552) with different sensitivities to azole fungicides and the bacterial metabolite fengycin were compared to determine the mechanisms responsible for these differences. Antifungal activity tests showed that Rs552 had reduced sensitivity to tebuconazole and tetraconazole, as well as to fengycin alone or in a binary mixture with other lipopeptides (iturin A, pumilacidin, lichenysin). S755 was highly sensitive to fengycin, whose activity was close to that of tebuconazole. Unlike fengycin, lipopeptides from the iturin family (mycosubtilin, iturin A) had similar activity on both strains, while those from the surfactin family (lichenysin, pumilacidin) were not active, except in binary mixtures with fengycin. The activity of lipopeptides varies according to their family and structure. Analyses to determine the difference in sensitivity to azoles (which target the CYP51 enzyme involved in the ergosterol biosynthesis pathway) showed that the reduced sensitivity in Rs552 is linked to (i) a constitutive increased expression of the Cyp51A gene caused by insertions in the upstream region and (ii) greater efflux by membrane pumps with the involvement of ABC transporters. Microscopic observations revealed that fengycin, known to interact with plasma membranes, induced morphological and cytological changes in cells from both strains. Sterol and phospholipid analyses showed a higher level of ergosta-7,22-dien-3-ol and a lower level of PI(C16:0/C18:1) in Rs552 compared with S755. These differences could therefore influence the composition of the plasma membrane and explain the differential sensitivity of the strains to fengycin. However, the similar antifungal activities of mycosubtilin and iturin A in the two strains indirectly indicate that sterols are probably not involved in the fengycin resistance mechanism. This leads to the conclusion that different mechanisms are responsible for the difference in susceptibility to azoles or fengycin in the strains studied.
Asunto(s)
Ascomicetos , Azoles , Lipopéptidos , Malus , Enfermedades de las Plantas , Lipopéptidos/farmacología , Malus/microbiología , Enfermedades de las Plantas/microbiología , Ascomicetos/efectos de los fármacos , Ascomicetos/metabolismo , Ascomicetos/genética , Azoles/farmacología , Farmacorresistencia Fúngica/genética , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Antifúngicos/metabolismo , Fungicidas Industriales/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
The medicinal chemistry of ferrocene has gained its momentum after the discovery of biological activities of ferrocifen and ferroquine. These ferrocenyl drugs have been designed by replacing the aromatic moiety of the organic drugs, tamoxifen and chloroquine respectively, with a ferrocenyl unit. The promising biological activities of these ferrocenyl drugs have paved a path to explore the medicinal applications of several ferrocenyl conjugates. In these conjugates, the ferrocenyl moiety has played a vital role in enhancing or imparting the anticancer activity to the molecule. The ferrocenyl conjugates induce the cytotoxicity by generating reactive oxygen species and thereby damaging the DNA. In medicinal chemistry, the five membered nitrogen heterocycles (azoles) play a significant role due to their rigid ring structure and hydrogen bonding ability with the biomolecules. Several potent drug candidates with azole groups have been in use as chemotherapeutics. Considering the importance of ferrocenyl moiety and azole groups, several ferrocenyl azole conjugates have been synthesized and screened for their biological activities. Hence, in the view of a wide scope in the development of potent drugs based on ferrocenyl azole conjugates, herein we present the details of synthesis and the anticancer activities of ferrocenyl compounds bearing azole groups such as imidazole, triazoles, thiazole and isoxazoles.
Asunto(s)
Antineoplásicos , Azoles , Compuestos Ferrosos , Compuestos Heterocíclicos , Metalocenos , Azoles/química , Azoles/farmacología , Azoles/síntesis química , Compuestos Ferrosos/química , Compuestos Ferrosos/farmacología , Compuestos Ferrosos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Humanos , Metalocenos/química , Metalocenos/farmacología , Metalocenos/síntesis química , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/síntesis químicaRESUMEN
BACKGROUND: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. OBJECTIVES: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. METHODS: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. RESULTS: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. CONCLUSIONS: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.
Asunto(s)
Antifúngicos , Azoles , Candida , Candidiasis , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Azoles/farmacología , Candida/efectos de los fármacos , Candida/genética , Candida/clasificación , Candida/aislamiento & purificación , Candidiasis/microbiología , Humanos , Farmacorresistencia Fúngica Múltiple/genética , Colombia , Anfotericina B/farmacología , Farmacorresistencia Fúngica/genética , Mutación Puntual , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Análisis de Secuencia de ADN , Proteínas de Saccharomyces cerevisiaeRESUMEN
BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.
Asunto(s)
Antifúngicos , Aspergillus flavus , Azoles , Sistema Enzimático del Citocromo P-450 , Farmacorresistencia Fúngica , Larva , Pruebas de Sensibilidad Microbiana , Voriconazol , Aspergillus flavus/efectos de los fármacos , Aspergillus flavus/genética , Antifúngicos/farmacología , Farmacorresistencia Fúngica/genética , Animales , Voriconazol/farmacología , Azoles/farmacología , Sistema Enzimático del Citocromo P-450/genética , Larva/microbiología , Triazoles/farmacología , Proteínas Fúngicas/genética , Mariposas Nocturnas/microbiología , Aspergilosis/microbiología , Aspergilosis/tratamiento farmacológico , Virulencia , Aptitud Genética , Modelos Animales de EnfermedadRESUMEN
Cancer exhibits heterogeneity that enables adaptability and remains grand challenges for effective treatment. Chemotherapy is a validated and critically important strategy for the treatment of cancer, but the emergence of multidrug resistance which may lead to recurrence of disease or even death is a major hurdle for successful chemotherapy. Azoles and sulfonamides are important anticancer pharmacophores, and azole-sulfonamide hybrids have the potential to simultaneously act on dual/multiple targets in cancer cells, holding great promise to overcome drug resistance. This review outlines the current scenario of azole-sulfonamide hybrids with the anticancer potential, and the structure-activity relationships as well as mechanisms of action are also discussed, covering articles published from 2020 onward.
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Asunto(s)
Antineoplásicos , Azoles , Neoplasias , Sulfonamidas , Humanos , Sulfonamidas/química , Sulfonamidas/farmacología , Azoles/química , Azoles/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Relación Estructura-Actividad , Estructura MolecularRESUMEN
Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.
Asunto(s)
Antifúngicos , Aspergillus fumigatus , Azoles , Proteínas Fúngicas , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Antifúngicos/farmacología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Azoles/farmacología , Ergosterol/metabolismo , Ergosterol/biosíntesis , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Monoterpenos Bicíclicos/farmacología , Monoterpenos Bicíclicos/metabolismo , Pruebas de Sensibilidad Microbiana , Esterol 14-Desmetilasa/metabolismo , Esterol 14-Desmetilasa/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Escualeno-Monooxigenasa/metabolismo , Escualeno-Monooxigenasa/genética , Lanosterol/análogos & derivadosRESUMEN
Aim: To evaluate the antifungal activity of mangiferin against Candida spp. resistant to fluconazole.Materials & methods: The antifungal activity of mangiferin was assessed using broth microdilution and its interaction with azoles and amphotericin B was evaluated by checkerboard. The activity of mangiferin against Candida spp. biofilms was assessed using the MTT colorimetric assay and its possible mechanism of action was evaluated using flow cytometry.Results: Mangiferin showed activity against Candida albicans, Candida tropicalis and Candida parapsilosis resistant to fluconazole and showed synergism with azoles and amphotericin B. Mangiferin increased the activity of antifungals against Candida biofilms and caused depolarization of the mitochondrial membrane and externalization of phosphatidylserine, suggesting apoptosis.Conclusion: mangiferin combined with antifungals has potential against Candida spp.
Candida is a type of fungus that can make people ill. Over time, many species of Candida have found ways to resist the drugs used to kill them. It is important to find new drugs. We decided to see if a substance called mangiferin works against Candida. We found that mangiferin works against Candida and may help other drugs to work better. We still need to do more studies to find out whether mangiferin can help prevent diseases caused by Candida in the future.
Asunto(s)
Anfotericina B , Antifúngicos , Biopelículas , Candida , Farmacorresistencia Fúngica , Sinergismo Farmacológico , Fluconazol , Pruebas de Sensibilidad Microbiana , Xantonas , Antifúngicos/farmacología , Xantonas/farmacología , Fluconazol/farmacología , Biopelículas/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Anfotericina B/farmacología , Candida/efectos de los fármacos , Humanos , Apoptosis/efectos de los fármacos , Candida albicans/efectos de los fármacos , Azoles/farmacologíaRESUMEN
Superficial infections of the skin, hair, and nails by fungal dermatophytes are the most prevalent of human mycoses, and many infections are refractory to treatment. As current treatment options are limited, recent research has explored drug synergy with azoles for dermatophytoses. Bisphosphonates, which are approved to treat osteoporosis, can synergistically enhance the activity of azoles in diverse yeast pathogens but their activity has not been explored in dermatophytes or other molds. Market bisphosphonates risedronate, alendronate, and zoledronate (ZOL) were evaluated for antifungal efficacy and synergy with three azole antifungals: fluconazole (FLC), itraconazole (ITR), and ketoconazole (KET). ZOL was the most active bisphosphonate tested, displaying moderate activity against nine dermatophyte species (MIC range 64-256 µg/mL), and was synergistic with KET in eight of these species. ZOL was also able to synergistically improve the anti-biofilm activity of KET and combining KET and ZOL prevented the development of antifungal resistance. Rescue assays in Trichophyton rubrum revealed that the inhibitory effects of ZOL alone and in combination with KET were due to the inhibition of squalene synthesis. Fluorescence microscopy using membrane- and ROS-sensitive probes demonstrated that ZOL and KET:ZOL compromised membrane structure and induced oxidative stress. Antifungal activity and synergy between bisphosphonates and azoles were also observed in other clinically relevant molds, including species of Aspergillus and Mucor. These findings indicate that repurposing bisphosphonates as antifungals is a promising strategy for revitalising certain azoles as topical antifungals, and that this combination could be fast-tracked for investigation in clinical trials. IMPORTANCE: Fungal infections of the skin, hair, and nails, generally grouped together as "tineas" are the most prevalent infectious diseases globally. These infections, caused by fungal species known as dermatophytes, are generally superficial, but can in some cases become aggressive. They are also notoriously difficult to resolve, with few effective treatments and rising levels of drug resistance. Here, we report a potential new treatment that combines azole antifungals with bisphosphonates. Bisphosphonates are approved for the treatment of low bone density diseases, and in fungi they inhibit the biosynthesis of the cell membrane, which is also the target of azoles. Combinations were synergistic across the dermatophyte species and prevented the development of resistance. We extended the study to molds that cause invasive disease, finding synergy in some problematic species. We suggest bisphosphonates could be repurposed as synergents for tinea treatment, and that this combination could be fast-tracked for use in clinical therapy.
Asunto(s)
Antifúngicos , Arthrodermataceae , Difosfonatos , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Arthrodermataceae/efectos de los fármacos , Humanos , Difosfonatos/farmacología , Azoles/farmacología , Biopelículas/efectos de los fármacos , Farmacorresistencia Fúngica , Hongos/efectos de los fármacosRESUMEN
More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.