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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-ɑ-methylergosta-8,24(28)-dien-3β,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.

Acquired resistance in Candida albicans brings about a serious challenge to the clinical application of azoles, so it is urgent to elucidate the mechanisms of azole resistance to improve the therapeutic efficiency. In the aim of searching for the potential targets mediating fluconazole resistance, we screened a mutant library of 48 transcription factor deletion Candida albicans strains. The screening results showed that hap2Δ/Δ mutants were significantly more susceptible to azoles, especially to miconazole (MCZ). Under MCZ treatment, the intracellular reactive oxygen species (ROS) were significantly higher in hap2Δ/Δ mutants compared to the control strain SN250. The addition of antioxidants reversed the MCZ-sensitive phenotype caused by the deletion of HAP2. Consistently, the expression of antioxidases responsible for scavenging ROS was shown to decrease in hap2Δ/Δ mutants, suggesting that the transcription factor Hap2p is involved in the regulation of oxidative stress responses in C. albicans. In addition, HAP2 deficiency also resulted in impaired mitochondrial function and affected cellular energy supply, which may be related to the iron deficiency regulated by HAP complex. HAP2 disruption also decreased efflux-mediated resistance of C. albicans, as demonstrated by a significant decrease in Cdr1p expression and a slight decrease in Mdr1p expression in hap2Δ/Δ strains under the action of MCZ. The above results indicate that the transcription factor Hap2p was required for the resistance of C. albicans to azoles, which could provide a new strategy to solve the clinical azoles resistance. [ABSTRACT FROM AUTHOR]

Although the mitochondrial genome is an attribute of all eukaryotes, some yeast species (called petite-positive) can replicate without mitochondrial DNA (mtDNA). Strains without mtDNA (known as rho− mutants or petite mutants) are respiration-deficient and require fermentable carbon sources (such as glucose) for their metabolism. However, they are compromised in many aspects of fitness and competitiveness. Nevertheless, a few research groups have reported that some petite mutants of Candida glabrata and Saccharomyces cerevisiae manifested higher levels of tolerance to the antifungal fluconazole than their wild-type (WT) counterparts. In this study, we show that elevated tolerance to two or three out of four tested antifungals is a generic feature of at least five petite-positive species of yeasts including C. glabrata (higher tolerance of petites to clotrimazole and miconazole), S. bayanus (tolerance to clotrimazole, fluconazole, and miconazole), S. cerevisiae (tolerance to clotrimazole and fluconazole), S. paradoxus (tolerance to clotrimazole, fluconazole, and miconazole), and S. pastorianus (tolerance to clotrimazole and fluconazole). Comparing the levels of tolerance to the antifungals in WT and petite mutants was based on measuring the diameters of the zones of inhibition (ZOIs) using disc diffusion assays. The mode of inhibition in the majority of WT strains by all antifungals was fungicidal; most of the rho− mutants manifested fungistatic inhibition. We observed partial (not complete) inhibition in WT, with four different types of ZOI patterns that were species- and antifungal-specific. The partial inhibition was characterised by the presence of antifungal-tolerant colonies within ZOI areas. The inability of these colonies selected from ZOIs to grow on glycerol, as a single source of carbon, proved that they were rho− mutants spontaneously generated in the WT populations. The results on the elevated tolerance of petite strains to antifungals are discussed in terms of the prospective positive selection of respiratory-deficient mutants and the various implications of such selection. [ABSTRACT FROM AUTHOR]

A series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives–hydrazones, N-ethylhydrazones, pyrrole, pyrazole, oxadiazole, and triazole were synthesized and evaluated for their anticancer activity using human A549 pulmonary epithelial cells (ATCC CCl-185). The in vitro viability inhibitory effects of the compounds were assessed using the MTT assay. The characterization of the anticancer activity of the synthesized compounds showed that the incorporation of 1,3,4-oxadiazolethione and 4-aminotriazolethione rings into the molecular structures obviously enhances the anticancer activity against human A549 lung epithelial cells, reducing their viability to 28.0% and 29.6%, respectively. The anticancer activity of these azole derivatives was significantly higher than that of cytarabine. Further studies are needed to better optimize 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives and enhance their in vitro anticancer activity. [ABSTRACT FROM AUTHOR]

Pyrazole and tetrazole are among the most important heterocyclic members of the azole family. Over the past decade, these N-heterocycles and their derivatives have demonstrated specific properties that give them potent applications in several fields such as pharmacology, technology, and agriculture. Combining these two azoles in single hybrid architecture has given rise to highly potent molecules in terms of efficacy and specificity, with enhanced and scalable properties. In this context, the present paper deals with the literature of the last 10 years describing the synthesis protocols for pyrazole-tetrazole-based molecules. Their biological activities as well as their energetic properties are also reported. [ABSTRACT FROM AUTHOR]

The efficacy and safety of venetoclax in newly diagnosed pediatric acute myeloid leukemia (AML) are not well-established as they are in adults. Children newly diagnosed with AML were recommended for induction therapy with venetoclax and chemotherapy or hypomethylating agents (HMAs) as per for the ChiCTR1900027146 trial. Venetoclax was administered at a consistent dose of 200 mg/m2/day for 28 days, with adjustments when used concurrently with azoles. The study measured both the remission rates and the safety assessments of venetoclax. We enrolled 45 newly diagnosed pediatric patients with AML. The complete remission rates were 94.7% in the low/middle-risk group and 80.8% in the high-risk group; MRD-negative rates were 52.6% and 38.5% in the low/middle-risk group and high-risk group, respectively. Venetoclax based combination therapy was well tolerated by the majority of patients. The median duration of venetoclax dosing was 18 days (range 9–28), with hematological toxicity and infection being the most common adverse events. Venetoclax-based induction regimens demonstrated a high response rate and safety profile in newly diagnosed pediatric AML cases. This underscores the significance of venetoclax as a viable treatment option for untreated AML, extending beyond its role as salvage therapy for refractory/relapsed AML. [ABSTRACT FROM AUTHOR]

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.

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.

Invasive fungal infections are rapidly increasing, and the opportunistic pathogenic Candida species are the fourth most common cause of nosocomial systemic infections. The current antifungal classes, of which azoles are the most widely used, all have shortcomings. Azoles are generally considered fungistatic rather than fungicidal, they do not actively kill fungal cells and therefore resistance against azoles can be rapidly acquired. Combination therapies with azoles provide an interesting therapeutic outlook and agents limiting iron are excellent candidates. We summarize how iron is acquired by the host and transported towards both storage and iron‐utilizing organelles. We indicate whether these pathways alter azole susceptibility and/or tolerance, to finally link these transport mechanisms to mitochondrial iron availability. In this review, we highlight putative novel intracellular iron shuffling mechanisms and indicate that mitochondrial iron dynamics in relation to azole treatment and iron limitation is a significant knowledge gap. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance poses a major threat to global health as the number of efficient antimicrobials decreases and the number of resistant pathogens rises. Our research group has been actively involved in the design of novel antimicrobial drugs. The blueprints of these compounds were azolic heterocycles, particularly thiazole. Starting with oxadiazolines, our research group explored, one by one, the other five-membered heterocycles, developing more or less potent compounds. An overview of this research activity conducted by our research group allowed us to observe an evolution in the methodology used (from inhibition zone diameters to minimal inhibitory concentrations and antibiofilm potential determination) correlated with the design of azole compounds based on results obtained from molecular modeling. The purpose of this review is to present the development of in-house azole compounds with antimicrobial activity, designed over the years by this research group from the departments of Pharmaceutical and Therapeutical Chemistry in Cluj-Napoca. [ABSTRACT FROM AUTHOR]

Fungal resistance is a public health concern due to the limited availability of antifungal resources and the complexities associated with treating persistent fungal infections. Azoles are thus far the primary line of defense against fungi. Specifically, azoles inhibit the conversion of lanosterol to ergosterol, producing defective sterols and impairing fluidity in fungal plasmatic membranes. Studies on azole resistance have emphasized specific point mutations in CYP51/ERG11 proteins linked to resistance. Although very insightful, the traditional approach to studying azole resistance is time-consuming and prone to errors during meticulous alignment evaluation. It relies on a reference-based method using a specific protein sequence obtained from a wild-type (WT) phenotype. Therefore, this study introduces a machine learning (ML)-based approach utilizing molecular descriptors representing the physiochemical attributes of CYP51/ERG11 protein isoforms. This approach aims to unravel hidden patterns associated with azole resistance. The results highlight that descriptors related to amino acid composition and their combination of hydrophobicity and hydrophilicity effectively explain the slight differences between the resistant non-wild-type (NWT) and WT (nonresistant) protein sequences. This study underscores the potential of ML to unravel nuanced patterns in CYP51/ERG11 sequences, providing valuable molecular signatures that could inform future endeavors in drug development and computational screening of resistant and nonresistant fungal lineages. [ABSTRACT FROM AUTHOR]

Candida parapsilosis is one of the most commen causes of life-threatening candidaemia, particularly in premature neonates, individuals with cancer of the haematopoietic system, and recipients of organ transplants. Historically, drug-susceptible strains have been linked to clonal outbreaks. However, worldwide studies started since 2018 have reported severe outbreaks among adults caused by fluconazole-resistant strains. Outbreaks caused by fluconazole-resistant strains are associated with high mortality rates and can persist despite strict infection control strategies. The emergence of resistance threatens the efficacy of azoles, which is the most widely used class of antifungals and the only available oral treatment option for candidaemia. The fact that most patients infected with fluconazole-resistant strains are azole-naive underscores the high potential adaptability of fluconazole-resistant strains to diverse hosts, environmental niches, and reservoirs. Another concern is the multidrug-resistant and echinocandin-tolerant C parapsilosis isolates, which emerged in 2020. Raising awareness, establishing effective clinical interventions, and understanding the biology and pathogenesis of fluconazole-resistant C parapsilosis are urgently needed to improve treatment strategies and outcomes.

From the standpoint of chemical structures, the organic backbones of energetic materials can be classified into aromatic rings, nonaromatic rings, and open chains. Although the category of aromatic energetic compounds exhibits several advantages in the regulation of energetic properties, the nonaromatic heterocycles, assembling nitramino explosophores with simple alkyl bridges, still have prevailed in benchmark materials. The methylene bridge plays a pivotal role in the constructions of the classic nonaromatic heterocycle-based energetic compounds, e.g., hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), whereas ethylene bridge is the core moiety of state-of-the-art explosive 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20). In this context, it is of great interest to employ simple and practical bridges to assemble aromatic and nonaromatic nitrogen-rich heterocycles, thereby expanding the structural diversity of energetic materials, e.g., bridged and fused nitrogen-rich poly-heterocycles. Furthermore, alkyl-bridged poly-heterocycles highlight the potential for the open chain type of energetic materials. In this review, the development of alkyl bridges in linking nitrogen-rich heterocycles is presented, and the perspective of the newly constructed energetic backbones is summarized for the future design of advanced energetic materials.

Introduction. Vulvovaginal candidiasis is an extremely common pathology of the female genital organs, leading to a long-term recurrent course and multiple complications. Although currently it is widely known about developing antibiotic resistance of bacterial pathogens, it is necessary to remember about similar phenomenon observed in other groups of infectious agents. In this regard, fungal infection also requires development of new therapeutic techniques and medicinal antifungal drugs, such as ibrexafungerp. Aim: to analyze available publications revealing the mechanism of action, efficacy, antifungal spectrum and results of clinical trials for a new oral antifungal drug ibrexafungerp. Materials and Methods. A search for publications in the electronic databases PubMed, eLibrary and ClinicalTrials.gov, published over the last 25 years was conducted using the following keywords in Russian and English: “candidiasis”, “vulvovaginal candidiasis”, “antifungal drugs”, “ibrexafungerp”, “clinical trials”, “mechanism of action”. Articles were assessed according to PRISMA guidelines. The titles and abstracts of identified publications were independently reviewed to retrieve relevant full text studies. After the selection procedure, 46 articles were included in the review. Results. This review provides information on the creation of the drug ibrexafungerp, its mechanism of action, the activity against a relatively wide range of pathogens, as well as the results from 13 ongoing and completed clinical trials in patients with fungal infection. Conclusion. The analysis of ibrexafungerp-related clinical studies showed its good oral bioavailability, high antifungal efficacy, so that its one-day dosage may further eliminate a need for unnecessarily long hospitalization and complex dosing schedules, thereby increasing adherence to therapy and odds for treatment success.

Azole fungicides are used for spraying crops and also in seed treatments of corn, wheat and other important agricultural crops, in which seeds are dressed with a plastic-like coat containing an azole fungicide and other seed and seedling protection agents. In this study, the effect of tebuconazole in corn seed treatment on selecting for tebuconazole-resistant A. flavus isolates was investigated. Seed-borne A. flavus isolates growing during seed germination were tested for tebuconazole resistance. When seeds were treated with increasing dosages of tebuconazole, the relative abundance of resistant isolates increased. At the recommended dosage, up to 72.1 % of the seed borne A. flavus isolates that emerged from germinating seeds were resistant to tebuconazole. Resistance increased to 83.4 and 95.1 %, when dosages were doubled or quadrupled, respectively. Application of tebuconazole also increased the abundance of aflatoxin-producing isolates of A. flavus, from 32.2 % in untreated seeds to 67.4 % in seeds receiving the highest dosage. Results from this study suggest that seed treatment with tebuconazole should be included in the list of hotspots that induce resistance to azole antifungals and that measures and strategies, such as alternative fungicides with different metabolic targets, should be considered for reducing this risk.

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-3β,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. Azole antifungals inhibit the ergosterol biosynthesis enzyme CYP51, but their effects on fungal viability and growth vary greatly among fungal species. Here, the authors provide evidence that the mode of action of azoles against Aspergillus fumigatus relies on accumulation of the CYP51 substrate eburicol, which exerts fungicidal effects by triggering cell-wall carbohydrate patch formation. [ABSTRACT FROM AUTHOR]

Introduction: Fungal diseases are frequently associated with elevated mortality rates in elasmobranchs. Currently, there is a notable absence of scientifically validated therapeutic medications that can ensure both effectiveness and safety when administered to this group of animals. The empirical prescription of azole antifungal agents, particularly voriconazole, has been posited as a potentially efficacious treatment approach for addressing most common mycoses in sharks and rays. However, there are still no published pharmacokinetic studies supporting its use in elasmobranchs and there is a lack of scientific base for its utilization in elasmobranchs. Methods: For this study, voriconazole was administered intravenously (IV) and intramuscularly (IM), at a single dose of 4 mg/kg to six adult undulate skates (Raja undulata). A washout period of 8 weeks was left between each route of administration. Blood samples were collected both before and at ten predetermined intervals after each dosing (0.25, 0.5, 1, 1.5, 2, 4, 8, 12, 24, and 36 h after drug administration). Plasma concentrations were quantified using a validated high-performance liquid chromatography method, and pharmacokinetic (PK) data was analyzed through non-compartmental methods. Results: The mean extrapolated concentration at 0 h (C0) after IV administration was 27.19 ± 7.15 µg/mL and the mean peak plasma concentrations (Cmax) ± SEM after IM administration resulted 2.98 ± 0.28 µg/mL at a mean time to maximum concentration (T max) of 1.33 ± 0.17 h. Terminal half-lives were calculated and resulted 11.18 ± 1.32 h for IV injections and 9.59 ± 1.38 h for IM injections. The area under the curve extrapolated to infinity was determined as 58.14 ± 2.79 h·µg/ml following IV injections and 37.60 ± 6.67 h·µg/ml following IM injections. The IM-administered voriconazole exhibited a mean absolute bioavailability of 64.67 ± 11.47%. Discussion: These discoveries provide backing for the possible application of voriconazole through the intramuscular route in undulate skates and support using lower dosage regimens compared to those required for oral administration, emphasizing the importance of conducting further pharmacokinetic studies with antifungals in elasmobranchs. [ABSTRACT FROM AUTHOR]

A series of hybrid compounds with triazole and thiazolidine nuclei connected by a linker has been synthesized and extensively studied. Various synthetic methods for the target compounds have been tested. A microbiological assessment of the obtained compounds was carried out on strains of pathogenic fungi C. albicans, C. non-albicans, multidrug-resistant C. auris, Rhizopus arrhizus, Aspergillus spp. and some dermatophytes and other yeasts. The lowest obtained MIC values for target compounds lie between 0.003 µg/mL and 0.5 µg/mL and therefore the compounds are not inferior or several times better than commercial azole drugs. The length of the acylpiperazine linker has a limited effect on antifungal activity. Some bioisosteric analogues were tested in microbiological analysis, but turned out to be weaker than the leader in activity. The highest activity was demonstrated by a compound with para-chlorobenzylidene substituent in the thiazolidine fragment. Molecular modelling was used to predict binding modes of synthesized molecules and rationalize experimentally observed SAR. The leader compound is twice more effective in inhibiting the formation of germ tubes by Candida albicans yeast cells compared to voriconazole. An increased level of Pdr5, an azoles drug efflux pump was observed, but the increase is lower than that caused by azoles. The results can be useful for further development of more powerful and safe antifungal agents. [ABSTRACT FROM AUTHOR]

Microbial contamination poses a threat to both the preservation of library and archival collections and the health of staff and users. This study investigated the microbial communities and potential health risks associated with the UNESCO-classified Norwegian Sea Trade Archive (NST Archive) collection exhibiting visible microbial colonization and staff health concerns. Dust samples from book surfaces and the storage environment were analysed using culturing methods, qPCR, Next Generation Sequencing, and mycotoxin, cytotoxicity, and azole resistance assays. Penicillium sp., Aspergillus sp., and Cladosporium sp. were the most common fungi identified, with some potentially toxic species like Stachybotrys sp., Toxicladosporium sp., and Aspergillus section Fumigati. Fungal resistance to azoles was not detected. Only one mycotoxin, sterigmatocystin, was found in a heavily contaminated book. Dust extracts from books exhibited moderate to high cytotoxicity on human lung cells, suggesting a potential respiratory risk. The collection had higher contamination levels compared to the storage environment, likely due to improved storage conditions. Even though overall low contamination levels were obtained, these might be underestimated due to the presence of salt (from cod preservation) that could have interfered with the analyses. This study underlines the importance of monitoring microbial communities and implementing proper storage measures to safeguard cultural heritage and staff well-being. [ABSTRACT FROM AUTHOR]

Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies. Antifungal azoles inhibit ergosterol biosynthesis, but how that leads to fungistatic or fungicidal activities in many pathogenic fungi is poorly understood. Here, Schuster, Kilaru & Steinberg show that azole lethality in the plant pathogens Zymoseptoria tritici and Magnaporthe oryzae is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. [ABSTRACT FROM AUTHOR]

General, catalytic and enantioselective construction of chiral α,α-dialkyl indoles represents an important yet challenging objective to be developed. Herein we describe a cobalt catalyzed enantioselective anti-Markovnikov alkene hydroalkylation via the remote stereocontrol for the synthesis of α,α-dialkyl indoles and other N-heterocycles. This asymmetric C(sp3)−C(sp3) coupling features high flexibility in introducing a diverse set of alkyl groups at the α-position of chiral N-heterocycles. The utility of this methodology has been demonstrated by late-stage functionalization of drug molecules, asymmetric synthesis of bioactive molecules, natural products and functional materials, and identification of a class of molecules exhibiting anti-apoptosis activities in UVB-irradiated HaCaT cells. Ligands play a vital role in controlling the reaction regioselectivity. Changing the ligand from bi-dentate L6 to tridentate L12 enables CoH-catalyzed Markovnikov hydroalkylation. Mechanistic studies disclose that the anti-Markovnikov hydroalkylation involves a migratory insertion process while the Markovnikov hydroalkylation involves a MHAT process. Catalytic and enantioselective construction of chiral α,α-dialkyl indoles represents an important yet challenging objective to be developed. Herein the authors describe a cobalt catalyzed enantioselective anti-Markovnikov alkene hydroalkylation via the remote stereocontrol for the synthesis of α,α-dialkyl indoles and other N-heterocycles. [ABSTRACT FROM AUTHOR]

The wheat scab caused by Fusarium graminearum (F. graminearum) has seriously affected the yield and quality of wheat in China. In this study, gallic acid (GA), a natural polyphenol, was used to synthesize three azole-modified gallic acid derivatives (AGAs1–3). The antifungal activity of GA and its derivatives against F. graminearum was studied through mycelial growth rate experiments and field efficacy experiments. The results of the mycelial growth rate test showed that the EC50 of AGAs–2 was 0.49 mg/mL, and that of AGAs–3 was 0.42 mg/mL. The biological activity of AGAs–3 on F. graminearum is significantly better than that of GA. The results of field efficacy tests showed that AGAs–2 and AGAs–3 significantly reduced the incidence rate and disease index of wheat scab, and the control effect reached 68.86% and 72.11%, respectively. In addition, preliminary investigation was performed on the possible interaction between AGAs–3 and F. graminearum using density functional theory (DFT). These results indicate that compound AGAs–3, because of its characteristic of imidazolium salts, has potential for use as a green and environmentally friendly plant-derived antifungal agent for plant pathogenic fungi. [ABSTRACT FROM AUTHOR]

A stereoselective N-alkenylation of azoles with alkynes and iodine(III) electrophile is reported. The reaction between various azoles and internal alkynes is mediated by benziodoxole triflate as the electrophile in a trans-fashion, affording azole-bearing vinylbenziodoxoles in moderate to good yields. The tolerable azole nuclei include pyrazole, indazole, 1,2,3-triazole, benzotriazole, and tetrazole. The iodanyl group in the product can be leveraged as a versatile synthetic handle, allowing for the preparation of hitherto inaccessible types of densely functionalized N-vinylazoles.

Although the mitochondrial genome is an attribute of all eukaryotes, some yeast species (called petite-positive) can replicate without mitochondrial DNA (mtDNA). Strains without mtDNA (known as rho− mutants or petite mutants) are respiration-deficient and require fermentable carbon sources (such as glucose) for their metabolism. However, they are compromised in many aspects of fitness and competitiveness. Nevertheless, a few research groups have reported that some petite mutants of Candida glabrata and Saccharomyces cerevisiae manifested higher levels of tolerance to the antifungal fluconazole than their wild-type (WT) counterparts. In this study, we show that elevated tolerance to two or three out of four tested antifungals is a generic feature of at least five petite-positive species of yeasts including C. glabrata (higher tolerance of petites to clotrimazole and miconazole), S. bayanus (tolerance to clotrimazole, fluconazole, and miconazole), S. cerevisiae (tolerance to clotrimazole and fluconazole), S. paradoxus (tolerance to clotrimazole, fluconazole, and miconazole), and S. pastorianus (tolerance to clotrimazole and fluconazole). Comparing the levels of tolerance to the antifungals in WT and petite mutants was based on measuring the diameters of the zones of inhibition (ZOIs) using disc diffusion assays. The mode of inhibition in the majority of WT strains by all antifungals was fungicidal; most of the rho− mutants manifested fungistatic inhibition. We observed partial (not complete) inhibition in WT, with four different types of ZOI patterns that were species- and antifungal-specific. The partial inhibition was characterised by the presence of antifungal-tolerant colonies within ZOI areas. The inability of these colonies selected from ZOIs to grow on glycerol, as a single source of carbon, proved that they were rho− mutants spontaneously generated in the WT populations. The results on the elevated tolerance of petite strains to antifungals are discussed in terms of the prospective positive selection of respiratory-deficient mutants and the various implications of such selection.

A series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives–hydrazones, N-ethylhydrazones, pyrrole, pyrazole, oxadiazole, and triazole were synthesized and evaluated for their anticancer activity using human A549 pulmonary epithelial cells (ATCC CCl-185). The in vitro viability inhibitory effects of the compounds were assessed using the MTT assay. The characterization of the anticancer activity of the synthesized compounds showed that the incorporation of 1,3,4-oxadiazolethione and 4-aminotriazolethione rings into the molecular structures obviously enhances the anticancer activity against human A549 lung epithelial cells, reducing their viability to 28.0% and 29.6%, respectively. The anticancer activity of these azole derivatives was significantly higher than that of cytarabine. Further studies are needed to better optimize 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives and enhance their in vitro anticancer activity.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue. [ABSTRACT FROM AUTHOR]

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants. Whole-genome analysis of 876 Aspergillus fumigatus strains including 171 strains from Japan highlights the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. [ABSTRACT FROM AUTHOR]

The incidence of infections caused by Candida species, specifically by drug-resistant isolates, is a major health concern as they can disseminate to and colonize most vital organs, enhancing morbidity and mortality. Several molecular mechanisms have been reported to be involved in drug resistance. These are mostly drug- and isolate-specific. Here, we characterized three different genetically modified strains of C. albicans that were multi-drug-resistant (MDR) and deciphered a uniform mechanism responsible for resistance. DNA polymerase epsilon (Polε) is a leading strand-specific polymerase consisting of four subunits, namely, Pol2, Dpb2, Dpb3, and Dpb4. The deletion of one or both of the Dpb3 and Dpb4 subunits in C. albicans rendered multi-drug resistance. A detailed characterization of these strains revealed that acquired mutagenesis, drug efflux pumps, and other known mechanisms did not play a significant role because the complemented strain showed drug sensitivity. More importantly, the function of heat shock protein 90 (Hsp90) in these knockout strains is critical for reducing susceptibility to several antifungal drugs. Cell wall deformity and composition in these strains can add to such a phenotype. The inhibition of Hsp90 function by geldanamycin and tricostatin A sensitized the MDR strains to antifungals. Considering our earlier research and this report, we suggest that replication stress induces Hsp90 expression and activity in order to orchestrate a cellular stress response circuit and thus develop fungal drug resistance. Thus, Hsp90 is an important drug target for use in combinatorial therapy. [ABSTRACT FROM AUTHOR]

Electrolytes are considered the lifeblood of lithium batteries, facilitating ion transfer between the cathode and anode. However, the flammability, volatility and corrosiveness of organic electrolytes impede the sustainable development of lithium batteries. Deep eutectic electrolytes (DEEs) have emerged as a promising alternative, exhibiting high stability, non-flammability and wide electrochemical window. Herein, a series of DEEs with varying molar ratios of azoles and LiTFSI were facilely prepared through the interaction of Li⋯N. These DEEs demonstrate unique advantages, including non-flammability, high conductivity and a favourable Li+ transference number. In particular, the DEEs ImL-4:1 and PyL-4:1 (Imidazole:LiTFSI = 4:1 and Pyrazole:LiTFSI = 4:1, respectively) achieve high ionic conductivities of 4.5 × 10−3 and 5.8 × 10−3 S cm−1 at 80 °C, respectively. Notably, the electrochemical window of PyL-4:1 is stable, making it a suitable electrolyte for the lithium metal-free Li 4 Ti 5 O 12 /LiFePO 4 full battery. This system exhibits an initial specific capacity of 147 mAh g−1 and maintains stable cycling performance over 200 cycles. This study offers valuable insights into the design of electrolytes, addressing the challenges posed by traditional organic electrolytes and paving the way for sustainable lithium battery development. [ABSTRACT FROM AUTHOR]

(1) Background: Despite being considered a non-pathogenic yeast, recently, a growing occurrence of Saccharomyces cerevisiae infections has been noted. There is little knowledge about the drug susceptibility of this species. Therefore, the objective of this research was to expand it and determine the drug susceptibility profile of a local collection of clinical isolates of this species. (2) Methods: This study contained 55 clinical isolates identified as Saccharomyces cerevisiae using the MALDI-TOF method. The susceptibility of Saccharomyces cerevisiae was tested to 10 antifungals (amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, micafungin, anidulafungin, caspofungin, and itraconazole) using MICRONAUT-AT tests and manogepix, a new drug, using the microdilution method according to EUCAST. (3) Results: Overall, most strains were classified as sensitive to amphotericin B and flucytosine (MIC ranges of ≤0.03–1 and ≤0.06–0.125, respectively) and also to echinocandins. However, five isolates expressed high MIC values for all of the tested azoles, indicating cross-resistance. The MIC range for manogepix was 0.001–0.125 mg/L, with an MIC50 of 0.03 mg/L and an MIC90 of 0.06 mg/L. (4) Conclusions: The occurrence of resistance to azoles may be a concerning problem and therefore should be investigated further. However, the new antifungal manogepix appears to be an interesting new therapeutic option for treating such infections. [ABSTRACT FROM AUTHOR]

We have developed an intermolecular aminothiolation of simple olefins using Bunte salt as a thiolating agent. This protocol produces thiyl free radicals under PIDA oxidation conditions, eliminating the need for transition-metal catalysts. The method has a wide range of substrate applicability and is suitable for large-scale preparation and late-stage modification of drug molecules. [ABSTRACT FROM AUTHOR]

Antifungal therapy, especially with the azoles, could promote the incidence of less susceptible isolates of Cryptococcus neoformans and C. gattii species complexes (SC), mostly in developing countries. Given that these species affect mostly the immunocompromised host, the infections are severe and difficult to treat. This review encompasses the following topics: 1. infecting species and their virulence, 2. treatment, 3. antifungal susceptibility methods and available categorical endpoints, 4. genetic mechanisms of resistance, 5. clinical resistance, 6. fluconazole minimal inhibitory concentrations (MICs), clinical outcome, 7. environmental influences, and 8. the relevance of host factors, including pharmacokinetic/pharmacodynamic (PK/PD) parameters, in predicting the clinical outcome to therapy. As of now, epidemiologic cutoff endpoints (ECVs/ECOFFs) are the most reliable antifungal resistance detectors for these species, as only one clinical breakpoint (amphotericin B and C. neoformans VNI) is available. [ABSTRACT FROM AUTHOR]

Birds are highly susceptible to aspergillosis, which can manifest as a primary infection in both domestic and wild birds. Aspergillosis in wild birds causes mortalities ranging in scale from single animals to large-scale epizootic events. However, pathogenicity factors associated with aspergillosis in wild birds have not been examined. Specifically, it is unknown whether wild bird-infecting strains are host-adapted (i.e. phylogenetically related). Similarly, it is unknown whether epizootics are driven by contact with clonal strains that possess unique pathogenic or virulence properties, or by distinct and equally pathogenic strains. Here, we use a diverse collection of Aspergillus fumigatus isolates taken from aspergillosis-associated avian carcasses, representing 24 bird species from a wide geographic range, and representing individual bird mortalities as well as epizootic events. These isolates were sequenced and analyzed along with 130 phylogenetically diverse human clinical isolates to investigate the genetic diversity and phylogenetic placement of avian-associated A. fumigatus, the geographic and host distribution of avian isolates, evidence for clonal outbreaks among wild birds, and the frequency of azole resistance in avian isolates. We found that avian isolates were phylogenetically diverse, with no clear distinction from human clinical isolates, and no sign of host or geographic specificity. Avian isolates from the same epizootic events were diverse and phylogenetically distant, suggesting that avian aspergillosis is not contagious among wild birds and that outbreaks are likely driven by environmental spore loads or host comorbidities. Finally, all avian isolates were susceptible to Voriconazole and none contained the canonical azole resistance gene variants.

In the recent past, the incidence of Candidiasis has witnessed a concerning upsurge, resulting in a significant healthcare challenge. These infections are further exacerbated by factors like the widespread use of broad-spectrum antimicrobials, chemotherapy-induced neutropenia, and the presence of medical devices. The present study is designed to address the critical need for identifying the Candida species responsible for clinical infections and assessing their susceptibility to key antifungal drugs Fluconazole, Voriconazole, and Itraconazole. Two hundred clinical samples from Rohilkhand Medical College & Hospital, Bareilly were analyzed. Using Vitek-2 Compact (Biomerieux, France), the Candida spp. and the antifungal drug sensitivities were identified for Fluconazole and Voriconazole. E-test was done to identify Itraconazole sensitivity. This study found that C. albicans accounted for 21.5% while Non-albicans Candida (NAC) constituted 78.5%. Prolonged medication was the most common factor making susceptible for Candidiasis (43.5%), followed by indwelling biomedical devices (23%), Diabetes mellitus (16%), surgical causes (5.5%), trauma (5%), pregnancy (5%), and HIV (2%). Antifungal susceptibility testing showed that 68.5%, 72%, and 69.5% of Candida spp. isolates were sensitive to Fluconazole, Voriconazole, and Itraconazole, respectively. In conclusion, non-albicans Candida infections are increasing due to predisposing conditions, and some of these species are inherently resistant to the routinely used antifungal drugs. The study emphasizes the importance of identifying Candida spp. and their susceptibility to antifungals. This can limit the indiscriminate use of antifungal drugs, aid in selecting appropriate treatments, and reduce treatment costs, hospital stays, and patient morbidity and mortality.

The research was conducted to synthesize and study fungicidal activity of synthesized chemical compounds of various classes, triazole and imidazole, and their mode of action due to the wide spectrum of action and low application rates. The developed synthesis methods resulted in several groups of nitrogen-containing heterocyclic compounds and evaluated their fungicidal activity. Inhibitory activity of compounds to strains of Fusarium solani (medium resistance to fungicides) and Sclerotinia sclerotiorum (susceptible to most fungicides) from the collection of phytopathogenic microorganisms of Agrobiotechnological Department, RUDN University, was tested. In the synthesis of new chemical compounds with fungicidal activity, urea derivatives 1-(3-(Difluoromethyl)-1-methyl-1H-pyrazole-5-yl)-3-(2-chlorophenyl) urea were identified and structurally confirmed. All target compounds were evaluated for their antifungal activity to inhibit mycelium growth. Preliminary screening results showed that all synthesized compounds have good fungicidal activity against S. sclerotiorum . The compound 1-(3-(Difluoromethyl)-1-methyl-1H-pyrazole-5-yl)-3-(3-fluorophenyl) urea showed antifungal activity against S. sclerotiorum . At concentration of 100 ppm, the compound suppressed growth of S. sclerotiorum strain by 90.5 %. An in vitro experiment revealed that the compound 1-(3-(Difluoromethyl)1-methyl-1H-pyrazole-5-yl)-3-(3-fluorophenyl) urea was effective for suppressing white mold - S . sclerotiorum , at the dose of 100 mg/L. The significance of the research lies in the fact that production of eco-safe products in agroindustry is impossible without development of new biologically active compounds with low application rates and toxicity indicators, controlled persistence, corresponding to the world level. The results obtained can be implemented in real sector of economy engaged in production of chemical plant protection products. Synthesis and use of new fungicides are relevant in agricultural production as an element of development and intensification of existing agricultural technologies.

Abstract Background Lenvatinib is a multitargeted tyrosine kinase inhibitor used in the treatment of a variety of solid tumors. This study aims to investigate the potential pharmacokinetic interactions between lenvatinib and various azoles (ketoconazole, voriconazole, isavuconazole and posaconazole) when orally administered to rats. Methods A total of 30 Sprague–Dawley rats were randomly allocated into five groups and administered 20 mg/kg of ketoconazole, voriconazole, isavuconazole and 30 mg/kg of posaconazole and 0.5% CMC‐Na, through gavage for a duration of 7 days prior to the commencement of the experiment. On the final day, the rats were given 10 mg/kg of lenvatinib. The blood concentration of lenvatinib was determined using UPLC‐MS–MS. In vitro lenvatinib were incubated with azoles and rat liver microsomes (RLMs) or human liver microsomes (HLMs). Molecular docking was lastly used to examine the binding strength of the enzymes and ligands with Autodock Vina. Results AUC and Cmax of lenvatinib significantly increased with each of the azoles (p

Proteasome-addicted neoplastic malignancies present a considerable refractory and relapsed phenotype with patients exhibiting drug resistance and high mortality rates. To counter this global problem, novel proteasome-based therapies are being developed. In the current study, we extensively characterize TIR-199, a syrbactin-class proteasome inhibitor derived from a plant virulence factor of bacterium Pseudomonas syringae pv syringae. We report that TIR-199 is a potent constitutive and immunoproteasome inhibitor, capable of inducing cell death in multiple myeloma, triple-negative breast cancer, (TNBC) and non-small cell lung cancer lines. TIR-199 also effectively inhibits the proteasome in primary myeloma cells of patients, and bypasses the PSMB5 A49T+A50V bortezomib-resistant mutant. TIR-199 treatment leads to accumulation of canonical proteasome substrates in cells, it is specific, and does not inhibit 50 other enzymes tested in vitro. The drug exhibits synergistic cytotoxicity in combination with proteasome-activating kinase DYRK2 inhibitor LDN192960. Furthermore, low-doses of TIR-199 exhibits in vivo activity by delaying myeloma-mediated bone degeneration in a mouse xenograft model. Together, our data indicates that proteasome inhibitor TIR-199 could indeed be a promising next-generation drug within the repertoire of proteasome-based therapeutics.

Antimicrobial resistance poses a major threat to global health as the number of efficient antimicrobials decreases and the number of resistant pathogens rises. Our research group has been actively involved in the design of novel antimicrobial drugs. The blueprints of these compounds were azolic heterocycles, particularly thiazole. Starting with oxadiazolines, our research group explored, one by one, the other five-membered heterocycles, developing more or less potent compounds. An overview of this research activity conducted by our research group allowed us to observe an evolution in the methodology used (from inhibition zone diameters to minimal inhibitory concentrations and antibiofilm potential determination) correlated with the design of azole compounds based on results obtained from molecular modeling. The purpose of this review is to present the development of in-house azole compounds with antimicrobial activity, designed over the years by this research group from the departments of Pharmaceutical and Therapeutical Chemistry in Cluj-Napoca.

Introduction: Acanthamoeba encompasses several species of free-living ameba encountered commonly throughout the environment. Unfortunately, these species of ameba can cause opportunistic infections that result in Acanthamoeba keratitis, granulomatous amebic encephalitis, and occasionally systemic infection.Areas covered: This review discusses relevant literature found through PubMed and Google scholar published as of January 2021. The review summarizes current common Acanthamoeba keratitis treatments, drug discovery methodologies available for screening potential anti-Acanthamoeba compounds, and the anti-Acanthamoeba activity of various azole antifungal agents.Expert opinion: While several biguanide and diamidine antimicrobial agents are available to clinicians to effectively treat Acanthamoeba keratitis, no singular treatment can effectively treat every Acanthamoeba keratitis case.Efforts to identify new anti-Acanthamoeba agents include trophozoite cell viability assays, which are amenable to high-throughput screening. Cysticidal assays remain largely manual and would benefit from further automation development. Additionally, the existing literature on the effectiveness of various azole antifungal agents for treating Acanthamoeba keratitis is incomplete or contradictory, suggesting the need for a systematic review of all azoles against different pathogenic Acanthamoeba strains.

ABSTRACTHeteroresistance to antifungal agents poses a significant challenge in the treatment of fungal infections. Currently, the absence of established methods for detecting and measuring heteroresistance impedes progress in understanding this phenomenon in fungal pathogens. In response to this gap, we present a comprehensive set of new and optimized methods designed to detect and quantify azole heteroresistance in Candida albicans. Here, we define two primary assays for measuring heteroresistance: population analysis profiling, based on growth on solid medium, and single-cell assays, based on growth in liquid culture. We observe good correlations between the measurements obtained with liquid and solid assays, validating their utility for studying azole heteroresistance. We also highlight that disk diffusion assays could serve as an additional tool for the rapid detection of heteroresistance. These methods collectively provide a versatile toolkit for researchers seeking to assess heteroresistance in C. albicans. They also serve as a critical step forward in the characterization of antifungal heteroresistance, providing a framework for investigating this phenomenon in diverse fungal species and in the context of other antifungal agents. Ultimately, these advancements will enhance our ability to effectively measure antifungal drug responses and combat fungal infections.IMPORTANCEHeteroresistance involves varying antimicrobial susceptibility within a clonal population. This phenomenon allows the survival of rare resistant subpopulations during drug treatment, significantly complicating the effective management of infections. However, the absence of established detection methods hampers progress in understanding this phenomenon in human fungal pathogens. We propose a comprehensive toolkit to address this gap in the yeast Candida albicans, encompassing population analysis profiling, single-cell assays, and disk diffusion assays. By providing robust and correlated measurements through both solid and liquid assays, this work will provide a framework for broader applications across clinically relevant Candida species. These methods will enhance our ability to understand this phenomenon and the failure of antifungal therapy.

Introduction: Azole resistance has been increasingly reported and become an issue for clinical managements of invasive mycoses. New strategy with combination therapy arises as a valuable and promising alternative option. The aim of the present study is to investigate the in vitro combinational effect of proton pump inhibitors (PPIs) and azoles against pathogenic fungi. Methods: In vitro interactions of PPIs including omeprazole (OME), lansoprazole (LAN), pantoprazole (PAN), and rabeprazole (RAB), and commonly used azoles including itraconazole (ITC), posaconazole (POS), voriconazole (VRC) and fluconazole(FLC), were investigated via broth microdilution chequerboard procedure adapted from the CLSI M27-A3 and M38-A2. A total of 67 clinically isolated strains, namely 27 strains of Aspergillus spp., 16 strains of Candida spp., and 24 strains of dematiaceous fungi, were studied. C. parapsilosis (ATCC 22019) and A. flavus (ATCC 204304) was included to ensure quality control. Results: PPIs individually did not exert any significant antifungal activity. The combination of OME with ITC, POS, or VRC showed synergism against 77.6%, 86.6%, and 4% strains of tested pathogenic fungi, respectively, while synergism of OME/FLC was observed in 50% strains of Candida spp. Synergism between PAN and ITC, POS, or VRC was observed against 47.8%, 77.6% and 1.5% strains of tested fungi, respectively, while synergism of PNA/FLC was observed in 50% strains of Candida spp. Synergism of LAN with ITC, POS, or VRC was observed against 86.6%, 86.6%, and 3% of tested strains, respectively, while synergism of LAN/FLC was observed in 31.3% strains of Candida spp. Synergy of the combination of RAB with ITC, POS, or VRC was observed against 25.4%, 64.2%, and 4.5% of tested strains, respectively, while synergism of RAB/FLC was observed in 12.5% of Candida spp.. Among PPIs, synergism was least observed between RAB and triazoles, while among triazoles, synergism was least observed between VRC and PPIs. Among species, synergy was much more frequently observed in Aspergillus spp. and dematiaceous fungi as compared to Candida spp. Antagonism between PPIs with ITC or VRC was occasionally observed in Aspergillus spp. and dematiaceous fungi. It is notable that PPIs combined with azoles showed synergy against azole resistant A. fumigatus, and resulted in category change of susceptibility of ITC and POS against Candida spp. Discussion: The results suggested that PPIs combined with azoles has the potential to enhance the susceptibilities of azoles against multiple pathogenic fungi and could be a promising strategy to overcome azole resistance issues. However, further investigations are warranted to study the combinational efficacy in more isolates and more species, to investigate the underlying mechanism of interaction and to evaluate the potential for concomitant use of these agents in human. [ABSTRACT FROM AUTHOR]

The structures and energetics of the minima and the transition states corresponding to the migration of hydrogen and halogen atoms (F, Cl, Br) in imidazole and in cyclopentadiene rings have been calculated using the composite method Gaussian-4 (G4), a quantum chemical method for the calculation of energies of molecular species containing first-row, second-row, and third row main group atoms. The heats of formation obtained from the G4 values were found to be reliable by comparing them with the NIST Chemistry WebBook values of cyclopentadiene, imidazole and related compounds. The new G4 values thus obtained have been discussed using correlation analyses. In summary, this work was carried out because there is no clear evidence for intramolecular halogen migrations in azoles. [ABSTRACT FROM AUTHOR]

A new series of trifluoromethylated pyrimido[1,2-b]indazol-4(1H)-one derivatives was synthesized with good to excellent yields through a simple condensation of 3-aminoindazole derivatives with ethyl 4,4,4-trifluoro 3-oxobutanoate. The functionalization of the corresponding chlorinated fused tricyclic scaffolds via Suzuki-Miyaura and aromatic nucleophilic substitution reactions led to the synthesis of highly diverse trifluoromethylated pyrimido[1,2-b]indazole derivatives with good yields. [ABSTRACT FROM AUTHOR]

In order to look for the origins of tautomeric preferences in neutral creatinine in vacuo, we examined prototropic conversions for model azoles, namely mono-hydroxy and mono-amino imidazoles, and also for their selected 1-methyl derivatives. All possible isomeric forms of creatinine and model compounds, resulting from intramolecular proton transfer (prototropy), conformational isomerism about –OH, and configurational isomerism about =NH, were studied in the gas phase (model of non-polar environment) by means of quantum-chemical methods. Because the bond-length alternation is a consequence of the resonance phenomenon, it was measured for all DFT-optimized structures by means of the harmonic oscillator model of electron delocalization (HOMED) index. Important HOMED analogies were discussed for investigated azoles and compared with those for previously studied cyclic azines, including pyrimidine nucleic acid bases. The internal effects were taken into account, and the stabilities of the investigated tautomers-rotamers were analyzed. Significant conclusions on the favored factors that can dictate the tautomeric preferences in creatinine were derived. [ABSTRACT FROM AUTHOR]

Fluconazole resistance is commonly encountered in Candida auris, and the yeast frequently displays resistance to other standard drugs, which severely limits the number of effective therapeutic agents against this emerging pathogen. In this study, we aimed to investigate the effect of acquired azole resistance on the viability, stress response, and virulence of this species. Fluconazole-, posaconazole-, and voriconazole- resistant strains were generated from two susceptible C. auris clinical isolates (0381, 0387) and compared under various conditions. Several evolved strains became pan-azole-resistant, as well as echinocandin-cross-resistant. While being pan-azole-resistant, the 0381-derived posaconazole-evolved strain colonized brain tissue more efficiently than any other strain, suggesting that fitness cost is not necessarily a consequence of resistance development in C. auris. All 0387-derived evolved strains carried a loss of function mutation (R160S) in BCY1, an inhibitor of the PKA pathway. Sequencing data also revealed that posaconazole treatment can result in ERG3 mutation in C. auris. Despite using the same mechanisms to generate the evolved strains, both genotype and phenotype analysis highlighted that the development of resistance was unique for each strain. Our data suggest that C. auris triazole resistance development is a highly complex process, initiated by several pleiotropic factors. [ABSTRACT FROM AUTHOR]

BCR-ABL inhibition is an effective therapeutic approach for the treatment of chronic myeloid leukaemia (CML). Herein, we report the discovery of AKE-72 (5), a diarylamide 3-aminoindazole, as a potent pan-BCR-ABL inhibitor, including the imatinib-resistant mutant T315I. A focussed array of compounds 4a, 4b, and 5 has been designed based on our previously reported indazole I to improve its BCR-ABLT315I inhibitory activity. Replacing the morpholine moiety of I with the privileged tail (4-ethylpiperazin-1-yl)methyl afforded 5 (AKE-72) with IC50 values of < 0.5 nM, and 9 nM against BCR-ABLWT and BCR-ABLT315I, respectively. Moreover, AKE-72 potently inhibited a panel of other clinically important mutants in single-digit nanomolar IC50 values. AKE-72 elicited remarkable anti-leukemic activity against K-562 cell line (GI50 < 10 nM, TGI = 154 nM). In addition, AKE-72 strongly inhibited the proliferation of Ba/F3 cells expressing native BCR-ABL or its T315I mutant. Overall, AKE-72 may serve as a promising candidate for the treatment of CML, including those harbouring T315I mutation. [ABSTRACT FROM AUTHOR]