Development Of Antifungal Drugs Research Articles

Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1.

Current Updates on Pathogenesis, Systemic Therapy, and Treatment of Invasive Fungal Infections.

Numerous health hazards are associated with fungal infections, ranging from asymptomatic cases to potentially fatal invasive diseases that are especially dangerous for those with impaired immune systems. The main causes behind these diseases are opportunistic fungi, namely Aspergillus, Candida, and Cryptococcus. Invasive fungal infections (IFIs) require a global response that includes the development of vaccines, standardized protocols for diagnosis, potent antifungal medications, and strategies to stop drug-resistant strains. Improving high-risk group diagnosis and treatment is essential to lowering death rates. This review highlights the substantial health concerns associated with fungal infections, especially in immunocompromised individuals, and identifies Aspergillus, Candida, and Cryptococcus as the main pathogens. It highlights the necessity of international efforts, such as the development of novel diagnostic instruments, imaging methods, and antifungal drugs, to combat these invasive infections. The review also addresses the increasing need for novel treatment approaches in light of the developing resistance to widely used antifungal medications. Furthermore, the significance of secretory proteins in fungal pathogenicity and the potential of combination therapy are investigated. It is also suggested that a multimodal strategy be used to fight these illnesses, given the promise of multivalent vaccinations. Overall, this study emphasizes how critical it is to develop better diagnostic and treatment strategies in order to successfully control and lessen the impact of invasive fungal diseases on the health of the world.

Critical roles of Dpb3-Dpb4 sub-complex of DNA polymerase epsilon in DNA replication, genome stability, and pathogenesis of Candida albicans.

This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development.

Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii.

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.

Comparative analysis of polysaccharide and cell wall structure in Aspergillus nidulans and Aspergillus fumigatus by solid-state NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

Disruption to de novo uridine biosynthesis alters β-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

Cryo-EM structures of Candida albicans Cdr1 reveal azole-substrate recognition and inhibitor blocking mechanisms

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.

Review and Current Perspectives on DNA Topoisomerase I and II Enzymes of Fungi as Study Models for the Development of New Antifungal Drugs.

Fungal infections represent a growing public health problem, mainly stemming from two phenomena. Firstly, certain diseases (e.g., AIDS and COVID-19) have emerged that weaken the immune system, leaving patients susceptible to opportunistic pathogens. Secondly, an increasing number of pathogenic fungi are developing multi-drug resistance. Consequently, there is a need for new antifungal drugs with novel therapeutic targets, such as type I and II DNA topoisomerase enzymes of fungal organisms. This contribution summarizes the available information in the literature on the biology, topology, structural characteristics, and genes of topoisomerase (Topo) I and II enzymes in humans, two other mammals, and 29 fungi (including Basidiomycetes and Ascomycetes). The evidence of these enzymes as alternative targets for antifungal therapy is presented, as is a broad spectrum of Topo I and II inhibitors. Research has revealed the genes responsible for encoding the Topo I and II enzymes of fungal organisms and the amino acid residues and nucleotide residues at the active sites of the enzymes that are involved in the binding mode of topoisomerase inhibitors. Such residues are highly conserved. According to molecular docking studies, antifungal Topo I and II inhibitors have good affinity for the active site of the respective enzymes. The evidence presented in the current review supports the proposal of the suitability of Topo I and II enzymes as molecular targets for new antifungal drugs, which may be used in the future in combined therapies for the treatment of infections caused by fungal organisms.

Antifungal Activity of Disalt of Epipyrone A from Epicoccum nigrum Likely via Disrupted Fatty Acid Elongation and Sphingolipid Biosynthesis.

Multidrug-resistant fungal pathogens and antifungal drug toxicity have challenged our current ability to fight fungal infections. Therefore, there is a strong global demand for novel antifungal molecules with the distinct mode of action and specificity to service the medical and agricultural sectors. Polyenes are a class of antifungal drugs with the broadest spectrum of activity among the current antifungal drugs. Epipyrone A, a water-soluble antifungal molecule with a unique, linear polyene structure, was isolated from the fungus Epiccocum nigrum. Since small changes in a compound structure can significantly alter its cell target and mode of action, we present here a study on the antifungal mode of action of the disalt of epipyrone A (DEA) using chemical-genetic profiling, fluorescence microscopy, and metabolomics. Our results suggest the disruption of sphingolipid/fatty acid biosynthesis to be the primary mode of action of DEA, followed by the intracellular accumulation of toxic phenolic compounds, in particular p-toluic acid (4-methylbenzoic acid). Although membrane ergosterol is known to be the main cell target for polyene antifungal drugs, we found little evidence to support that is the case for DEA. Sphingolipids, on the other hand, are known for their important roles in fungal cell physiology, and their biosynthesis has been recognized as a potential fungal-specific cell target for the development of new antifungal drugs.

Comparative Analysis of Polysaccharide and Cell Wall Structure in Aspergillus nidulans and Aspergillus fumigatus by Solid-State NMR.

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

Discovery of petroselinic acid with in vitro and in vivo antifungal activity by targeting fructose-1,6-bisphosphate aldolase

BackgroundThe incidence of invasive fungal diseases (IFDs), represented by Candida albicans infection, is increasing year by year. However, clinically available antifungal drugs are very limited and encounter challenges such as limited efficacy, drug resistance, high toxicity, and exorbitant cost. Therefore, there is an urgent need for new antifungal drugs. PurposeThis study aims to find new antifungal compounds from plants, preferably those with good activity and low toxicity, and reveal their antifungal targets. MethodsIn vitro antifungal activities of compounds were investigated using broth microdilution method, spot assay, hyphal growth assay and biofilm formation assay. Synergistic effects were assessed using broth microdilution checkerboard technique. In vivo antifungal activities were evaluated using Galleria mellonella and murine candidiasis models. Cytotoxicity of compounds was investigated using Cell Counting Kit-8 (CCK-8). Discovery and validation of antifungal targets of compounds were conducted by using monoallelic knockout library of C. albicans, haploinsufficiency profiling (HIP), thermal shift assay (TSA), enzyme inhibitory effect assay, molecular docking, and in vitro and in vivo antifungal studies. Results814 plant products were screened, among which petroselinic acid (PeAc) was found as an antifungal molecule. As a rare fatty acid isolated from coriander (Coriandrum sativum), carrot (Daucus carota) and other plants of the Apiaceae family, PeAc had not previously been found to have antifungal effects. In this study, PeAc was revealed to inhibit the growth of various pathogenic fungi, exhibited synergistic effects with fluconazole (FLC), inhibited the formation of C. albicans hyphae and biofilms, and showed antifungal effects in vivo. PeAc was less toxic to mammalian cells. Fructose-1,6-bisphosphate aldolase (Fba1p) was identified as a target of PeAc by using HIP, TSA, enzyme inhibitory effect assay and molecular docking methods. PeAc exerted antifungal effects more effectively on fba1Δ/FBA1 than wild-type (WT) strain both in vitro and in vivo. ConclusionsPeAc is an effective and low toxic antifungal compound. The target of PeAc is Fba1p. Fba1p is a promising target for antifungal drug development.

Saturation transposon mutagenesis enables genome-wide identification of genes required for growth and fluconazole resistance in the human fungal pathogen Cryptococcus neoformans.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the Cryptococcus neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that 5' insertions of transposons can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C–13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C–13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

Antifungal activity of propafenone on Candida spp. strains: interaction with antifungals and possible mechanism of action.

Introduction. The development of new antifungal drugs has become a global priority, given the increasing cases of fungal diseases together with the rising resistance to available antifungal drugs. In this scenario, drug repositioning has emerged as an alternative for such development, with advantages such as reduced research time and costs.Gap statement. Propafenone is an antiarrhythmic drug whose antifungal activity is poorly described, being a good candidate for further study.Aim. This study aims to evaluate propafenone activity against different species of Candida spp. to evaluate its combination with standard antifungals, as well as its possible action mechanism.Methodology. To this end, we carried out tests against strains of Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei based on the evaluation of the MIC, minimum fungicidal concentration and tolerance level, along with checkerboard and flow cytometry tests with clinical strains and cell structure analysis by scanning electron microscopy (SEM).Results. The results showed that propafenone has a 50% MIC ranging from 32 to 256 µg ml-1, with fungicidal activity and positive interactions with itraconazole in 83.3% of the strains evaluated. The effects of the treatments observed by SEM were extensive damage to the cell structure, while flow cytometry revealed the apoptotic potential of propafenone against Candida spp.Conclusion. Taken together, these results indicate that propafenone has the potential for repositioning as an antifungal drug.

Glutathione-mediated redox regulation in Cryptococcus neoformans impacts virulence.

The fungal pathogen Cryptococcus neoformans is well adapted to its host environment. It has several defence mechanisms to evade oxidative and nitrosative agents released by phagocytic host cells during infection. Among them, melanin production is linked to both fungal virulence and defence against harmful free radicals that facilitate host innate immunity. How C. neoformans manipulates its redox environment to facilitate melanin formation and virulence is unclear. Here we show that the antioxidant glutathione is inextricably linked to redox-active processes that facilitate melanin and titan cell production, as well as survival in macrophages and virulence in a murine model of cryptococcosis. Comparative metabolomics revealed that disruption of glutathione biosynthesis leads to accumulation of reducing and acidic compounds in the extracellular environment of mutant cells. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Unveiling the anti-Candida albicans activity of bioactive fractions from mining soil-dwelling Streptomyces sp. DS104

Due to an increase in the incidence of Candida albicans infection and its growing resistance to currently used antifungal drugs, further development of more potent antimicrobials is needed. This study sought to assess the antifungal properties of Streptomyces sp. DS104, isolated from a mining soil, against C. albicans and identify the bioactive substances that are produced by this strain. Ethyl acetate supernatant's crude extract showed a high inhibition against C. albicans, with a minimal inhibitory concentration value as low as 4 µg/mL. Two bioactive fractions (F5 and F6) have been isolated using silica gel column chromatography, demonstrating zones of inhibition against C. albicans measuring 28.33 ± 01.52 to 29.00 ± 02.64 mm. The GC–MS analysis was carried out on the purified fractions and allowed the identification of 21 compounds predominantly consisting of carboxylic acids and pyrrolopyrazine derivatives. In Fraction F5, the major compounds detected are pentadecanoic and hexadecanoic acids, constituting the highest area percentages of 41.99 % and 33.77 %, respectively. As for fraction F6, the predominant compounds include pyrrolo[1,2-a]pyrazine-1,4‑dione, hexahydro-3-(2-methylpropyl) and pyrrolo[1,2-a]pyrazine-1,4‑dione, hexahydro-3-(methylphenyl), accounting for area percentages of 20.18 % and 12.16 %, respectively. Various other bioactive compounds, such as isocoumarins, and phenolic compounds, had previously been identified as components with antifungal activity, were also detected. It's worth highlighting that three compounds specifically, 2-hydrazino-8‑hydroxy-4-phenylquinoline, hydroxyphenylacetic acid, and 3-carboxaldehyde-1H-indole have been reported for the first time to be detected within the Streptomyces genus. These results suggested that Streptomyces sp. DS104 is a promising strain and thus appears to be a potential candidate for the development of antifungal drugs.

Antifungal pipeline: Is there light at the end of the tunnel?

The misuse and overuse of classic antifungals have accelerated the development of resistance mechanisms, diminishing the efficacy of established therapeutic pathways and necessitating a shift towards alternative targets. Despite this pressing need for new treatments, the antifungal drug pipeline has been largely stagnant for the past three decades, primarily due to the high risks and costs associated with antifungal drug development, compounded by uncertain market returns. Extensive research durations, special patient populations and rigorous regulatory demands pose significant barriers to bringing novel antifungal agents to market. In response, the “push-pull” incentive model has emerged as a vital strategy to invigorate the pipeline and encourage innovation. This editorial critically examines the current clinical landscape and spotlights emerging antifungal agents, such as Fosmanogepix, Ibrexafungerp, and Olorofim, while also unraveling the multifaceted challenges faced in new antifungal drug development. The generation of novel antifungals offers a beacon of hope in the battle against antimicrobial resistance, but it is premature to declare them as definitive solutions. Their future role hinges on thorough clinical validation, cost-effectiveness assessments, and continuous post-marketing surveillance. Only through strategic implementation and integration with market strategies we can transform the landscape of antifungal development, addressing both the resistance crisis and the treatment challenges.

QbD Approach to Method Development, Validation and Degradation Profiling of Antifungal Drugs by RP-HPLC

For the simultaneous estimation of Itraconazole (ITZ) and Terbinafine hydrochloride (TBF HCl) in tablet dosage form, a simple, novel, cost effective and rapid RP-HPLC method was developed. Method development becomes quick, accurate, sensitive, and robust by using quality by design (QbD). RP-HPLC was performed using a C18 (agilent, 4.6 x 250mm, 5µm particle size) column. Methanol and 0.05% OPA (pH 3.5) was used as mobile phase in 84:16 ratio. The analysis was conducted in gradient mode with a flow rate of 0.95mL/min. Detection wavelength was set at 270nm. Retention time for TBF and ITZ was at 2.93 and 6.96min, respectively. The accuracy, precision, repeatability, linearity, LOD, LOQ and robustness parameters have been validated as per the ICH Q2 (R1) guideline: Validation of analytical procedures–text and methodology. Values for LOD and LOQ were found to be 0.131 and 0.398µg/mL and 0.350 and 1.061µg/mL respectively for ITZ and TBF HCl. Thus, the technique can be employed for regular QC analysis of bulk ITZ and TBF HCl and their pharmaceutical formulations. Degradative stress conditions like acid, base, neutral, oxidation applied and degradations were studied.