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The steep increase in acquired drug resistance in Candida isolates has posed a great challenge in the clinical management of candidiasis globally. Information of genes and codon sites that are positively selected during evolution can provide insights into the mechanisms driving antifungal resistance in Candida. This study aimed to create a manually curated list of genes of Candida spp. reported to be associated with antifungal resistance in literature, and further investigate the structure-function implications of positively selected genes and mutation sites. Sequence analysis of antifungal drug resistance associated gene sequences from various species and strains of Candida revealed that ERG11 and MRR1 of C. albicans were positively selected during evolution. Four sites in ERG11 and two sites in MRR1 of C. albicans were positively selected and associated with drug resistance. These four sites (132, 405, 450, and 464) of ERG11 are predictive markers for azole resistance and have evolved over time. A well-characterized crystal structure of sterol-14-α-demethylase (CYP51) encoded by ERG11 is available in PDB. Therefore, the stability of CYP51 in complex with fluconazole was evaluated using MD simulations and molecular docking studies for two mutations (Y132F and Y132H) reported to be associated with azole resistance in literature. These mutations induced high flexibility in functional motifs of CYP51. It was also observed that residues such as I304, G308, and I379 of CYP51 play a critical role in fluconazole binding affinity. The insights gained from this study can further guide drug design strategies addressing antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Cercospora leaf spot, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugarbeet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated with synonymous and nonsynonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/L144F_C) correlated with resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study offers an understanding of the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decisionmaking. [ABSTRACT FROM AUTHOR]

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription‐quantitative PCR and RNA‐seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild‐type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles. [ABSTRACT FROM AUTHOR]

Fungicide resistance is often conferred through the mutation of genes encoding fungicide targets or proteins that remove fungicides from cells, but mechanisms can vary widely between taxa. Discovering the specific resistance alleles present in pathogen populations is essential for monitoring the evolution and movement of resistant genotypes. In this study, we explored the genomic basis of demethylase inhibitor (DMI) resistance in Leptosphaeria maculans, the main pathogen of the canola crop Brassica napus. Using an international collection of over 200 genome‐sequenced isolates, we assayed in vitro sensitivity to the DMI tebuconazole and conducted a genome‐wide association study on a variant set including single‐nucleotide polymorphisms (SNPs), small indels and structural variants. The main resistance allele identified was a 237 bp remnant transposable element insertion in the promoter of the erg11/CYP51 DMI target gene in a large proportion of isolates from Europe, an allele known to confer DMI resistance in Australia. Several associated loci were identified, none of which are commonly linked to DMI resistance in other phytopathogens. We also found little to no relationship between DMI tolerance and baseline growth rate, suggesting minimal fitness effects of fungicide resistance in these isolates. This study indicates common DMI resistance alleles in L. maculans are shared across continents and erg11/CYP51 coding mutations, which are near‐ubiquitous in other fungal pathogens, may not underpin DMI resistance in this species. Furthermore, that resistance occurs frequently in numerous canola‐growing regions suggests management is essential for growers. [ABSTRACT FROM AUTHOR]

Plenodomus lingam (Leptosphaeria maculans) and P. biglobosus (L. biglobosa) are fungi causing Phoma leaf spot/stem canker, an international damaging disease of oilseed rape (Brassica napus) and other brassicas. In Europe, fungicides used for disease management are mainly sterol 14α‐demethylase (CYP51) inhibitors (DMIs/azoles); quinone‐outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are also used. Decreased DMI sensitivity has emerged in Australian and eastern European P. lingam populations and is mediated by CYP51 promoter inserts resulting in target site overexpression. In this study using in vitro sensitivity testing, we report decreased DMI (prothioconazole‐desthio, mefentrifluconazole) sensitivity in modern western European P. lingam isolates (collected 2022–2023) compared to older baseline (1992–2005) isolates. Around 85% of modern western European P. lingam isolates collected, for which the CYP51 promoter region was sequenced, carried a promoter insert, but target site alterations were not detected. Six different CYP51 promoter inserts were identified, most commonly a 237 bp fragment of the Sahana transposable element. Inserts were associated with an approximately 3‐ to 10‐fold decrease in sensitivity to the DMIs tested. In contrast to P. lingam, PCR screening revealed CYP51 promoter inserts were absent in modern western European P. biglobosus isolates (2021–2023). Combined data indicate P. lingam isolates lacking an insert were similarly (or slightly more) sensitive to the DMIs tested for P. biglobosus, whereas those carrying an insert were slightly less sensitive than P. biglobosus. No evidence for substantive sensitivity shifts to the QoI (pyraclostrobin) or SDHI (boscalid) fungicides tested was obtained for either Plenodomus species. [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]

Background: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals ‐ CYP 51 A, B and C ‐ are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. Objectives: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. Methods: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. Results: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. Conclusions: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus. [ABSTRACT FROM AUTHOR]

Pyrenophora teres f. teres (Ptt) is a severe pathogen to spring barley in Northern Europe. Ptt with relevant mutations in fungicide target proteins, sterol 14α-demethylase (CYP51A), cytochrome b (Cyt b), and succinate dehydrogenase (SDH) would put efficient disease control at risk. In the growing seasons of 2021 and 2022, 193 Ptt isolates from Estonia were analysed. In this study, mutation detection and in vitro fungicide sensitivity assays of single-spore isolates were carried out. Reduced sensitivity phenotype to mefentrifluconazole was evident in Ptt isolates with a F489L mutation in CYP51A or with 129 bp insert in the Cyp51A gene-promoter region. However, sensitivity to a prothioconazole-desthio remained high regardless of these molecular changes. The Ptt population was mostly sensitive to bixafen, fluxapyroxad, pyraclostrobin, and azoxystrobin. The sensitivity of fluxapyroxad and bixafen has been affected by two mutations, C-S135R and D-H134R, found in SDH subunits. The F129L mutation in Cyt b influenced azoxystrobin but not pyraclostrobin sensitivity. In total, 30 isolates from five fields had relevant mutations in three target protein genes simultaneously. Most of these isolates had a reduced sensitivity phenotype to mefentrifluconazole, fluxapyroxad, and azoxystrobin, while sensitivity to other tested fungicides remained high. Furthermore, possible sexual reproduction may enhance the pathogen's fitness and help it adapt to fungicides. [ABSTRACT FROM AUTHOR]

Sterol biosynthesis via the mevalonate-isoprenoid pathway produces ergosterol (24β-methyl cholesta-5,7-dienol) necessary for growth in a wide-range of eukaryotic pathogenic organisms in eukaryotes, including the fungi, trypanosomes and amoebae, while their animal hosts synthesize a structurally less complicated product—cholesterol (cholest-5-enol). Because phyla-specific differences in sterol metabolizing enzyme architecture governs the binding and reaction properties of substrates and inhibitors while the order of sterol metabolizing enzymes involved in steroidogenesis determine the positioning of crucial chokepoint enzymes in the biosynthetic pathway, the selectivity and effectiveness of rationally designed ergosterol biosynthesis inhibitors toward ergosterol-dependent infectious diseases varies greatly. Recent research has revealed an evolving toolbox of mechanistically distinct tight-binding inhibitors against two crucial methylation-demethylation biocatalysts—the C24 sterol methyl transferase (absent from humans) and the C14-sterol demethylase (present generally in humans and their eukaryotic pathogens). Importantly for rational drug design and development, the activities of these enzymes can be selectively blocked in ergosterol biosynthesis causing loss of ergosterol and cell killing without harm to the host organism. Here, we examine recent advances in our understanding of sterol biosynthesis and the reaction differences in catalysis for sterol methylation-demethylation enzymes across kingdoms. In addition, the novelties and nuances of structure-guided or mechanism-based approaches based on crystallographic mappings and substrate specificities of the relevant enzyme are contrasted to conventional phenotypic screening of small molecules as an approach to develop new and more effective pharmacological leads. [ABSTRACT FROM AUTHOR]

ABSTRACT Alternaria alternata is a ubiquitous soil-borne fungus capable of causing diseases in a variety of plants and occasionally in humans. While populations of A. alternata from infected plants have received significant attention, relatively little is known about its soil populations, including its population genetic structure and antifungal susceptibilities. In addition, over the last two decades, greenhouses have become increasingly important for food and ornamental plant production throughout the world, but how greenhouses might impact microbial pathogens such as A. alternata populations remains largely unknown. Different from open crop fields, greenhouses are often more intensively cultivated, with each greenhouse being a relatively small and isolated space where temperature and humidity are higher than surrounding environments. Previous studies have shown that greenhouse populations of two common molds, Aspergillus fumigatus and A. alternata, within a small community in southwestern China were variably differentiated. However, the relative contribution of physical separation among local greenhouses to the large-scale population structure remains unknown. Here, we isolated strains of A. alternata from seven greenhouses in Shijiazhuang, northeast China. Their genetic diversity and triazole susceptibilities were analyzed and compared with each other and with 242 isolates from nine greenhouses in Kunming, southwest China. Results showed that the isolation of greenhouses located

BACKGROUND: Septoria tritici blotch caused by Zymoseptoria tritici continues to be one of the most economically destructive diseases of winter wheat in north‐western Europe. Control is heavily reliant on the application of fungicides, in particular those belonging to the azole group. Here we describe the sensitivity of European Z. tritici populations to the novel azole mefentrifluconazole and the analysis of associated mechanisms of resistance. RESULTS: A wide range of sensitivity to mefentrifluconazole was observed amongst the Z. tritici collections examined, with strong cross‐resistances also observed between mefentrifluconazole, difenoconazole and tebuconazole. Overall, the Irish population displayed the lowest sensitivity to all azoles tested. Further detailed analysis of the Irish population in 2021 demonstrated differences in sensitivity occurred between sampling sites, with these differences associated with the frequencies of key resistance mechanisms (CYP51 alterations and MFS1 promoter inserts linked to overexpression). Under glasshouse conditions reductions in the efficacy of mefentrifluconazole were observed towards those strains exhibiting the lowest in vitro sensitivities. CONCLUSIONS: This study demonstrates that a large range of sensitivity to mefentrifluconazole exists in European Z. tritici populations. Those strains exhibiting the lowest sensitivity to the azoles tested had the most complex CYP51 haplotypes in combination with the 519 bp insert, associated with enhanced activity of MFS1. The future use of mefentrifluconazole should take these findings into consideration to minimise the selection of these strains. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Context: Given the elusive nature of Primary Amoebic Meningoencephalitis (PAM), caused by Naegleria fowleri, early detection is vital, yet challenging due to limited clinical indicators. This research leverages Indonesia’s rich biodiversity to explore novel sources of traditional medicine. Aims: To evaluate the potential compounds from Indonesian plants that possess antiamoebic and antifungal properties for inhibiting the N. fowleri CYP51 protein, crucial for cell integrity. Methods: Initially, 92 compounds were screened, and six were shortlisted following ADMETox evaluation. Subsequent steps encompassed QSAR analysis, molecular docking, and molecular dynamics simulations. Results: The QSAR analysis verified the activity potential of these six compounds, progressing them to molecular docking analysis. Among these, curcumenol from Curcuma longa emerged as a promising contender, displaying the lowest binding affinity at -9.2 kcal/mol, indicative of superior binding compared to other ligands. Molecular dynamics simulations underscored the stability of all compounds, with root mean square fluctuation (RMSF) values within 1-3 Å. Conclusions: Consequently, employing a comprehensive approach spanning ADMETox, QSAR, molecular docking, and dynamics simulations, curcumenol emerged as the prime candidate for inhibiting the N. fowleri CYP51 protein, suggesting its potential as a PAM therapeutic agent.

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.

Candida albicans, an opportunistic pathogen, is the most common type of fungus and represents a substantial source of human invasive disease (nosocomial infection). This category of fungi are part of our microbiota, and given the appropriate environmental conditions, it has the potential to cause both superficial and systemic infections. There is a soaring resistance against the available anticandidal agents. The purpose of this research is to investigate the activity of certain previously synthesized benzo[g]quinazolines against C. albicans in vitro by using the cup-plate diffusion method. There was a marked difference in the effectiveness of the target compounds 1–6 against the sample of C. albicans that was tested. Benzo[g]quinazolines 1 (inhibition zone = 20 mm) and 2 (inhibition zone = 22 mm) had good effects in comparison to fluconazole (inhibition zone = 26 mm). A docking study was conducted between benzo[g]quinazolines 1–6 and Candida spp. CYP51 to establish the binding mode compared with fluconazole and VT-1161 (oteseconazole) as reference medicines, and it was determined that binding at the active site of Candida spp. CYP51 occurred in the same manner. Quantitative structure–activity relationship (QSAR) investigation was performed to further characterize the identified anticandidal agents and recognize the major regulatory components governing such activity. In future studies, the benzo[g]quinazoline scaffold could serve as a model for the design and development of novel derivatives with antifungal potential.

A series of novel triazole derivatives containing aryl-propanamide side chains was designed and synthesised. In vitro antifungal activity studies demonstrated that most of the compounds inhibited the growth of six human pathogenic fungi. In particular, parts of phenyl-propionamide-containing compounds had excellent, broad-spectrum antifungal activity against Candida albicans SC5314, Cryptococcus neoformans 22-21, Candida glabrata 537 and Candida parapsilosis 22-20 with MIC values in the range of ≤0.125 µg/mL–4.0 µg/mL. In addition, compounds A1, A2, A6, A12 and A15 showed inhibitory activities against fluconazole-resistant Candida albicans and Candida auris. Preliminary structure-activity relationships (SARs) are also summarised. Moreover, GC-MS analysis demonstrated that A1, A3, and A9 interfered with the C. albicans ergosterol biosynthesis pathway by inhibiting Cyp51. Molecular docking studies elucidated the binding modes of A3 and A9 with Cyp51. These compounds with low haemolytic activity and favourable ADME/T properties are promising for the development of novel antifungal agents.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential. [ABSTRACT FROM AUTHOR]

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme. [ABSTRACT FROM AUTHOR]

Acclimatized fungal diseases in wheat crops pose a severe threat to world wheat production. Zymoseptoria tritici, which causes Septoria tritici blotch (STB), is known as a devastating latent necrotrophic fungal pathogen that causes a significant yield loss, especially in Europe and in other wheat-growing countries. This fungal pathogen developed rapid resistance against new and old fungicides and has become a major threat to future wheat crops. Strobilurines and Azoles are the main group of contact and systemic fungicides along with succinate dehydrogenase inhibitors with reliable results to control the Z. tritici all over the world. However, many reports from Europe described the presence of resistance in Z. tritici against these fungicides. In the present study, a total of 102 isolates of Z. tritici were collected from five regions (Central Anatolia, Southeastern Anatolia, Marmara, Aegean, and the Mediterranean) of 19 provinces of Turkey to check the presence of target-site mutation at a molecular level to these two main fungicides. Azole target-site mutation in the CYP51 gene was checked by sanger sequencing while the strobilurin resistance mutation in the Cytb gene was confirmed by using PCR–RFLP using the FnuHI restriction enzyme. The obtained results from the two genes revealed some target-site mutations, however no known amino acid changes that cause resistance against these two main group fungicides in Turkish isolates have been detected. We concluded that the severe infection of STB pathogen in Turkish wheat growing may be still manageable with these fungicides although the resistance tests must be conducted for certainity. [ABSTRACT FROM AUTHOR]

The biotrophic basidiomycete Phakopsora pachyrhizi is the causal agent of Asian soybean rust (ASR), which has become a serious soybean disease in South America. Control of this disease is mainly based on fungicide applications, with demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) representing a large market. DMIs have been the key component for ASR-control for many years. This ongoing selection pressure has led to an adaptation of P. pachyrhizi resulting in a continuous shift of the population towards reduced sensitivity. The objective of the present study was to characterize the resistance of P. pachyrhizi to DMIs and the underlying mutations in the target gene (CYP51), using single spore isolates for the first time. The fungal populations from which these isolates were obtained, originated from infected soybean leaves from South America. Sequence analyses of the CYP51-gene confirmed three newly identified mutations (V130A, I145V, F154Y), occurring in one allele in combination with previously described mutations. A total of nine mutations were observed in CYP51 of P. pachyrhizi (F120L, V130A, Y131F/H, K142R, I145V/F, F154Y, I475T), present in different combinations. Their locations in the enzyme were shown by CYP51-protein modeling. The newly identified mutation combinations F120L + V130A + Y131F, F120L + Y131H + I145V and F120L + Y131H + F154Y are associated with different sensitivities to DMIs. The sensitivity studies also demonstrated incomplete cross-resistance of P. pachyrhizi to DMIs caused by different mutation combinations. Furthermore, using an isolate mixture (defined population), it could be shown that different DMIs select different mutation combinations. P. pachyrhizi produces dikaryotic uredospores, and moreover, this study confirmed that each spore contains a total of 6 CYP51-copies. [ABSTRACT FROM AUTHOR]

P450 family, especially CYP51 protein, is a common target for the design of antifungal and antiprotozoal drugs. Designing new effective drugs against these pathogens is a necessity and a challenge for the scientific community. To this end, they are evaluated by molecular docking of five schemes of aryl-substituted imidazoles and pseudopeptic imidazoles against CYP51 proteins from different pathogens and against the similar human protein to estimate their selectivity. Once these calculations have been carried out, none of the compounds studied appears to be an effective inhibitor against CYP51- L.infantum. However, for all the remaining proteins lower normalized coupling scores are obtained, fundamentally for schemes 1 and 3. Given the geometry of the proteinlinked complexes formed, schemes 2 and 4 appear to be more selective than schemes 1, 3 and 5. However, the highest estimated selectivity values are obtained for schemes 1 and 3 against CYP51-C.glabrata and for scheme 1 against CYP51-N.fowleri. In general, the direct relationship between the stability of the protein-ligated complex with the direct interaction of the ligand with the Fe2+ cation of the heme group, which provides stability to the union. [ABSTRACT FROM AUTHOR]

CYtochrome P450, family 51 (CYP51) is an important enzyme for de novo cholesterol synthesis in mammalian cells. In the present study, we found that the expression of CYP51 positively correlated with CD4+ T cell activation both in vivo and in vitro. The addition of ketoconazole, a pharmacological inhibitor of CYP51, prevented the proliferation and activation of anti-CD3/CD28-expanded mouse CD4+ T cells in a dose-dependent fashion. Liquid chromatography-tandem mass spectrometry indicated an increase in levels of lanosterol in T cells treated with ketoconazole during activation. Ketoconazole-induced blockade of the cholesterol synthesis pathway also caused Sterol regulatory element binding protein 2 (SREBP2) activation in CD4+ T cells. Additionally, ketoconazole treatment elicited an integrated stress response in T cells that up-regulated activating transcription factor 4 (ATF4) and DNA-damage inducible transcript 3 (DDIT3/CHOP) at the translational level. Furthermore, treatment with ketoconazole significantly decreased the amount of CD4+ T cells infiltrating lesions in the submandibular glands of NOD/Ltj mice. In summary, our results suggest that CYP51 plays an essential role in the proliferation and survival of CD4+ T cells, which makes ketoconazole an inhibitor of CD4+ T cell proliferation and of the SS-like autoimmune response through regulating the biosynthesis of cholesterol and inducing the integrated stress response. [ABSTRACT FROM AUTHOR]

Fungal infections are posing serious threat to healthcare system due to emerging resistance among available antifungal agents. Among available antifungal agents in clinical practice, azoles (diazole, 1,2,4‐triazole and tetrazole) remained most effective and widely prescribed antifungal agents. Now their associated side effects and emerging resistance pattern raised a need of new and potent antifungal agents. Lanosterol 14α‐demethylase (CYP51) is responsible for the oxidative removal of 14α‐methyl group of sterol precursors lanosterol and 24(28)‐methylene‐24,25‐dihydrolanosterol in ergosterol biosynthesis hence an essential component of fungal life cycle and prominent target for antifungal drug development. This review will shed light on various azole‐ as well as non‐azoles‐based derivatives as potential antifungal agents that target fungal CYP51. Review will provide deep insight about structure activity relationship, pharmacological outcomes, and interactions of derivatives with CYP51 at molecular level. It will help medicinal chemists working on antifungal development in designing more rational, potent, and safer antifungal agents by targeting fungal CYP51 for tackling emerging antifungal drug resistance. [ABSTRACT FROM AUTHOR]

BackgroundPseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes.ResultsWe observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross-sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild-type isolates from unsprayed bananas fields did not contain any promoter insertions.ConclusionThe presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pyrenophora teres f. teres (Ptt) is a severe pathogen to spring barley in Northern Europe. Ptt with relevant mutations in fungicide target proteins, sterol 14α-demethylase (CYP51A), cytochrome b (Cyt b), and succinate dehydrogenase (SDH) would put efficient disease control at risk. In the growing seasons of 2021 and 2022, 193 Ptt isolates from Estonia were analysed. In this study, mutation detection and in vitro fungicide sensitivity assays of single-spore isolates were carried out. Reduced sensitivity phenotype to mefentrifluconazole was evident in Ptt isolates with a F489L mutation in CYP51A or with 129 bp insert in the Cyp51A gene-promoter region. However, sensitivity to a prothioconazole-desthio remained high regardless of these molecular changes. The Ptt population was mostly sensitive to bixafen, fluxapyroxad, pyraclostrobin, and azoxystrobin. The sensitivity of fluxapyroxad and bixafen has been affected by two mutations, C-S135R and D-H134R, found in SDH subunits. The F129L mutation in Cyt b influenced azoxystrobin but not pyraclostrobin sensitivity. In total, 30 isolates from five fields had relevant mutations in three target protein genes simultaneously. Most of these isolates had a reduced sensitivity phenotype to mefentrifluconazole, fluxapyroxad, and azoxystrobin, while sensitivity to other tested fungicides remained high. Furthermore, possible sexual reproduction may enhance the pathogen’s fitness and help it adapt to fungicides.

Candida albicans , an opportunistic pathogen, is the most common type of fungus and represents a substantial source of human invasive disease (nosocomial infection). This category of fungi are part of our microbiota, and given the appropriate environmental conditions, it has the potential to cause both superficial and systemic infections. There is a soaring resistance against the available anticandidal agents. The purpose of this research is to investigate the activity of certain previously synthesized benzo[ g ]quinazolines against C. albicans in vitro by using the cup-plate diffusion method. There was a marked difference in the effectiveness of the target compounds 1–6 against the sample of C. albicans that was tested. Benzo[ g ]quinazolines 1 (inhibition zone = 20 mm) and 2 (inhibition zone = 22 mm) had good effects in comparison to fluconazole (inhibition zone = 26 mm). A docking study was conducted between benzo[ g ]quinazolines 1–6 and Candida spp. CYP51 to establish the binding mode compared with fluconazole and VT-1161 (oteseconazole) as reference medicines, and it was determined that binding at the active site of Candida spp. CYP51 occurred in the same manner. Quantitative structure–activity relationship (QSAR) investigation was performed to further characterize the identified anticandidal agents and recognize the major regulatory components governing such activity. In future studies, the benzo[ g ]quinazoline scaffold could serve as a model for the design and development of novel derivatives with antifungal potential. [ABSTRACT FROM AUTHOR]

A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different chemical approaches were then developed. The first one, in seven steps, involved the synthesis of the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole and the final opening of oxiranes by imidazole or 1H-1,2,4-triazole. The second route allowed access to the target compounds in only three steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested against Candida albicans and other Candida species. The enantiomers of the best anti-Candida compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g), were analyzed by X-ray diffraction to determine their absolute configuration. The (-)-8g enantiomer (Minimum inhibitory concentration (MIC) = IC80 = 0.000256 µg/mL on C. albicans CA98001) was found with the S-absolute configuration. In contrast the (+)-8g enantiomer was found with the R-absolute configuration (MIC = 0.023 µg/mL on C. albicans CA98001). By comparison, the MIC value for FLC was determined as 0.020 µg/mL for the same clinical isolate. Additionally, molecular docking calculations and molecular dynamics simulations were carried out using a crystal structure of Candida albicans lanosterol 14α-demethylase (CaCYP51). The (-)-(S)-8g enantiomer aligned with the positioning of posaconazole within both the heme and access channel binding sites, which was consistent with its biological results. All target compounds have been also studied against human fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.

Abstract The watermelon and muskmelon productions are important agriculture pillar industries of Zhejiang Province in China. Difenoconazole is an imidazole-class sterol, 14-demethylase inhibitor (DMI), which has been in use for several years to control gummy stem blight (GSB) caused by Stagonosporopsis species. However, the detection and characterization of difenoconazole resistance in GSB have not been clarified. In this study, a total of 112 isolates were collected from samples of GSB on watermelon and muskmelon in five locations of Zhejiang Province, China. All of the isolates were identified as Stagonosporopsis citrulli through morphology and multiplex PCR analysis. The determination of their resistance to difenoconazole via the discriminatory dosage method showed that the total resistance frequency was 89.3%. Among the resistant sub-population, 36.6% had high-level resistance to difenoconazole (DifHR), while 46.4% and 6.3% had low- (DifLR) and moderate-level resistance (DifMR), respectively. Additionally, the difenoconazole showed a positive cross-resistance with four DMIs, i.e., tebuconazole, prochloraz, metconazole, and mefentrifluconazole, but not hexaconazole. The phenotypic analysis found that the difenoconazole resistant (DifR) isolates demonstrated attenuated ability in both the mycelial growth and sporulation compared with the difenoconazole sensitive (DifS) isolates, while there was no significant difference in pathogenicity on watermelon leaves between the DifR and DifS isolates. Further exploration of the mechanism related to difenoconazole resistance of S. citrulli isolates revealed that the resistance to difenoconazole involved four types of mutations in CYP51, i.e., G463S for DifLR, I444M, Y446H, and A464G for DifHR. No over-expression of the cyp51 gene was found in the tested DifR isolates. Furthermore, it was found that 5% of the DifR isolates were significantly more sensitive to difenoconazole after being treated with 20 μg/mL chlorpromazine hydrochloride, indicating that the efflux mechanism may be involved in these difenoconazole-resistant isolates. Together, our study results suggested that S. citrulli had a strong resistance to difenoconazole on watermelon and muskmelon, and the mutations in cyp51and changes in fungicide efflux were responsible for the emergence of difenoconazole resistance in S. citrulli.

Azole antifungals, including fluconazole, have long been the first-line antifungal agents in the fight against fungal infections. The emergence of drug-resistant strains and the associated increase in mortality from systemic mycoses has prompted the development of new agents based on azoles. We reported a synthesis of novel monoterpene-containing azoles with high antifungal activity and low cytotoxicity. These hybrids demonstrated broad-spectrum activity against all tested fungal strains, with excellent minimum inhibitory concentration (MIC) values against both fluconazole-susceptible and fluconazole-resistant strains of Candida spp. Compounds 10a and 10c with cuminyl and pinenyl fragments demonstrated up to 100 times lower MICs than fluconazole against clinical isolates. The results indicated that the monoterpene-containing azoles had much lower MICs against fluconazole-resistant clinical isolates of Candida parapsilosis than their phenyl-containing counterpart. In addition, the compounds did not exhibit cytotoxicity at active concentrations in the MTT assay, indicating potential for further development as antifungal agents. [ABSTRACT FROM AUTHOR]

Preventing antifungal resistance development and identifying pathogens with high, medium, and low risk of resistance development to a particular fungicide or fungicide class is crucial in the fight against phytopathogens. We characterized the sensitivity of potato wilt-associated Fusarium oxysporum isolates to fludioxonil and penconazole and assessed the effect of these fungicides on the expression of fungal sterol-14-α-demethylase (CYP51a) and histidine kinase (HK1) genes. Penconazole stunted the growth of F. oxysporum strains at all concentrations used. While all isolates were susceptible to this fungicide, concentrations of up to 1.0 μg/mL were insufficient to cause a 50% inhibition. At low concentrations (0.63 and 1.25 μg/mL), fludioxonil stimulated growth in F. oxysporum. With an increase in the concentration of fludioxonil, only one strain (F. oxysporum S95) exhibited moderate sensitivity to the fungicide. Interaction of F. oxysporum with penconazole and fludioxonil leads to respective elevated expressions of the CYP51a and HK1 genes, which upsurge with increasing concentration of the fungicides. The data obtained indicate that fludioxonil may no longer be suitable for potato protection and its continuous use could only lead to an increased resistance with time. [ABSTRACT FROM AUTHOR]

Background: Fusarium fujikuroi is the pathogenic agent of rice bakanae disease and has developed serious resistance to prochloraz, a 14α‐demethylase inhibitor (DMI). Prochloraz resistance in F. fujikuroi is caused by cooperation between FfCyp51B with Cyp51A and shows cross‐resistance only to prothioconazole but not to tebuconazole, difenoconazole, propiconazole, metconazole, hexaconazole, and triadimefon. This study aimed to analyze the functions of the three Cyp51s in F. fujikuroi, especially their role in determining sensitivity to DMIs. Results: The respective deletion of FfCyp51A, Cyp51B, and Cyp51C had no obvious effect on morphology, conidium germination, or pathogenicity. The involvement of growth, growth and ergosterol biosynthesis, and conidium production and ergosterol biosynthesis was observed for FfCyp51A, Cyp51B, and Cyp51C, respectively. Compared with the sensitive isolate of F. fujikuroi, the effect on sensitivity to the tested DMIs was divided into four groups: (i) both of Cyp51A and Cyp51B positively regulate the sensitivity to prochloraz and prothioconazole; (ii) Cyp51B positively regulate the sensitivity to tebuconazole and metconazole, but negatively regulate the sensitivity to difenoconazole; (iii) Cyp51A and Cyp51B play opposite roles in the sensitivity to triadimefon. Therefore, deletion of Cyp51A in F. fujikuroi confers a higher sensitivity to triadimefon, while deletion of Cyp51B results in a triadimefon‐resistant mutant isolate; (iv) deletion of Cyp51B yielded a mutant isolate that was more resistant to propiconazole and hexaconazole. Conclusion: Sophisticated interactions exist within the three Cyp51 genes to DMIs fungicides sensitivity in F. fujikuroi, and Cyp51B probably plays a more critical role than Cyp51A and Cyp51C. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Sterol biosynthesis via the mevalonate-isoprenoid pathway produces ergosterol (24β-methyl cholesta-5,7-dienol) necessary for growth in a wide-range of eukaryotic pathogenic organisms in eukaryotes, including the fungi, trypanosomes and amoebae, while their animal hosts synthesize a structurally less complicated product—cholesterol (cholest-5-enol). Because phyla-specific differences in sterol metabolizing enzyme architecture governs the binding and reaction properties of substrates and inhibitors while the order of sterol metabolizing enzymes involved in steroidogenesis determine the positioning of crucial chokepoint enzymes in the biosynthetic pathway, the selectivity and effectiveness of rationally designed ergosterol biosynthesis inhibitors toward ergosterol-dependent infectious diseases varies greatly. Recent research has revealed an evolving toolbox of mechanistically distinct tight-binding inhibitors against two crucial methylation-demethylation biocatalysts—the C24 sterol methyl transferase (absent from humans) and the C14-sterol demethylase (present generally in humans and their eukaryotic pathogens). Importantly for rational drug design and development, the activities of these enzymes can be selectively blocked in ergosterol biosynthesis causing loss of ergosterol and cell killing without harm to the host organism. Here, we examine recent advances in our understanding of sterol biosynthesis and the reaction differences in catalysis for sterol methylation-demethylation enzymes across kingdoms. In addition, the novelties and nuances of structure-guided or mechanism-based approaches based on crystallographic mappings and substrate specificities of the relevant enzyme are contrasted to conventional phenotypic screening of small molecules as an approach to develop new and more effective pharmacological leads.

Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review is to describe the antifungal mechanism action from Piper crocatum and its phytochemical profiling against lanosterol 14a demethylase CYP51. The methods used to search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. From 1.150.000 results searched by database, there is 73 final results article to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol, especially lanosterol 14a demethylase (CYP51) inhibition, which is one of the main target sites for antifungal activity because it functions to maintain the integrity and function of cell membranes in Candida. P. crocatum has an antifungal activity through its phytochemical profiling against fungal by inhibiting the lanosterol 14a demethylase, make damaging cell membranes, fungal growth inhibition, and fungal cell lysis.

Mycoses or fungal infections are general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review is to describe the antifungal mechanism action from Piper crocatum and its phytochemical profiling against lanosterol 14a demethylase CYP51. The methods used to search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. From 1.150.000 results searched by database, there is 73 final results article to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol especially lanosterol 14a demethylase (CYP51) inhibition, which is one of the main target sites for antifungal activity because it functions to maintain the integrity and function of cell membranes in Candida. P. crocatum has an antifungal activity through its phytochemical profiling against fungal by inhibiting the lanosterol 14a demethylase, make damaging cell membranes, fungal growth inhibition, and fungal cell lysis.

Arome Solomon Odiba,1,2 Olanrewaju Ayodeji Durojaye,3– 5 Ifeoma Maureen Ezeonu,6 Anthony Christian Mgbeahuruike,7 Bennett Chima Nwanguma1,2 1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 2Department of Molecular Genetics and Biotechnology, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 3Department of Chemical Sciences, Coal City University, Emene, Enugu State, Nigeria; 4Department of Molecular and Cell Biology, University of Science and Technology of China, Hefei, Anhui, 230026, People’s Republic of China; 5MOE Key Laboratory of Membraneless Organelle and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China; 6Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 7Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, 410001, NigeriaCorrespondence: Bennett Chima Nwanguma; Anthony Christian Mgbeahuruike, Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Enugu State, Nsukka, 410001, Nigeria, Tel +234 8063655062, Email bennett.nwanguma@unn.edu.ng; anthony.mgbeahuruike@unn.edu.ngBackground: Resistance to antifungal drugs for treating Candida infections remains a major concern globally despite the range of medications available. Most of these drugs target key proteins essential to the life cycle of the organism. An enzyme essential for fungal cell membrane integrity, lanosterol 14–α demethylase (CYP51), is encoded by the ERG11 gene in Candida species. This enzyme is the target of azole–based drugs. The organism has, however, devised molecular adaptations to evade the activity of these drugs.Materials and Methods: Classical methods were employed to characterize clinical isolates sampled from women and dogs of reproductive age. For fluconazole efficacy studies, CLSI guidelines on drug susceptibility testing were used. To understand the susceptibility pattern, various molecular and structural analytic approaches, including sequencing, in silico site-directed mutagenesis, and protein-ligand profiling, were applied to the ERG11 gene and CYP51 protein sequences. Several platforms, comprising Clustal Omega, Pymol plugin manager, Pymol molecular visualizer, Chimera–curated Dynameomics rotamer library, protein–ligand interaction profiler, Charmm36 force field, GROMACS, Geneious, and Mega7, were employed for this analysis.Results: The following Candida species distribution was obtained: 37.84% C. albicans, 8.12% C. glabrata, 10.81% C. krusei, 5.41% C. tropicalis, and 37.84% of other unidentified Candida species. Two codons in the nucleotide sequence of the wild-type (CTC and CCA) coding for LEU– 370 and PRO– 375, respectively, were mutated to L370S and P375H in the resistant strain. The mutation stabilized the protein at the expense of the heme moiety. We found that the susceptible isolate from dogs (Can–iso– 029/dog) is closely related to the most resistant isolate from humans.Conclusion: Taken together, our results showed new mutations in the heme-binding pocket of caCYP51 that explain the resistance to fluconazole exhibited by the Candida isolates. So far, the L370S and P375H resistance-linked mutations have not been previously reported.Keywords: Candida infection, CYP51, drug resistance, ERG11, fluconazole, mutation

Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review is to describe the antifungal mechanism action from Piper crocatum and its phytochemical profiling against lanosterol 14a demethylase CYP51. The methods used to search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. From 1.150.000 results searched by database, there is 73 final results article to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol, especially lanosterol 14a demethylase (CYP51) inhibition, which is one of the main target sites for antifungal activity because it functions to maintain the integrity and function of cell membranes in Candida. P. crocatum has an antifungal activity through its phytochemical profiling against fungal by inhibiting the lanosterol 14a demethylase, make damaging cell membranes, fungal growth inhibition, and fungal cell lysis.

The watermelon and muskmelon productions are important agriculture pillar industries of Zhejiang Province in China. Difenoconazole is an imidazole-class sterol, 14-demethylase inhibitor (DMI), which has been in use for several years to control gummy stem blight (GSB) caused by Stagonosporopsis species. However, the detection and characterization of difenoconazole resistance in GSB have not been clarified. In this study, a total of 112 isolates were collected from samples of GSB on watermelon and muskmelon in five locations of Zhejiang Province, China. All of the isolates were identified as Stagonosporopsis citrulli through morphology and multiplex PCR analysis. The determination of their resistance to difenoconazole via the discriminatory dosage method showed that the total resistance frequency was 89.3%. Among the resistant sub-population, 36.6% had high-level resistance to difenoconazole (DifHR), while 46.4% and 6.3% had low- (DifLR) and moderate-level resistance (DifMR), respectively. Additionally, the difenoconazole showed a positive cross-resistance with four DMIs, i.e., tebuconazole, prochloraz, metconazole, and mefentrifluconazole, but not hexaconazole. The phenotypic analysis found that the difenoconazole resistant (DifR) isolates demonstrated attenuated ability in both the mycelial growth and sporulation compared with the difenoconazole sensitive (DifS) isolates, while there was no significant difference in pathogenicity on watermelon leaves between the DifR and DifS isolates. Further exploration of the mechanism related to difenoconazole resistance of S. citrulli isolates revealed that the resistance to difenoconazole involved four types of mutations in CYP51, i.e., G463S for DifLR, I444M, Y446H, and A464G for DifHR. No over-expression of the cyp51 gene was found in the tested DifR isolates. Furthermore, it was found that 5% of the DifR isolates were significantly more sensitive to difenoconazole after being treated with 20 μg/mL chlorpromazine hydrochloride, indicating that the efflux mechanism may be involved in these difenoconazole-resistant isolates. Together, our study results suggested that S. citrulli had a strong resistance to difenoconazole on watermelon and muskmelon, and the mutations in cyp51and changes in fungicide efflux were responsible for the emergence of difenoconazole resistance in S. citrulli. [ABSTRACT FROM AUTHOR]

Background: Management of rice false smut is based mainly on chemical control, which poses many safety and environmental challenges. The other option, biological control with biofungicides, does not have such problems but is not as reliable because of low systemic ability, and lower and unstable efficacy. Therefore, it is necessary to combine application of chemical fungicides and biological control agents (BCAs) and elucidate their synergistic mechanism. Results: A combination of tebuconazole and a proven BCA, Bacillus subtilis H158, was evaluated for control of rice false smut. Tebuconazole at low application rates stimulated growth of B. subtilis, prolonged the effective period of B. subtilis by enhancing its persistence on the surface of rice plants, accelerated biofilm formation of the BCA to facilitate colonization, promoted induced systemic resistance in rice by regulating defense‐related enzymes and genes, and reduced the natural resistance of the pathogen by suppressing the key gene for fungal resistance to tebuconazole. However, at high application rates, tebuconazole had adverse effects on these factors and showed antagonistic combination effects with B. subtilis. The combination of B. subtilis with tebuconazole at low application rates showed great synergistic effects, but at high application rates showed only antagonistic effects in field experiments over two consecutive years. Conclusion: The combination of B. subtilis with tebuconazole had significant synergistic effects at low application rates. The synergistic effects are the result of multiple mechanisms involved in BCA, rice and the fungal pathogen. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery. [ABSTRACT FROM AUTHOR]

Candida albicans is a common human fungal pathogen that colonizes mucosa and develops biofilm in the oral cavity that causes oral candidiasis. It has been reported that cytochrome P450 enzyme (CYP51), a vital part of the ergosterol synthesis cascade, is associated with Candida infections and its biofilm formation. Thidiazuron, a phenyl-urea cytokinin, exhibits anti-senescence and elicitor activity against fungal infection in plants. However, how Thidiazuron impacts C. albicans biofilm formation is still uncertain. Here, we aimed to investigate the effects of a Thidiazuron against the growth and biofilm formation properties of C. albicans using in silico and in vitro experimental approaches. A preliminary molecular docking study revealed potential interaction between Thidiazuron and amino acid residues of CYP51. Further in vitro antifungal susceptibility test, scanning electron microscopy (SEM) and time kill analysis revealed the anti-fungal activity of Thidiazuron in both dose and time-dependent manner. Crystal violet staining, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay revealed 50% inhibition in C. albicans biofilm by Thidiazuron at concentrations 11 and 19 µM respectively. Acridine orange staining assay visually confirmed the biofilm inhibitory potential of Thidiazuron. The gene expression study showed that Thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis (ERG3, ERG11, ERG25), cell adhesion (ASL3, EAP1), and hyphae development (EFG1, HWP1, SAP5) in C. albicans. Wherease, the expression of negative transcription regulator of hyphae (NRG1) was upregulated (5.7-fold) by Thidiazuron treatment. Collectively, our data suggest that Thidiazuron is a robust antifungal compound and an outstanding biofilm inhibitor, which may promise further therapeutic development due to CYP51 binding and inhibition of ergosterol formation against C. albicans. [ABSTRACT FROM AUTHOR]

[Display omitted] • Inhibitors of Leishmania CYP51 and CYP5122A1 were synthesized and evaluated. • CYP inhibitors possessing in vitro antileishmanial activity were identified. • A CYP5122A1 inhibitor causes lanosterol accumulation in Leishmania. CYP5122A1, an enzyme involved in sterol biosynthesis in Leishmania , was recently characterized as a sterol C4-methyl oxidase. Screening of a library of compounds against CYP5122A1 and CYP51 from Leishmania resulted in the identification of two structurally related classes of inhibitors of these enzymes. Analogs of screening hit N -(3,5-dimethylphenyl)-4-(pyridin-4-ylmethyl)piperazine-1-carboxamide (4a) were generally strong inhibitors of CYP51 but were less potent against CYP5122A1 and typically displayed weak inhibition of L. donovani promastigote growth. Analogs of screening hit N -(4-(benzyloxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18a) were stronger inhibitors of both CYP5122A1 and L. donovani promastigote proliferation but also remained selective for inhibition of CYP51. Two compounds in this series, N -(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18e) and N -(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18i) showed modest selectivity for inhibiting L. donovani promastigote proliferation compared to J774 macrophages and were effective against intracellular L. donovani with EC 50 values in the low micromolar range. Replacement of the 4-pyridyl ring present in 18e with imidazole resulted in a compound (4-(2-(1 H -imidazol-1-yl)ethyl)- N -(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)piperazine-1-carboxamide, 18p) with approximately fourfold selectivity for CYP5122A1 over CYP51 that inhibited both enzymes with IC 50 values ≤ 1 µM, although selective potency against L. donovani promastigotes was lost. Compound 18p also inhibited the proliferation of L. major promastigotes and caused the accumulation of 4-methylated sterols in L. major membranes, indicating that this compound blocks sterol demethylation at the 4-position in Leishmania parasites. The molecules described here may therefore be useful for the future identification of dual inhibitors of CYP51 and CYP5122A1 as potential antileishmanial drug candidates and as probes to shed further light on sterol biosynthesis in Leishmania and related parasites. [ABSTRACT FROM AUTHOR]

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.

Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, has recently become widespread in Europe. Succinate dehydrogenase inhibitor (SDHI) and demethylation inhibitor (DMI) fungicides are mainly applied for disease control on barley fields, but pathogen isolates with a reduced sensitivity can cause difficulties. There is an urgent need for new spring barley cultivars that are more resistant to RLS development and can inhibit R. collo-cygni epidemics.

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either 1 or 2 of 3 Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across fungi. We found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota, and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in fungi. Our results suggest Cyp51 is present in all fungi with 3 paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups. [ABSTRACT FROM AUTHOR]

Fusarium keratoplasticum is the Fusarium species most commonly associated with human infections (fusariosis). Antifungal treatment of fusariosis is often hampered by limited treatment options due to resistance towards azole antifungals. The mechanisms of antifungal resistance and sterol biosynthesis in fusaria are poorly understood. Therefore, in this study we assessed the transcriptional response of F. keratoplasticum when exposed to voriconazole. Our results revealed a group of dramatically upregulated ergosterol biosynthesis gene duplicates, most notably erg6A (912-fold), cyp51A (52-fold) and ebp1 (20-fold), which are likely part of an alternative ergosterol biosynthesis salvage pathway. The presence of human cholesterol biosynthesis gene homologs in F. keratoplasticum (ebp1, dhcr7 and dhcr24_1, dhcr24_2 and dhcr24_3) suggests that additional sterol biosynthesis pathways may be induced in fusaria under other growth conditions or during host invasion. Voriconazole also induced the expression of a number of ABC efflux pumps. Further investigations suggested that the highly conserved master regulator of ergosterol biosynthesis, FkSR, and the pleiotropic drug resistance network that induces zinc-cluster transcription factor FkAtrR coordinate the response of FSSC species to azole antifungal exposure. In-depth genome mining also helped clarify the ergosterol biosynthesis pathways of moulds and provided a better understanding of antifungal drug resistance mechanisms in fusaria. [ABSTRACT FROM AUTHOR]

Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review was to describe antifungal activity from Piper crocatum and its phytochemical profiling against lanosterol 14 alpha demethylase CYP51. The methods used search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow diagram as a clinical information retrieval method. From 1,150,000 results search by database, there were 73 selected articles to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol especially lanosterol 14 alpha demethylase CYP51 inhibition as a result of 5,6 desaturase (ERG3) downregulation. P. crocatum has an antifungal activity by its phytochemical profiling that act against fungi by inhibiting the fungal cytochrome P 450 pathway, make damaging cell membranes, fungal growth inhibition, morphological changes, and fungal cell lysis.