CYP51 Protein Research Articles

Design, Synthesis, and Antimicrobial Activity of Amide Derivatives Containing Cyclopropane.

As an important small organic molecule, cyclopropane is widely used in drug design. In this paper, fifty-three amide derivatives containing cyclopropane were designed and synthesized by introducing amide groups and aryl groups into cyclopropane through the active splicing method, and their antibacterial and antifungal activities were evaluated in vitro. Among them, thirty-five compounds were new compounds, and eighteen compounds were known compounds (F14, F15, F18, F20-F26, F36, and F38-F44). Bioassay results disclosed that four, three, and nine of the compounds showed moderate activity against Staphylococcus aureus, Escherichia coli, and Candida albicans, respectively. Three compounds were sensitive to Candida albicans, with excellent antifungal activity (MIC80 = 16 μg/mL). The molecular docking results show that compounds F8, F24, and F42 have good affinity with the potential antifungal drug target CYP51 protein.

Molecular Mechanisms and Biological Characteristics of Botrytis cinerea Field Isolate Resistance to Pyrisoxazole in Liaoning Province.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in 14 cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 value of 0.151 μg/ml. Three highly resistant isolates, XD-5, DG-4, and GQ-3, were screened, and the EC50 values were 0.734, 0.606, and 0.639 μg/ml with corresponding resistance factors of 12.88, 10.63, and 11.21, respectively. Compared with field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits, including mycelial growth, conidial production, and pathogenicity, but DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the non-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam, and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3, and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared with those in the sensitive isolates, whereas the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, whereas no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.

Epoxiconazole altered hepatic metabolism in adult zebrafish based on transcriptomic analysis

Epoxiconazole (EPX) is a triazole fungicide, which has been widely used in pest control of cereal crops. However, its extensive use has led to concerning levels of residue in water bodies, posing substantial risks to aquatic life. In this study, we characterized the toxicological effects of EPX on 6-month-old male and female zebrafish at 70 and 700 μg/L, respectively. The results revealed that EPX exposure markedly increased both body length and weight in zebrafish of both sexes, consequently elevating their condition factor. Besides, EPX exposure resulted in notable alterations in hepatic histopathology. These changes included loosened hepatocyte structure, ballooning degeneration, nucleolysis, and disappearance of cell line, with male zebrafish exhibiting more severe damage. High concentration of EPX also significantly increased hepatic lipid accumulation in male zebrafish, as well as increased hepatic triglyceride (TG) levels. Correspondingly, there was a notable alteration in the transcription of genes including cyp51, hmgcr, and PPAR-γ, which associated with cholesterol and lipid metabolism. Interestingly, with the hepatic transcriptomic analysis, high concentration of EPX produced 195 upregulated and 107 downregulated differential expression genes. Both KEGG and GO analyses identified significant enrichment of these genes in lipid and amino acid metabolism pathways. Notably, some key genes involved in the steroid synthesis pathway were marked upregulated. In addition, molecular docking study confirmed that EPX could bind CYP51 protein well (△G = −7.7 kcal/mol). Taken together, these findings demonstrated the multiple toxic effects of EPX on adult zebrafish.

Trans-kingdom fungal pathogens infecting both plants and humans, and the problem of azole fungicide resistance.

Azole antifungals are abundantly used in the environment and play an important role in managing fungal diseases in clinics. Due to the widespread use, azole resistance is an emerging global problem for all applications in several fungal species, including trans-kingdom pathogens, capable of infecting plants and humans. Azoles used in agriculture and clinics share the mode of action and facilitating cross-resistance development. The extensive use of azoles in the environment, e.g., for plant protection and wood preservation, contributes to the spread of resistant populations and challenges using these antifungals in medical treatments. The target of azoles is the cytochrome p450 lanosterol 14-α demethylase encoded by the CYP51 (called also as ERG11 in the case of yeasts) gene. Resistance mechanisms involve mainly the mutations in the coding region in the CYP51 gene, resulting in the inadequate binding of azoles to the encoded Cyp51 protein, or mutations in the promoter region causing overexpression of the protein. The World Health Organization (WHO) has issued the first fungal priority pathogens list (FPPL) to raise awareness of the risk of fungal infections and the increasingly rapid spread of antifungal resistance. Here, we review the main issues about the azole antifungal resistance of trans-kingdom pathogenic fungi with the ability to cause serious human infections and included in the WHO FPPL. Methods for the identification of these species and detection of resistance are summarized, highlighting the importance of these issues to apply the proper treatment.

1,3,4-oxadiazole derivatives: synthesis, characterization, antifungal activity, DNA binding investigations, TD-DFT calculations, and molecular modelling

1,3,4-Oxadiazole-based heterocyclic analogs (3a–3m) were synthesized via cyclization of Schiff bases with substituted aldehydes in the presence of bromine and acetic acid. The structural clarification of synthesized molecules was carried out with various spectroscopic techniques such as FT-IR,1H and 13C-NMR, UV–visible spectroscopy, and mass spectrometry. In-vitro antifungal activity was performed against C. albicans, C. glabrata and C. tropicalis and analogs 3g, 3i, and 3m showed potent MIC at 200 µg/ml and excellent ZOI measurements of 17–21 nm. The cell viability on Huh7 for lead molecules 3g, 3i, and 3m was found to be 99.5%, 92.3%, and 86.9% at 20, 10, and 20 μM, respectively. The antioxidant activity of molecules 3 g, 3i, and 3 m was estimated and exhibited great IC50 values of 0.104 ± 0.021, 0.145 ± 0.05, and 0.165 ± 0.018 μg/mL with DPPH and 0.107 ± 0.04, 0.191 ± 0.12, and 0.106 ± 0.08 with H2O2, respectively. The binding interaction mode for the lead molecules was also carried out with Ct-DNA using the absorption, emission, CV, CD, and Time resolve fluorescence techniques. The results showed good binding constant (Kb) values of 9.1 × 105, 9.94 × 105, and 9.32 × 105 M−1 for 3g, 3i, and 3m, respectively. TD-DFT study of compounds 3g, 3i, and 3m was done to find out HOMO/LUMO energy levels, surface study of the molecular electrostatic potential, Mulliken population analysis, and natural bond orbitals showing the linkages between the donors and acceptors.Molecular docking of three lead analogs with PDB ID: 1BNA and molecular modelling of compounds 3g, 3i, and 3m with C. albicans CYP51 protein (PDB ID: 5FSA) were carried out. Communicated by Ramaswamy H. Sarma

Genetic variation of drug target CYP51 conferring resistance to ergosterol biosynthesis inhibitors in Botrytis cinerea, causing lily gray mould

Gray mould caused by Botrytis cinerea poses a threat to Lily (Lilium spp.), an important plant with both medicinal and ornamental value, during pre- and post-harvest stages. Ergosterol biosynthesis inhibitors (EBIs) have been widely registered to control gray mould caused by B. cinerea in various plants over a prolonged period of time, yet no instances of resistance of B. cinerea to EBIs have been detected in the field. In this study, the field B. cinerea EBIs-resistant populations were detected from the diseased lily flowers. The CYP51 protein, which is the target of EBIs, was found to have G461S or R464K substitutions in these resistant strains. To further verify whether the G461S or R464K substitutions of CYP51 confer the resistance to EBIs in B. cinerea, the mutants carrying the G461S or R464K substitutions of CYP51 were generated from EBIs-sensitive wild type strains via site-directed mutagenesis. The G461S or R464K substitutions indeed resulted in decreased sensitivity of B. cinerea to EBIs. Meanwhile, the mutants conferring the S461G or K464R substitutions of CYP51 were generated from the field EBIs-resistant strains, and the significant increase in sensitivity to EBIs were observed in these mutants. Furthermore, the G461S or R464K substitutions suffered to fitness penalty in mycelial growth and virulence, but improved sporulation capability. Molecular docking analysis furtherly demonstrated that the CYP51 protein, carrying the G461S or R464K substitutions, exhibited reduced binding affinity with tebuconazole. To the best of our knowledge, it is the first report that G461S or R464K substitutions in CYP51 leading to field resistance to EBIs in B. cinerea. These findings not only provided valuable insights for managing lily gray mould caused B. cinerea, but also enhance our understanding of molecular mechanism underlying plant pathogen resistance to EBIs.

Enantioselective behavior and bioactivity of chiral tebuconazole in apples

Tebuconazole, a chiral triazole fungicide widely used in apple disease control, is commonly applied as a racemic mixture without differentiating between enantiomers. This study investigated the dissipation kinetics of tebuconazole enantiomers in various apple fruit varieties under field conditions using UPLC-MS/MS. Evidence of stereoselective degradation was found in Fuji and Hanfu apples, where (−)-R-tebuconazole showed faster degradation compared to (+)-S-tebuconazole, with half-lives of 15.59 and 19.12 d, and 16.91 and 21.02 d, respectively. In contrast, no enantioselective degradation was observed in Huahong variety apples. Furthermore, (−)-R-tebuconazole was found to exhibit 16.8−68.4 times more fungicidal activity than (+)-S-tebuconazole against three of the most common pathogens in apple groves, including Botryosphaeria berengeriana, Colletotrichum gloeosporioides, and Alternaria alternate. Molecular docking analysis revealed that (−)-R-tebuconazole had a shorter binding distance and lower energy with CYP51 protein than (+)-S-tebuconazole, shedding light on the mechanism behind these experimental findings. Therefore, the development of a pure (−)-R-tebuconazole stereoisomer appears to be a promising option in the near future, given its higher bioactivity and faster degradation rate compared to the racemic mixture. This study provided valuable insights into the stereoselective behavior and bioactivity of tebuconazole in apples. Regular monitoring and effective management of chiral pesticides are increasingly essential due to their potential impact on food safety, pesticide risk assessment, and environmental pollution.

In silico targeting CYP51 of Naegleria fowleri using bioactive compounds from Indonesian plants

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.

In silico prediction of Antifungal compounds from Natural sources towards Lanosterol 14-alpha demethylase (CYP51) using Molecular docking and Molecular dynamic simulation

An increase in the occurrence of fungal infections throughout the world, as well as the rise of novel fungal strains and antifungal resistance to commercially available drugs, suggests that new therapeutic choices for fungal infections are needed. The purpose of this research was to find new antifungal candidates or leads of secondary metabolites derived from natural sources that could effectively inhibit the enzymatic activity of Candida albicans lanosterol 14-alpha demethylase (CYP51) while also having good pharmacokinetics. In silico prediction of the drug-likeness, chemo-informatics and enzyme inhibition indicate that the 46 compounds derived from fungi, sponges, plants, bacteria and algae sources have a high novelty to meet all five requirements of Lipinski's rules and impede enzymatic function. Among the 15 candidate molecules with strong binding affinity to CYP51 investigated by molecular docking simulation, didymellamide A-E compounds demonstrated the strongest binding energy against the target protein at −11.14, −11.46, −11.98, −11.98, and −11.50 kcal/mol, respectively. Didymellamide molecules bind to comparable active pocket sites of antifungal ketoconazole and itraconazole medicines by hydrogen bonds forming to Tyr132, Ser378, Met508, His377 and Ser507, and hydrophobic interactions with HEM601 molecule. The stability of the CYP51-ligand complexes was further investigated using molecular dynamics simulations that took into account different geometric features and computed binding free energy. Using the pkCSM ADMET descriptors tool, several pharmacokinetic characteristics and the toxicity of candidate compounds were assessed. The findings of this study revealed that didymellamides could be a promising inhibitor against these CYP51 protein. However, there is still a need for further in vivo and in vitro studies to support these findings.

Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi.

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.

The importance of selecting crystal form for triazole fungicide tebuconazole to enhance its botryticidal activity

The growing evidences of resistant fungi stimulate fully understanding tebuconazole regarding its crystal structure on fungicidal activity. In this study, the crystal structures of six technical tebuconazoles (BX, HH, JP, QZ, SJ, and YT) were characterized by using high-resolution X-ray powder diffraction and three-dimensional crystal structure modeling. A structure-activity relationship of the tebuconazoles on the susceptible (HLS and YJS) or resistant (XHR) Botrytis cinerea isolates was analyzed, the differential tricarboxylic acid (TCA) cycle metabolism was determined, and molecular docking with sterol 14α-demethylase (CYP51) was performed. The results showed that tebuconazole existed in three types of crystal forms: an overlapping-pair conformation, a side-by-side-pair conformation, and a parallel-pair conformation. QZ with the parallel-pair conformation and the minimum crystal cell volume exhibited a higher activity and a lower resistant level. XHR possessed a higher content of TCA cycle metabolites and phosphate than YJS, but the exposure to QZ significantly reduced the contents of citrate, isocitrate, α-ketoglutarate and oxaloacetate in XHR, as did the exposure to other technical tebuconazoles. Moreover, the point mutations F487L, G464S, and G443S altered the binding properties of chiral stereoscopic R-QZ with CYP51 protein. Especially the G443S mutation promoted a weak linking of R-QZ with LEU380 and TYR126, and greatly slashed the binding action at lower docking score. In conclusion, our results evidenced an efficient crystal conformation of tebuconazole to improve botryticidal activity and a potential adaptability of B. cinerea to tebuconazole inhibition in TCA cycle metabolism and CYP51 protein mutation.

New s-Triazine/Tetrazole conjugates as potent antifungal and antibacterial agents: Design, molecular docking and mechanistic study

A novel hybrid N2-(tetrazol-5-yl)-6-substituted-5,6-dihydro-1,3,5-triazine-2,4-diamines have been synthesized in excellent yields through one-pot multi-component reaction of 5-amino-1,2,3,4-tetrazole, cyanamide and aromatic/heteroaromatic aldehydes in acetic acid promoted by controlled microwave heating. All the obtained heterocycles were screened for their antimicrobial and antifungal activities. New series (4a-j) were developed through binding of s-triazine and tetrazole in one compact structure. The tested compounds showed excellent antibacterial activity against the Gram-negative bacterial strains, P. aeruginosa and E. coli compared to ciprofloxacin. The same derivatives experienced high outstanding activity against C. albicans compared to Fluconazole. Investigation of the mode of action as antibacterial agents revealed that the most active compound 4a has good inhibitory effect for DNA gyrase and topoisomerase IV ATPase and E. coli TOPO IV and DNA gyrase supercoiling. Additionally, 4a and 4g showed good in vitro inhibitory activity to lanosterol 14-α-demethylase (CYP51 protein) and good fitting to the same protein as possible antifungal target. Similarly, the target compounds have acceptable ADME and drug-likeness properties without any violation. Hence, new highly potent antibacterial and antifungal candidates were introduced that require further optimization.

A New Variant of Mutational and Polymorphic Signatures in the ERG11 Gene of Fluconazole-Resistant Candida albicans.

BackgroundResistance 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 MethodsClassical 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.ResultsThe 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.ConclusionTaken 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.

CYP51 Mutations in the Fusarium solani Species Complex: First Clue to Understand the Low Susceptibility to Azoles of the Genus Fusarium.

Members of Fusarium solani species complex (FSSC) are cosmopolitan filamentous fungi responsible for invasive fungal infections in immunocompromised patients. Despite the treatment recommendations, many strains show reduced sensitivity to voriconazole. The objective of this work was to investigate the potential relationship between azole susceptibility and mutations in CYP51 protein sequences. Minimal inhibitory concentrations (MICs) for azole antifungals have been determined using the CLSI (Clinical and Laboratory Standards Institute) microdilution method on a panel of clinical and environmental strains. CYP51A, CYP51B and CYP51C genes for each strain have been sequenced using the Sanger method. Amino acid substitutions described in multiple azole-resistant Aspergillus fumigatus (mtrAf) strains have been sought and compared with other Fusarium complexes’ strains. Our results show that FSSC exhibit point mutations similar to those described in mtrAf. Protein sequence alignments of CYP51A, CYP51B and CYP51C have highlighted different profiles based on sequence similarity. A link between voriconazole MICs and protein sequences was observed, suggesting that these mutations could be an explanation for the intrinsic azole resistance in the genus Fusarium. Thus, this innovative approach provided clues to understand low azole susceptibility in FSSC and may contribute to improving the treatment of FSSC infection.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

Green tea phytocompounds targets Lansterol 14-α demethylase against ergosterol biosynthesis in Candida glabrata

Green tea is credited as one of the world’s healthiest drinks with enriched antioxidants. It is known for its multi-beneficial health benefits against diabetes, blood pressure, hypertension, gastro-intestinal upset and is bestowed with significant antimicrobial potential. There are previous scientific evidence highlighting the antifungal potential of green tea and has identified it as a potential inhibitor of non-albicans Candida species. Lansterol 14-α demethylase (Erg 11) or CYP51 protein belongs to the cytochrome P450 monooxygenase (CYP) superfamily. Erg 11 is involved in ergosterol biosynthesis and has a significant role in azole drug resistance in Candida glabrata. The present study attempted to identify the inhibitory potential of green tea phytocompounds against inhibition of Erg 11 in Candida glabrata using bioinformatics tool viz., autodock vina software. Out of 15 green tea phytocompounds investigated, the study identified, Rutin (-10.5 kcal) Kaempferitrin (-9.4kcal), Epigallocatechin gallate (-10kcal), Epicatechin gallate (-8.7kcal), and Coumaroylquinic acid (-8.6kcal) acid as the potent phytocompounds which showed significant molecular interaction with Erg 11 in Candida glabrata. In attribution to the constant emergence of azole-resistant isolates, this preliminary analysis therefore, indicated the potential of green tea phytocompounds against inhibition of non-albicans Candida specific candidiasis. However, further, in vitro antimicrobial efficacy of these phytocompounds, the dose regime, drug likeliness, and cytotoxic analysis are required to be investigated and validated.

Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC=0.01μg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC=0.02μg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising invivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Difenoconazole Resistance Shift in Botrytis cinerea From Tomato in China Associated With Inducible Expression of CYP51.

Gray mold caused by Botrytis cinerea is one of the most important diseases in tomato. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but their resistance status after long-term use in the field is unclear. The baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean effective concentration for 50% mycelial growth inhibition (EC50) of 0.97 ± 0.50 μg/ml. EC50 values for difenoconazole sensitivity of another 248 isolates collected in 2011 and 2016 ranged from 0.04 to 11.99 μg/ml, and the frequency of difenoconazole sensitivity formed a nonnormal distribution curve. Detached fruit studies revealed that isolates with EC50 values of approximately 6.00 μg/ml were not controlled effectively. The mean EC50 of the resistant isolates changed from 6.74 to 8.65 μg/ml between 2011 and 2016. Positive cross-resistance was only observed between difenoconazole and two DMIs. One dual resistant isolate and one triple resistant isolate were found among the difenoconazole-resistant isolates collected in 2016, associated with point mutations in corresponding target proteins of the fungicides azoxystrobin and fludioxonil. This indicated that B. cinerea not only showed higher difenoconazole resistance levels but gradually changed from single to multiple fungicide resistance over time. No amino acid variation was found in the CYP51 protein. In the absence of difenoconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. Induced expression of CYP51 is an important determinant of DMI resistance in B. cinerea from tomato. However, nucleotide variants found in the upstream region had no association with the fungicide resistance phenotype. These results will be helpful for the management of B. cinerea in the field.

Monitoring and analysis of rice pathogen Ustilaginoidea virens isolates with resistance to sterol demethylation inhibitors in China

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is an important fungal disease of rice. In China, sterol demethylation inhibitors (DMIs) are common fungicides used to control RFS. In a previous study, we detected two propiconazole-resistant U. virens isolates in 2015 in Huai’an city, Jiangsu Province, China. In the current study, we detected six propiconazole-resistant isolates out of 180 U. virens isolates collected from rice fields in Jiangsu Province in 2017, and found they were from three different places (Xuzhou, Huai’an and Jintan). All these six propiconazole-resistant isolates were cross-resistant to three other sterol demethylation inhibitor (DMI) fungicides, i.e. difenoconazole, tebuconazole, and epoxiconazole. Among them, two isolates (2017–61 and 2017–170) had high fitness. Through sequencing and RT-qPCR analysis, we found that the expression levels of CYP51 and its encoded protein were significantly increased in the propiconazole-resistant isolates with a “CC” insertion mutation upstream of the CYP51 coding region compared to the propiconazole-sensitive isolates. In addition, propiconazole stimulated CYP51 expression in all isolates. Propiconazole also stimulated the accumulation of CYP51 protein in propiconazole-sensitive isolates and propiconazole-resistant isolates without mutation, but not in propiconazole-resistant isolates with the “CC” mutation. According to JASPAR database analysis, the predicated functional binding sites for propiconazole-resistant isolates with a “CC” insertion mutation and propiconazole-sensitive isolates were different. Given the high fitness of the propiconazole-resistant isolates, the development of resistance to DMIs in U. virens should be monitored. Furthermore, we speculated that the over-expression of CYP51 may contribute to DMI resistance in U. virens with the “CC” insertion mutation.

Antifungal Susceptibility Profiles and Drug Resistance Mechanisms of Clinical Lomentospora prolificans Isolates.

Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC90>16 μg/ml), itraconazole (MIC90>16 μg/ml), posaconazole (MIC90>16 μg/ml), isavuconazole (MIC90>16 μg/ml), amphotericin B (MIC90>16 μg/ml), and terbinafine (MIC90>64 μg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC90>8 μg/ml), with the exception of miltefosine showing an MIC90 value of 4 μg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5' upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.