Search Results (129)

The sterol demethylation inhibitors (DMIs) are among the most widely used fungicides for controlling black Sigatoka (Mycosphaerella fijiensis) and yellow Sigatoka (Mycosphaerella musicola) in banana plantations in Brazil. Black Sigatoka is considered more important due to causing yield losses of up to 100% in commercial banana crops under predisposing conditions. In contrast, yellow Sigatoka is important due to its widespread occurrence in the country. This study aimed to determine the current sensitivity levels of Mf and Mm populations to DMI fungicides belonging to the chemical group of triazoles. Populations of both species were sampled from commercial banana plantations in Registro, Vale do Ribeira, São Paulo (SP), Ilha Solteira, Northwestern SP, and Janaúba, Northern Minas Gerais, and were further characterized phenotypically. Additionally, allelic variation in the CYP51 gene was analyzed in populations of these pathogens to identify and characterize major mutations and/or mechanisms potentially associated with resistance. Sensitivity to the triazoles propiconazole and tebuconazole was determined by calculating the 50% inhibitory concentration of mycelial growth (EC₅₀) based on dose–response curves ranging from 0 to 5 µg mL⁻¹. Variation in sensitivity to fungicides was evident with all nine Mf isolates showing moderate resistance levels to both propiconazole or tebuconazole, while 11 out of 42 Mm strains tested showed low to moderate levels of resistance to these triazoles. Mutations leading to CYP51 substitutions Y136F, Y461N/H, and Y463D in Mm and Y461D, G462D, and Y463D in Mf were associated with low or moderate levels of resistance to the triazoles. Interestingly, Y461H have not been reported before in Mm or Mf populations, and this alteration was found in combination with V106D and A446S. More complex CYP51 variants and CYP51 promoter inserts associated with upregulation of the target protein were not detected and can explain the absence of highly DMI-resistant strains in Brazil. Disease management programs that minimize reliance on fungicide sprays containing triazoles will be needed to slow down the further evolution and spread of novel CYP51 variants in Mf and Mm populations in Brazil. Full article

Adverse drug–drug interactions (DDIs) often arise from cytochrome P450 (CYP450) enzyme inhibition, which is vital for metabolism. The accurate identification of CYP450 inhibitors is crucial, but current machine learning models struggle to assess the importance of key inputs like ligand SMILES and protein sequences, limiting their biological insights. The proposed study developed LiSENCE, an artificial intelligence (AI) framework to identify CYP450 inhibitors. It aimed to enhance prediction accuracy and provide biological insights, improving drug development and patient safety regarding drug–drug interactions: The innovative LiSENCE AI framework comprised four modules: the Ligand Encoder Network (LEN), Sequence Encoder Network (SEN), classification module, and explainability (XAI) module. The LEN and SEN, as deep learning pipelines, extract high-level features from drug ligand strings and CYP protein target sequences, respectively. These features are combined to improve prediction performance, with the XAI module providing biological interpretations. Data were outsourced from three databases: ligand/compound SMILES strings from the PubChem and ChEMBL databases and protein target sequences from the Protein Data Bank (PDB) for five CYP isoforms: 1A2, 2C9, 2C19, 2D6, and 3A4. The model attains an average accuracy of 89.2%, with the LEN and SEN contributing 70.1% and 63.3%, respectively. The evaluation performance records 97.0% AUC, 97.3% specificity, 92.2% sensitivity, 93.8% precision, 83.3% F1-score, and 87.8% MCC. LiSENCE outperforms baseline models in identifying inhibitors, offering valuable interpretability through heatmap analysis, which aids in advancing drug development research. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is a major cause of cancer-related deaths among women. The Hedgehog (Hh) signaling pathway plays a critical role in tumor development, and targeting this pathway may provide new therapeutic opportunities for TNBC. TPB15 is a novel smoothened inhibitor of the Hh pathway, showing promising tumor reduction and low-toxicity properties in vivo/vitro. This study aims to evaluate TPB15’s protein binding rates, metabolic stability, and metabolism across different species, including mice, rats, dogs, monkeys, and humans. Methods: TPB15 was synthesized, and its pharmacokinetic profile was assessed. Plasma protein binding was determined using ultrafiltration across multiple species. Stability studies were conducted in plasma and liver microsomes from each species. Additionally, metabolic enzymes in human liver microsomes were characterized with selective CYP450 inhibitors, and high-resolution mass spectrometry was employed to identify metabolites. Results: Plasma protein binding of TPB15 was consistent across species, ranging from 81.5% to 82.4% in humans and rats. After 120 min, TPB15 remained stable in plasma, with retention rates of 97.2–98.3%. The elimination half-life (t_1/2) varied from 88 min in monkeys to 630 min in dogs. In human liver microsomes, metabolism was significantly inhibited by sulfaphenazole and ketoconazole, indicating the involvement of CYP3A4 and CYP2C9 enzymes. TPB15 underwent phase I metabolism, producing a major metabolite with a molecular weight of 468.9. Conclusions: TPB15 demonstrates stable pharmacokinetic properties across species, with consistent protein binding and significant variability in half-life. The observed differences in metabolism are primarily attributed to CYP2C9 and CYP3A4, offering valuable insights into its drug development potential. Full article

Background/Objectives: 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive metabolites produced from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively, by CYP450s. These metabolites are unstable and quickly metabolized by auto-oxidation, esterification, β-oxidation, or hydrolysis by soluble epoxide hydrolase (sEH). 17,18-EEQ or 19,20-EDP combined with a potent sEH inhibitor t-TUCB differentially activated brown adipose tissue in diet-induced obesity. In the current study, we investigated whether these n-3 epoxy fatty acids with t-TUCB directly promote brown adipocyte differentiation and their thermogenic capacities. Methods: Murine brown preadipocytes were treated with 17,18-EEQ or 19,20-EDP with t-TUCB during and post differentiation. Brown marker protein expression and mitochondrial respiration were measured. In addition, the activation of PPARγ and suppression of NFκB reporter by 17,18-EEQ or 19,20-EDP alone or with t-TUCB were assessed, and the roles of PPARγ were evaluated with PPARγ knockdown and GW9662. Results: 17,18-EEQ or 19,20-EDP with t-TUCB promoted brown adipogenesis and mitochondrial respiration and uncoupling. Moreover, with t-TUCB, both epoxides improved mitochondrial respiration, but only 17,18-EEQ with t-TUCB significantly increased mitochondrial uncoupling (and heat production) in the differentiated adipocytes. PPARγ may be required for the effects of epoxides on differentiation but not on the thermogenic function post differentiation. Conclusions: The results demonstrate that, with t-TUCB, 17,18-EEQ and 19,20-EDP promote brown adipogenesis and mitochondrial respiration and uncoupling. 17,18-EEQ also promotes thermogenesis in differentiated brown adipocytes. Together, the results suggest thermogenic potentials of tested n-3 epoxides, especially 17,18-EEQ with t-TUCB. Translational studies of these n-3 epoxides on human brown adipocyte differentiation and functions are warranted. Full article

It is well known that the host response to different human and animal coronaviruses infection is regulated by the aryl hydrocarbon receptor, a ligand-activated transcription factor. The present study investigates the expression of the aryl hydrocarbon receptor during bovine coronavirus infection, through in vitro and in silico investigations. The in vitro studies demonstrate that the aryl hydrocarbon receptor and as well as its targets, CYP1A1 and CYP1B1, were significantly activated by bovine coronavirus infection in bovine cells (MDBK). During infection, the pretreatment of cells with non-cytotoxic doses of CH223191, a selective inhibitor of the aryl hydrocarbon receptor, resulted in a significant reduction in virus yield and a downregulation in the viral spike protein expression. These findings occurred in the presence of the inhibition of aryl hydrocarbon receptor signaling. Our results reveal that the bovine coronavirus acts on viral replication, upregulating the aryl hydrocarbon receptor and its downstream target proteins, CYP1A1 and CYP1B1. In addition, following the in silico studies, the three-dimensional structural model of the bovine aryl hydrocarbon receptor in complex with the antagonist CH223191 indicates that the molecular mechanism, by which the PASB and TAD domains of the receptor interact with the inhibitor, is mainly driven by an extensive network of hydrophobic interactions, with a series of hydrogen bonds contributing to stabilizing the complex. Interestingly, bioinformatic analyses revealed that the PASB and TAD domains in the human and bovine aryl hydrocarbon receptor present high similarity at the primary sequence and three-dimensional structure levels. Taken together, these findings represent a fundamental step for the development of innovative drugs targeting AhR as a potential object for CoVs therapy. Full article

Feline coronavirus (FCoV) is an alphacoronavirus (αCoV) that causes moderate or chronic asymptomatic infection in cats. However, in a single infected cat, FCoV can modify its cellular tropism by acquiring the ability to infect macrophages, resulting in the development of feline infectious peritonitis (FIP). In this context, to restrain the impact of FCoV infection, scientific research has focused attention on the development of antiviral therapies involving novel mechanisms of action. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) signaling regulates the host response to different human and animal CoVs. Hence, the mechanism of action of AhR was evaluated upon FCoV infection in Crandell Feline Kidney (CRFK) and in canine fibrosarcoma (A72) cells. Following infection with feline enteric CoV (FECV), strain “München”, a significant activation of AhR and of its target CYP1A1, was observed. The selective AhR antagonist CH223191 provoked a reduction in FCoV replication and in the levels of viral nucleocapsid protein (NP). Furthermore, the effect of the AhR inhibitor on the acidity of lysosomes in infected cells was observed. Our findings indicate that FCoV acts on viral replication that upregulates AhR. CH223191 repressed virus yield through the inhibition of AhR. In this respect, for counteracting FCoV, AhR represents a new target useful for identifying antiviral drugs. Moreover, in the presence of CH223191, the alkalinization of lysosomes in FCoV-infected CRFK cells was detected, outlining their involvement in antiviral activity. Full article

Background: Tuberculosis (TB) is the second leading cause of death from infectious diseases, with 10.6 million cases and 1.3 million deaths. Conventional treatment faces difficulties due to the emergence of resistant strains, such as MDR and XDR-TB. M. tuberculosis uses host cholesterol as an energy source, via the CYP125A1 protein, which catalyses cholesterol oxidation, a process critical for the survival of the bacterium. Methods: This study used computational methods to identify selective inhibitors of the CYP125A1 enzyme. A total of 5968 structure-like compounds from the ASINEX database were evaluated for protein-binding affinity. In addition, docking tests were performed to verify whether the identified compounds could interact with other M. tuberculosis proteins, such as InhA and the human CYP3A4 protein to assess possible off-target effects. Results: The top ten compounds showed a good pharmacological profile and favourable binding energies. Compounds LAS 52160899 and LAS 7298627 served as a basis to search for others with known biological activity, with DB07463 and DB01081 selected as candidates. Conclusions: Potential new inhibitors of the CYP125A1 enzyme were identified. These findings highlight the importance of further research to develop new treatments against M. tuberculosis, especially to combat resistant strains. Full article

Irbesartan improves ventricular remodeling (VR) following myocardial infarction (MI). This study investigates whether irbesartan attenuates VR by reducing aldosterone production in the heart and its underlying mechanisms. MI was induced in male Sprague–Dawley rats through coronary artery ligation. The MI rats were randomly assigned to two groups: one received a vehicle, and the other received 100 mg/kg/day of irbesartan for 5 weeks. Cardiac function and myocardial fibrosis were assessed using echocardiography and Masson’s trichrome staining, respectively. The impact of angiotensin II (Ang II) stimulation on cardiac microvascular endothelial cells (CMECs) from commercial sources was determined using ELISA, real-time PCR, and Western blotting. Irbesartan reduced left ventricular mass index, collagen composition, and aldosterone levels while enhancing cardiac function in MI rats. In vitro, Ang II time-dependently stimulated aldosterone secretion and CYP11B2 mRNA expression in CMECs (p < 0.05). Additionally, Ang II significantly upregulated p-CREB protein levels. However, these effects were abrogated by irbesartan and partially attenuated by CaMK inhibitor KN93 (p < 0.05). In conclusion, our study demonstrated that improvement in VR by irbesartan coincided with reduced CREB phosphorylation in CMECs and reduced aldosterone synthesis in the non-infarcted tissue. These effects may be mediated by blocking the AT1 receptor. Full article

Background: Cytochromes P450 (CYPs) are heme-containing oxidoreductase enzymes with mono-oxygenase activity. Human CYPs catalyze the oxidation of a great variety of chemicals, including xenobiotics, steroid hormones, vitamins, bile acids, procarcinogens, and drugs. Findings: In our review article, we discuss recent data evidencing that the same CYP isoform can be involved in both bioactivation and detoxification reactions and convert the same substrate to different products. Conversely, different CYP isoforms can convert the same substrate, xenobiotic or procarcinogen, into either a more or less toxic product. These phenomena depend on the type of catalyzed reaction, substrate, tissue type, and biological species. Since the CYPs involved in bioactivation (CYP3A4, CYP1A1, CYP2D6, and CYP2C8) are primarily expressed in the liver, their metabolites can induce hepatotoxicity and hepatocarcinogenesis. Additionally, we discuss the role of drugs as CYP substrates, inducers, and inhibitors as well as the implication of nuclear receptors, efflux transporters, and drug–drug interactions in anticancer drug resistance. We highlight the molecular mechanisms underlying the development of hormone-sensitive cancers, including breast, ovarian, endometrial, and prostate cancers. Key players in these mechanisms are the 2,3- and 3,4-catechols of estrogens, which are formed by CYP1A1, CYP1A2, and CYP1B1. The catechols can also produce quinones, leading to the formation of toxic protein and DNA adducts that contribute to cancer progression. However, 2-hydroxy- and 4-hydroxy-estrogens and their O-methylated derivatives along with conjugated metabolites play cancer-protective roles. CYP17A1 and CYP11A1, which are involved in the biosynthesis of testosterone precursors, contribute to prostate cancer, whereas conversion of testosterone to 5α-dihydrotestosterone as well as sustained activation and mutation of the androgen receptor are implicated in metastatic castration-resistant prostate cancer (CRPC). CYP enzymatic activities are influenced by CYP gene polymorphisms, although a significant portion of them have no effects. However, CYP polymorphisms can determine poor, intermediate, rapid, and ultrarapid metabolizer genotypes, which can affect cancer and drug susceptibility. Despite limited statistically significant data, associations between CYP polymorphisms and cancer risk, tumor size, and metastatic status among various populations have been demonstrated. Conclusions: The metabolic diversity and dual character of biological effects of CYPs underlie their implications in, preliminarily, hormone-sensitive cancers. Variations in CYP activities and CYP gene polymorphisms are implicated in the interindividual variability in cancer and drug susceptibility. The development of CYP inhibitors provides options for personalized anticancer therapy. Full article

Mango stem-end rot caused by Lasiodiplodia theobromae is a major postharvest disease in China. Prochloraz is commonly used for disease control in mango orchards and in storage. However, prochloraz resistance has been detected in L. theobromae. This study aimed to explore the underlying mechanisms responsible for prochloraz resistance in L. theobromae. The results show that no point mutation in the target gene LtCYP51 of the prochloraz-resistant L. theobromae strain was detected, but the expression was upregulated significantly. Additionally, the full-length sequences of the cytochrome P450 gene CYP55A3 were successfully amplified and identified from L. theobromae, and the qRT-PCR results confirm that CYP55A3 was significantly upregulated after treatment with prochloraz. The knockout mutant of the CYP55A3 presented significantly lower gene expression levels than the wild-type strain HL02, with a 16.67-fold reduction, but a 1.34-fold reduction in P450 activities and a 1.72-fold increase in the accumulation of prochloraz in the mycelia. Treatment with the P450 enzyme inhibitor significantly synergized with the prochloraz toxicity. The wild-type strain was highly resistant to pyraclostrobin and carbendazim; similarly, the sensitivity of the knockout mutant to pyraclostrobin and carbendazim also notably increased. There was no significant difference between the wild-type strain and the gene-complemented strain. The homology model and molecular docking analysis provide evidence that prochloraz interacts with the protein structure of CYP55A3. These findings suggest that the overexpression of the target gene LtCYP51 and the detoxification gene CYP55A3 were involved in the molecular mechanisms of resistance to prochloraz by L. theobromae. Full article

Binding site flexibility and dynamics strongly affect the ability of proteins to accommodate substrates and inhibitors. The significance of these properties is particularly pronounced for proteins that are inherently flexible, such as cytochrome P450 enzymes (CYPs). While the research on human CYPs provides detailed knowledge on both structural and functional level, such analyses are still lacking for their plant counterparts. This study aims to bridge this gap. We developed a novel computational pipeline consisting of two steps. Firstly, we use molecular dynamics (MD) simulations to capture the full conformational ensemble for a certain plant CYP. Subsequently, we developed and applied a comprehensive methodology to analyze a number of binding site properties—size, flexibility, shape, hydrophobicity, and accessibility—using the fpocket and mdpocket packages on MD-generated trajectories. The workflow was validated on human CYPs 1A2, 2A6, and 3A4, as their binding site characteristics are well known. Not only could we confirm known binding site properties, but we also identified and named previously unseen binding site channels for CYPs 1A2 and 2A6. The pipeline was then applied to plant CYPs, leading to the first categorization of 15 chosen plant CYPs based on their binding site’s (dis)similarities. This study provides a foundation for the largely uncharted fields of plant CYP substrate specificity and facilitates a more precise understanding of their largely unknown specific biological functions. It offers new insights into the structural and functional dynamics of plant CYPs, which may facilitate a more accurate understanding of the fate of agrochemicals or the biotechnological design and exploitation of enzymes with specific functions. Additionally, it serves as a reference for future structural–functional analyses of CYP enzymes across various biological kingdoms. Full article

The regio- and stereoselectivity and the molecular mechanisms of the [3 + 2] cycloaddition reactions between Syn-propanethial S-oxide and selected conjugated nitroalkenes were explored theoretically in the framework of the Molecular Electron Density Theory. It was found that cycloadditions with the participation of nitroethene as well as its methyl- and chloro-substituted analogs can be realized via a single-step mechanism. On the other hand, [3 + 2] cycloaddition reactions between Syn-propanethial S-oxide and 1,1-dinitroethene can proceed according to a stepwise mechanism with a zwitterionic intermediate. Finally, we evaluated the affinity of model reaction products for several target proteins: cytochrome P450 14α-sterol demethylase CYP51 (RSCB Database PDB ID: 1EA1), metalloproteinase gelatinase B (MMP-9; PDB ID: 4XCT), and the inhibitors of cyclooxygenase COX-1 (PDB:3KK6) and COX-2 (PDB:5KIR). Full article

Sterol biosynthesis requires the oxidative removal of two methyl groups from the C-4 position by sterol C-4-demethylase and one methyl group from the C-14 position by sterol C-14-demethylase. In Leishmania donovani, a CYP5122A1 (Cytochrome P450 family 5122A1) protein was recently identified as the bona fide sterol C-4 methyl oxidase catalyzing the initial steps of C-4-demethylation. Besides CYP5122A1, Leishmania parasites possess orthologs to ERG25 (ergosterol pathway gene 25), the canonical sterol C-4 methyl oxidase in Saccharomyces cerevisiae. To determine the contribution of CYP5122A1 and ERG25 in sterol biosynthesis, we assessed the essentiality of these genes in Leishmania major, which causes cutaneous leishmaniasis. Like in L. donovani, CYP5122A1 in L. major could only be deleted in the presence of a complementing episome. Even with strong negative selection, L. major chromosomal CYP5122A1-null mutants retained the complementing episome in both promastigote and amastigote stages, demonstrating its essentiality. In contrast, the L. major ERG25-null mutants were fully viable and replicative in culture and virulent in mice. Deletion and overexpression of ERG25 did not affect the sterol composition, indicating that ERG25 is not required for C-4-demethylation. These findings suggest that CYP5122A1 is the dominant and possibly only sterol C-4 methyl oxidase in Leishmania, and inhibitors of CYP5122A1 may have strong therapeutic potential against multiple Leishmania species. Full article

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α₁-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin–Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4. Full article

Background: Kidney transplantation is followed by immunosuppressive therapy involving calcineurin inhibitors (CNIs) such as cyclosporin A. However, long-term high CNIs doses can lead to vitamin D deficiency, and genetic variations influencing vitamin D levels can indirectly impact the necessary CNIs dosage. This study investigates the impact of genetic variations of vitamin D binding protein (DBP) rs2282679 and CYP2R1 hydroxylase rs10741657 polymorphisms on the cyclosporin A dosage in kidney transplant recipients. Additional polymorphisims of genes that are predicted to influence the pharmacogenetic profile were included. Methods: Gene polymorphisms in 177 kidney transplant recipients were analyzed using data mining techniques, including the Random Forest algorithm and Classification and Regression Trees (C&RT). The relationship between the concentration/dose (C/D) ratio of cyclosporin A and genetic profiles was assessed to determine the predictive value of DBP rs2282679 and CYP2R1 rs10741657 polymorphisms. Results: Polymorphic variants of the DBP (rs2282679) demonstrated a strong predictive value for the cyclosporin A C/D ratio in post-kidney transplantation patients. By contrast, the CYP2R1 polymorphism (rs10741657) did not show predictive significance. Additionally, the immune response genes rs231775 CTLA4 and rs1800896 IL10 were identified as predictors of cyclosporin A response, though these did not result in statistically significant differences. Conclusions:DBP rs2282679 polymorphisms can significantly predict the cyclosporin A C/D ratio, potentially enhancing the accuracy of CNI dosing. This can help identify patient groups at risk of vitamin D deficiency, ultimately improving the management of kidney transplant recipients. Understanding these genetic influences allows for more personalized and effective treatment strategies, contributing to better long-term outcomes for patients. Full article