Development of a natural dihydrofolate reductase receptor binding assay for detecting the sulfonamide synergists group in foodstuff samples.

Human adenovirus (HAdV) is one of the most infectious pathogens that can cause diseases affecting multiple organ systems, posing a significant threat to public health. Currently, there are no specific antiviral therapies available for HAdV infection. In this study, three folate antagonists aminopterin (AMT), pralatrexate (PDX), and methotrexate (MTX) with potent antiviral activity against HAdV infection have been identified through a drug repurposing screening strategy. The three drugs showed broad and potent antiviral efficiency against multiple clinically prevalent HAdV serotypes, including HAdV-B3, HAdV-B7, HAdV-B55, HAdV-C2, and HAdV-C5 with half-maximal effective concentration (EC50) values ranging from 0.31 to 79.74 nM, and PDX further showed similar antiviral efficiency against HAdV-B55 in a 3D lung organoid model (EC50 = 0.51 nM). Mechanistic studies revealed that PDX achieved its antiviral activity through suppression of dihydrofolate reductase (DHFR). These findings support the potential of folate antagonists as repurposed therapeutic agents against HAdV infection and provide a rationale for the development of host-directed antiviral strategies.

Sepiapterin: From sapropterin to next-generation therapy.

Replication stress (RS) and oxidative stress (OS) are two main types of endogenous stress (ES) which, by inducing various forms of DNA damage lead to genome destabilization and disruption of cell division control mechanisms. Methotrexate (MTX) is a compound that inhibits dihydrofolate reductase (DHFR), thereby blocking DNA replication and exhibiting antiproliferative effects. The aim of the study was to investigate how 72-h exposure to 0.75mM MTX on meristematic cells of Vicia faba roots affects the morphology of cell nuclei and mitotic chromosomes, population change of cells in interphase, DNA replication dynamics, cell viability, and hydrogen peroxide (H2O2) production. Furthermore, in order to assess epigenetic changes induced by MTX, associated with DNA damage and the replication process, histone H3 acetylation at lysine 56 (H3K56Ac) and histone H4 acetylation at lysine 5 (H4K5Ac) were examined. It was demonstrated that root meristematic cells treated with MTX exhibited abnormal chromosome structure, sustained DNA biosynthesis, and elevated intracellular H2O2 levels. Immunocytochemical studies revealed an increased number of fluorescent H3K56Ac foci. It was observed that in the case of H4K5Ac, MTX significantly reduced the frequency of cell populations characterized by euchromatin immunofluorescence and limited the occurrence of heterochromatin-type nuclei. Furthermore, in cells treated with MTX, a significant increase in the number of nuclei with marked nucleoli was observed in addition to the gap 1 (G1) phase. In summary, continuous 3-day exposure to low concentrations of MTX induced a cellular response to reactive oxygen species and DNA replication stress conditions.

Folic acid (vitamin B9) plays a vital role in DNA synthesis, repair, and methylation, supporting both normal cellular functions and the proliferation of cancer cells. This review aims to explore the complex interplay between folic acid metabolism, antifolate chemotherapy (particularly methotrexate), and chemotherapy-induced malabsorption, with an emphasis on the need for nutritional interventions. A comprehensive literature review was conducted using databases such as PubMed, Scopus, and Google Scholar to identify studies addressing folic acid deficiency, chemotherapy-related malabsorption, methotrexate toxicity, and nutritional support strategies in cancer patients. Evidence indicates that methotrexate-induced inhibition of dihydrofolate reductase disrupts folate pathways, contributing to folic acid deficiency and malabsorption syndromes. This can lead to clinical complications such as anaemia, mucositis, immune suppression, and reduced treatment tolerance. Nutritional deficiencies further exacerbate drug toxicity and impair patient outcomes. While folic acid supplementation has shown benefit in reducing toxicity, its dosing must be carefully managed to avoid interference with chemotherapeutic efficacy. Folic acid deficiency and malabsorption are significant concerns in cancer therapy involving antifolate drugs. Targeted nutritional strategies, including judicious folic acid supplementation, are essential to improving patient outcomes and minimizing treatment-associated complications. Future clinical approaches should integrate personalized nutritional support alongside chemotherapy regimens.

In this research, we present the successful synthesis of benzimidazolium salts (2a-e) and their corresponding selenium-NHC adducts (3a-e), achieved with satisfactory yields ranging from 75% to 88%, and characterized using spectroscopic techniques, including NMR, FTIR, and mass spectrometry analysis. This work is the first to evaluate the antimicrobial activities and molecular docking studies of these novel compounds, shedding light on the limited enhancement provided by selenium incorporation and providing valuable insights into their enzyme inhibition mechanisms. The antimicrobial and antifungal activities of these compounds were evaluated against various bacterial and yeast strains using disk diffusion and minimum inhibitory concentration (MIC) methods. Benzimidazolium salts, particularly 2d and 2e, demonstrated superior antimicrobial efficacy against Staphylococcus aureus with inhibition zones of 26.73 mm and 18.10 mm and MIC values of 1.56 μg/mL, and against Candida albicans with inhibition zones of 18.10 mm and MIC values of 25 μg/mL and 12.5 μg/mL respectively, significantly outperforming the reference agents Ampicillin (15.33 mm inhibition zone; MIC: 25 μg/mL for S. aureus) and Caspofungin (14.30 mm inhibition zone; MIC: 25 μg/mL for C. albicans). Additionally, compounds 2d and 2e exhibited better activity against Escherichia coli with MIC values of 12.5 μg/mL compared to Ampicillin (25 μg/mL). Conversely, selenium-NHC compounds exhibited moderate to weak activity, with inhibition zones ranging from 8.35 to 11.93 mm and MIC values ranging from 100 to 800 μg/mL, and did not outperform the reference agents. To elucidate the potential mechanism of action, molecular docking studies were performed on compound 2d and its derivative 3d against three key bacterial enzymesDNA gyrase, dihydrofolate reductase (DHFR), and tyrosyl-tRNA synthetase (TyrRS)in addition to the fungal sterol 14-α-demethylase (CYP51) to assess possible antifungal interactions. The results revealed strong binding affinities for both bacterial and fungal enzymes. The compounds interact with crucial amino acids in the enzyme active sites, mirroring the interactions of native ligands. However, the presence of selenium in complex 3d did not enhance its inhibitory activity significantly compared to the salt 2d. The findings highlight the potential of benzimidazolium salts, particularly 2d and 2e, as promising therapeutic agents for microbial infections, with selenium incorporation offering limited enhancement in activity.

This study investigated the phytochemical profile and bioactivity of Hedychium coronarium J. Koenig rhizome methanolic extract and an isolated labdane diterpene. Qualitative and quantitative phytochemical analyses confirmed diverse secondary metabolites. FT-IR analysis indicated diverse functional groups, and GC-MS profiling identified 35 compounds in the crude extract. The extract exhibited concentration-dependent antioxidant (IC50 = 100.59 µg/mL) and antimicrobial activities, with the highest zone of inhibition against Staphylococcus aureus (28.73 ± 0.91 mm). Bioassay-guided isolation yielded (E)-Labda-8(17),12-diene-15,16-dial, structurally confirmed by FT-IR, GC-MS, and 1H/1³C NMR. The isolated compound demonstrated enhanced bioactivity at lower concentrations (antimicrobial: 30.40 ± 0.68 mm; antioxidant IC50 = 43.627 µg/mL). Molecular docking against seven Staphylococcus aureus proteins revealed highest binding affinities of −9.46 kcal/mol against dihydrofolate reductase. This integrated study highlights the therapeutic potential of Zingiberaceae rhizomes, combining phytochemical screening, in-vitro bioassays, and in silico modelling to advance the discovery of plant-based drug leads.

Eichhornia crassipes (water hyacinth), an invasive aquatic plant, has attracted interest as a potential source of pharmacologically active natural products despite its ecological impact. The present study investigated the petroleum ether soluble fraction (PSF) of the methanolic extract of E. crassipes flowers to identify bioactive constituents and evaluate their biological activities. Chromatographic isolation followed by ¹H NMR spectroscopy led to the identification of five compounds: Kaempferol (Compound 1), Luteolin (Compound 2), Stigmasterol (Compound 3), 4-carboxybenzyl alcohol (Compound 4), and 4-methoxybenzaldehyde (Compound 5). Antimicrobial activity was assessed using disc diffusion and minimum inhibitory concentration (MIC) assays, thrombolytic activity through an in vitro clot lysis method, and antidiarrheal activity using the castor oil–induced diarrhea model in mice. Molecular docking and ADMET analyses were performed to explore potential target interactions and pharmacokinetic characteristics. The PSF exhibited moderate antimicrobial activity, with the highest inhibition against Bacillus subtilis (20.33 ± 1.25 mm) and MIC values ranging from 15.6–500 μL/mL. The extract also produced 14% clot lysis in the thrombolytic assay. In vivo antidiarrheal testing showed dose-dependent inhibition, reaching 38.89% reduction in diarrheal count at 600 mg/kg, approaching the activity of loperamide. Molecular docking analysis revealed that the identified compounds exhibited potential binding affinities comparable to standard ligands, suggesting potential interactions with relevant biological targets. The stigmasterol showing the highest interaction toward the κ-opioid receptor (−10.3 kcal/mol), comparable to loperamide (−9.1 kcal/mol). Flavonoids such as kaempferol and luteolin demonstrated notable binding with antimicrobial targets including dihydrofolate reductase (DHFR) and β-ketoacyl-ACP synthase (KAS), as well as thrombolytic target tissue plasminogen activator (TPA). ADMET predictions indicated favorable drug-likeness for the flavonoids but highlighted lipophilicity-related limitations for stigmasterol. Collectively, these findings highlight the PSF of E. crassipes as a promising reservoir of multi-target phytochemicals with potential applications in gastrointestinal, infectious, and thromboembolic disorders.

AI-fragmented derivatives of methotrexate to design effective and safer DHFR inhibitor: A computational breakthrough for ectopic pregnancy therapy.

Lipophilic Pyrimethamine analogs with anti-toxoplasmosis activity.

Treatment of TMP with α-MnO2/CA film coupled with dielectric barrier discharge system: Mechanism and safety assessment.

Antibiotic resistance is a global threat requiring new potential antimicrobial sources. Secondary metabolites from cold-adapted microorganisms may provide unique antimicrobial compounds. Ethyl-acetate extract of Alcaligenes pakistanensis LTP10 from Passu glacier was explored for antimicrobial potential against clinical isolates. Cytotoxicity was determined by Brine shrimp lethality assay. The extract is analyzed by LC-MS/MS and the data is processed by MZmine. Important compounds were evaluated by molecular docking and in silico study. The extract demonstrated activity against clinical isolates Staphylococcus aureus, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Candida albicans with zone of inhibition ranging from 16mm to 24mm, with no killing of nauplii suggested non-cytotoxic nature. LC-MS/MS analysis established presence of important putative antimicrobial metabolites (E)-3-(acetyloxymethyl)-5-(2-formyl-4-hydroxy-5,5,8a-trimethyl-1,4,4a,6,7,8-hexahydronaphthalen-1-yl)pent-2-enoic acid, cyclizidine-F, neovasipyridone-G, paenibacillin-A, and tricholomenyn-A. Molecular docking study of these metabolites by AutoDock Vina against dihydrofolate reductase of S. aureus demonstrated binding affinities from -6.8kcal/mol to -8.5kcal/mol, while against enoyl-ACP reductase of E. coli showed binding affinity values from -6.3kcal/mol to -7.9kcal/mol. In silico analysis predicted considerable absorption, distribution, metabolism, excretion, safety, and druglikeness properties. These results suggest that metabolites from A. pakistanensis LTP10 possess antimicrobial potential and warrant advanced post-docking validation via molecular dynamics, free-energy, and mechanistic analyses for future antibiotic development.

Host DHFRL1 interacts with MtbEis and increases the intracellular survival of Mycobacterium tuberculosis by inhibiting autophagy.

A series of 2,3‐dihydrobenzo[b][1,4]thiazepine derivatives have been synthesized and characterized using IR, NMR, GC‐MS and microanalysis. The compounds were found to exhibit variable activity against Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis and Salmonella typhi with MIC values ranging from 1.88–15 mg/mL. The best activity was observed in compounds 4 and 6 with MIC of 1.875 mg/mL against E. faecalis. Compound 6 was also the most active against S. aureus with MIC of 1.875 mg/mL. The in silico computational docking revealed that the compounds have excellent binding to both multiple targets of E. coli and Staphylococcus aureus dihydrofolate reductase enzymes (PDB ID: 6XG5 and 6P9Z) and good binding to the S. aureus dihydropteroate synthase enzyme (PDB ID: 1ad4). The computational pharmacokinetic study showed that the highly orally bioavailable compounds were not P‐gp (p‐glycoprotein substrate). The compounds were found to be excellent hERG blockers.

Stenotrophomonas maltophilia is an opportunistic pathogen primarily associated with hospital-acquired infections, particularly in individuals who are immunocompromised. S. maltophilia infections pose a significant clinical challenge due to the bacterium's sophisticated intrinsic and acquired mechanisms, which render it naturally multidrug resistant. The management of such infections is thus difficult, as the availability of effective therapeutic agents is limited. Antibiotic therapy options include co-trimoxazole, minocycline, tigecycline, levofloxacin, cefiderocol, and ceftazidime-avibactam. Co-trimoxazole, which comprises a synergistic combination of trimethoprim and sulfamethoxazole, remains the recommended first-line therapy for S. maltophilia infections. In this review, we critically evaluate the current evidence on the efficacy of co-trimoxazole against S. maltophilia. The present global prevalence of co-trimoxazole resistance in S. maltophilia clinical isolates varies from <5% to approximately 44%, raising concerns about its long-term reliability. Resistance to co-trimoxazole arises through several mechanisms. Horizontalgene transfer can introduce sul genes, which encode sulfonamide-insensitive dihydropteroate synthase, or dfrA genes, which encode trimethoprim-insensitive dihydrofolate reductase. Bothenzymes function within the folate biosynthesis pathway, and their expression directly confers co-trimoxazole resistance. S. maltophilia can also acquire co-trimoxazole resistance through genetic mutations. Theoverexpression of efflux systems such as SmeVWX and SmeDEF, contributes to high-level resistanceto co-trimoxazole, often triggered by mutations in the transcriptional regulators. Resistant strains frequently emerge due to improper antimicrobial use, as environmental antibiotic residues can act as selection pressure, facilitating the emergence and persistence of resistant strains. Despite these challenges, co-trimoxazole continues to demonstrate substantial clinical utility. It remains effective in many settings, either as monotherapy or in combination with other antibiotics such as minocycline, tigecycline, cefiderocol, or levofloxacin, and often achieves favorable outcomes.

Pneumocystis jirovecii is an opportunistic fungal pathogen responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients. Antifolate drugs targeting the dihydrofolate reductase (DHFR), including trimethoprim (TMP), remain central to treatment, but studying the effects of mutations in DHFR on resistance to treatment is limited by our inability to culture this organism in vitro or in animal models. We expressed P. jirovecii DHFR (PjDHFR) in Saccharomyces cerevisiae and performed deep mutational scanning (DMS) on this protein to measure the effects of all single amino-acid substitutions on enzyme function and resistance to methotrexate (MTX), a model antifolate which shares structural features with TMP. We integrated experimental results with structural and evolutionary features from multiple biophysical modeling approaches, and by using an interpretable machine-learning framework, we trained a random forest model to classify MTX resistance-conferring mutations in PjDHFR. We then leveraged this framework as a prediction tool to model the effects of mutations on resistance to TMP, which cannot be directly assayed experimentally. Functional measurements from DMS were the strongest contributors to resistance prediction and generally outperformed purely computational features. Resistance-conferring mutations were constrained by function, revealing a functional–resistance trade-off within this essential protein. Feature contribution analyses highlighted key predictors such as distance to ligand, flexibility, stability, and functional trade-off as determinants of resistance. When extrapolated to TMP, the model identified candidate resistance mutations consistent with known biochemical constraints of DHFR. We demonstrate how experimentally measured functional landscapes can be combined with biophysical modeling to help understand and predict antifolate resistance in an unculturable fungal pathogen. Our results provide biological insight into the constraints affecting the evolution of resistance in PjDHFR, and support that resistance arises from mutations altering drug interactions while preserving function. We illustrate how DMS data can enable generalizable, mechanistically interpretable models of drug resistance across structurally related antifolates.

The dynamic and tissue-specific nature of protein secretion underlies a wide range of physiological and pathological processes, yet tools for profiling the in vivo secretome with high spatial and temporal resolution remain limited. Here, we present STePTag (Spatio-Temporal Protein Tagging), a conditional proximity labeling (PL) system for profiling secreted proteins in live animals. STePTag integrates the rapid labeling kinetics of the PL enzyme TurboID with a destabilized dihydrofolate reductase (DHFR) domain, enabling tight post-translational control of labeling activity through the small-molecule stabilizer trimethoprim (TMP). Targeting STePTag to the endoplasmic reticulum (ER) confines labeling to the secretory pathway and enables robust labeling within 10min of TMP administration. Furthermore, we design tissue-specific lipid nanoparticles (tsLNPs) to enable programmable, in vivo delivery of STePTag to the mouse liver. Using this approach, we identified 93 liver-derived secretory proteins under physiological conditions and uncovered 40 dynamically regulated proteins in a model of acetaminophen-induced acute liver injury (ALI), including Aldh1a1, which we functionally validated as a protective factor in ALI. Together, STePTag provides a versatile platform for spatiotemporally resolved secretome profiling, enabling the discovery of context-dependent biomarkers and tissue-derived signaling molecules in native physiological environments.

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of <Escherichia coli> dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligand protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct <cis>-to-<trans> transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pKa errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

Plant extract-mediated synthesis of metal oxide nanoparticles (MO NPs) offers a sustainable route for biomedical applications due to their biocompatibility and eco-friendly production. In this work, copper oxide (CuO), zinc oxide (ZnO), and novel CuO-ZnO nanocomposites were successfully synthesized using phytochemicals from Pistacia eurycarpa leaves. The nanostructures were comprehensively characterized by XRD, UV-Vis, SEM, EDX, and FTIR analyses, confirming crystalline phases with nanoscale spherical and rod-like morphologies. The optical band gaps (1.6-2.74eV) indicated suitability for diverse functional applications. The antibacterial activity of the synthesized nanomaterials was evaluated against Escherichia coli and Staphylococcus aureus, revealing significant inhibitory and bactericidal effects. Complementary density functional theory calculations provided insights into their electronic properties, while molecular docking studies demonstrated strong binding interactions with bacterial target proteins Staphylococcus aureus dihydrofolate reductase (2W9H), Escherichia coli DNA gyrase B (6F86) and Estrogen receptor alpha (ERα) (5GS4), supporting their potential bioactivity. Overall, this combined experimental and computational study highlights the promising antibacterial potential of green-synthesized CuO, ZnO, and CuO-ZnO nanostructures and underscores their applicability as eco-friendly agents for biomedical applications.

The rapid emergence of antimicrobial resistance (AMR) has become a global threat, limiting the clinical effectiveness of the drugs against microbial infections. As Methicillin-resistant Staphylococcus aureus (MRSA) and Extensively drug-resistant (XDR) Escherichia coli continue to evolve rapidly, it is critical to develop novel therapeutic agents and structurally unique antibacterial medicines. The selective bacterial folate biosynthesis pathway, which includes dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), represents an attractive therapeutic target; however, the alarming rise of microbial sulfonamides necessitates the development of novel inhibitors. Sulphonamide antibiotics are analogues of p-aminobenzoic acid (PABA), which serves as a substrate for folate biosynthesis. Taking this into account, we developed novel PABA-imidazole analogues to combat antimicrobial resistance. In this study we designed and synthesized seven novel PABA analogues and characterized them by various spectroscopy techniques. These synthesized PABA analogues show comparatively better efficiency against both the Gram-positive (S. aureus and S. pyogenes) and Gram-negative (E. coli and P. aeruginosa) strains. Furthermore, they also show potential activity against MRSA and XDR E. coli. The DHPS/DHFR enzyme assay demonstrated that the SB2 compound suppresses the sulfamethoxazole (SUL) in DHPS inhibition, and the SB5 compound shows comparatively increased DHFR inhibitory activity compared to the trimethoprim (TMP). These results have been validated by molecular dynamics simulations and free energy analyses. In addition, the lead compounds also showed strong antioxidant activity, low cytotoxicity, and good DNA nicking behaviour, which supports their therapeutic potential.