Molecules

BackgroundEpidemiological studies consistently report inverse associations between caffeinated coffee consumption and dementia risk. However, the molecular mechanisms linking coffee-derived phytochemicals to neuroprotection remain only partially understood. ObjectiveTo evaluate, through integrated in silico pharmacology, the relative contribution of adenosine receptor modulation versus direct amyloidogenic enzyme and kinase inhibition in mediating the putative neuroprotective effects of major coffee constituents. MethodsMolecular docking analyses were conducted for caffeine, paraxanthine, chlorogenic acid, trigonelline, cafestol, and kahweol against adenosine A2A and A1 receptors (A2AR, A1R), {beta}-secretase 1 (BACE1), glycogen synthase kinase-3{beta} (GSK-3{beta}), and NLRP3 inflammasome components. Docking was performed using the CB-Dock2 platform. Binding affinities, interaction patterns, and ligand efficiency metrics were assessed. Blood-brain barrier permeability and ADMET properties were predicted using pkCSM. ResultsCaffeine and paraxanthine demonstrated structurally coherent binding within the orthosteric pockets of A2AR and A1R, supported by favorable predicted blood-brain barrier penetration and high unbound fractions. Ligand efficiency analysis identified adenosine receptors as the most pharmacologically plausible targets for small xanthine derivatives. Although larger phytochemicals exhibited stronger absolute docking scores at BACE1, GSK-3{beta}, and NLRP3, predicted pharmacokinetic constraints suggest a small biological effect due to a limited central exposure. ConclusionsThese findings support an adenosine receptor-centered mechanism as the dominant molecular axis linking caffeinated coffee consumption to reduced dementia risk, favoring neuroinflammatory and signaling modulation over direct enzymatic inhibition. Experimental validation is warranted to confirm translational relevance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/723029v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a02629org.highwire.dtl.DTLVardef@129890dorg.highwire.dtl.DTLVardef@1e4c05corg.highwire.dtl.DTLVardef@110ec7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plant-derived bioactive compounds are recognised for their antioxidant potential and benefit for human diseases, including age-related diseases caused by oxidative stress. However, their antioxidant composition and safety profiles remain insufficiently understood. This study integrates phytochemical profiling, antioxidant evaluation, and in vivo toxicological assessment of Warrigal spinach (Tetragonia tetragonioides) and Kensington Pride mango (Mangifera indica). Spinach exhibited greater antioxidant capacity, and higher total phenolic and flavonoid content than mango: TPC (14.2 {+/-} 0.6 mg GAE/g vs 1.30 {+/-} 0.07 mg GAE/g) and TFC (9.61 {+/-} 0.39 mg QE/g vs 0.08 {+/-} 0.0 mg QE/g). LC-ESI-QTOF-MS/MS identified 187 metabolites dominated by flavonoids (53.5%) and phenolic acids (16%), with spinach showing greater chemical diversity. Quantitative analysis revealed higher levels of hydroxycinnamic acids and flavonoid glycosides in spinach, whereas mango contained distinct metabolites, including mangiferin and pyrogallol. Zebrafish embryo / larval assays demonstrated high safety margins, with LC50 values of 478.8 mg/L (spinach) and >480 mg/L (mango). At 480 mg/L spinach displayed developmental abnormalities and malformations. These findings demonstrate that antioxidant capacity is linked to phenolic composition, but does not predict toxicity. Thus, integrated phytochemical and safety evaluation for extracts with complex compound mixtures are critical to identify botanicals suitable for future drug development. HighlightsO_LIWarrigal spinach exhibited >10-fold higher phenolic content and antioxidant capacity than Kensington Pride mango. C_LIO_LILC-ESI-QTOF-MS/MS identified 187 metabolites, with flavonoids as the dominant phytochemical class. C_LIO_LIZebrafish assays confirmed high safety margins, demonstrating no direct correlation between antioxidant capacity and toxicity. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/720960v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c885dborg.highwire.dtl.DTLVardef@cbed43org.highwire.dtl.DTLVardef@460182org.highwire.dtl.DTLVardef@d1ec5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Numerous advances in quantum and computational chemistry over the last decades, well as the development of computer science, allowed utilisation of more precise and complex models, which can be now applied to much bigger systems than in the past. The authors used Gaussian, coupled with theoretical methods, to predict a new way of peptide bond formation, which could have taken place in prebiotic conditions. To better tackle this difficult task, the properties of substrates (glycine-derived radicals) were extensively analysed, using the aforementioned tool - Gaussian, paired with taking resonance and hybridisation into account, to better understand the stereochemistry and the very nature of processes taking place. The result is a series of reactions, which without any sophisticated catalysts and with relatively low energy thresholds ({inverted exclamation}20 kcal/mol) can lead to formation of dipeptides (and further, oligopeptides). The authors also hope, the other predicted properties of the investigated molecules can be of use to any researcher, who would like to utilise them in their experiments. Author summaryOur goal was to investigate a way first peptide bonds in prebiotic conditions could have been formed. This is an extremely important step in research into the beginning of life on Earth. We found a very promising series of reactions, which uses atomic hydrogen as its only catalyst and confirmed our expectations with theoretical calculations, using Gaussian. There are two radicals derived from glycine, which perform major roles in the process, so we investigated their properties with Gaussian and verified that the results are in agreement with our own theoretical considerations. This involved checking for possible geometric isomers and conformers and creating models which could explain their properties. We are well aware that such calculations have limitations and there is no model, which is 100% accurate, so our results should be further confirmed by empirical data in the future. However, we still to be as thorough as possible in how we approached the subject.

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous compounds, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction and inhibition can significantly alter the in vivo fate of xenobiotics i.e., levels of parent chemicals and/or metabolites, and thus toxicity, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. Utilizing available human in vivo pharmaceutical data, a successful validation was previously conducted on the in vitro HepaRG CYP induction test method for measurement of induction of three key human CYP enzymes CYP1A1/1A2, 2B6 and 3A4. However, further validation data was required to demonstrate applicability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals. Here we report on the supplementary validation of the HepaRG CYP enzyme induction test method carried out in two laboratories under the auspices of the EU Horizon2020-funded project "GOLIATH", to expand the chemical applicability domain beyond pharmaceutical chemicals. Successful transfer was demonstrated and reproducibility assessed for the original 10 selected proficiency pharmaceuticals, plus three reference inducers together with six additional non-pharmaceutical augmentation chemicals. The method and chemical selection were found to be reliable and relevant for the routine assessment of human CYP induction. For the augmentation chemicals being proposed as additional proficiency chemicals, the test method achieved a reasonable but not optimum reproducibility. Recommendations are proposed to improve the test methods specificity, reflecting the inherent uncertainty around borderline CYP inducing chemicals. Plain language summaryCytochrome P450 (CYP) enzymes help break down drugs and other chemicals in the body. Their activity can be increased (induced) or decreased (inhibited), which can change how toxic a chemical is and when it is excreted. Because of this, CYP data is important for chemical safety assessments. A laboratory-based method using HepaRG cells was previously validated to measure induction of key CYP enzymes (CYP1A1/1A2, CYP2B6 and CYP3A4) using pharmaceutical chemicals. This study aimed to show that it also works well for industrial and pesticidal chemicals. In the EU funded GOLIATH project, two laboratories tested 10 pharmaceutical and 6 non-pharmaceutical chemicals. The method showed good reliability overall and strong reproducibility for pharmaceuticals. For non-pharmaceutical chemicals, results were acceptable but less consistent. The study concludes that the method is useful for routine testing, but improvements are needed to increase accuracy and better handle chemicals that show weak or borderline CYP induction effects.

Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/733975v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@38d107org.highwire.dtl.DTLVardef@235a13org.highwire.dtl.DTLVardef@ee988aorg.highwire.dtl.DTLVardef@60cb13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

Chemotherapy treatment of colorectal cancers (CRC) using cisplatin (CDDP) encounters problems of drug resistance by the cancer cells and cytotoxicity to normal cells, highlighting the urgent need for joint therapeutical strategies. Selenium-enriched rapeseed extracts exhibit anti-cancer effects but the bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate the extracts from Selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells but no damage on normal cells. HPLC-ICP-MS analysis revealed that methylselenocysteine (MSC) and selenocystine (SeCys2) were the main selenium speciation in WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect when combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), Indole-3-carbinol (I3C), -linolenic acid (ALA) or linoleic acid (LA). We propose that the CDDP-WE combination treatment holds the promise for improving curative efficacy for chemo-refractory CRC patients in the future.

Glioblastoma is a very aggressive brain tumor with few therapeutics options. Type I and II Interferons (IFNs) co-formulation HeberFERON has been used in cancer treatment, with promising results in high grade brain tumors. High throughput techniques in easy-to-handle models have been important to interrogate biomolecules changes, describe mechanisms and find pharmacodynamic biomarkers. This study aims to elucidate the effect of HeberFERON over the cell proteome in comparison to its individual IFNs components. Proteomic changes with HeberFERON in the glioblastoma-derived cell line U-87MG, in comparison with individual IFN-2b and IFN-{gamma}, were studied using a nanoLC instrument EasyLC coupled to Velos Pro mass spectrometer; Maxquant and Perseus were also used. Several enrichment tools, networking analysis and canSAR for drug targets were employed. Translation, RNA processing, mitotic cell cycle, cytoskeleton and chromosome organization, apoptosis, autophagy, DNA repair are enriched to limit cellular growing together with changes in immune response components, supporting HeberFERON as a multitarget treatment. This co-formulation is distinguished at modulating RNA splicing with SMN complex, cytoskeleton organization and microtubule-based movement, nuclear envelope breakdown, DNA conformational changes, and oxidative phosphorylation, with a better drawing of effects over a variety of systems inside the tumoral cell. Together with previous microarray experiment, informative genes and proteins as pharmacodynamic biomarkers for antiproliferative effects showed up (ex. STAT1/2, CENPE, ATRIP, MAP1B, LIMA1, VCP, several ribosomal, spliceosome and proteasomal complexes proteins). This study complements transcriptomic and phosphoproteomic previous experiments in this model and underscore HeberFERON as a glioblastoma therapeutic.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Introduction: Thromboinflammation, which represents the pathological interplay between inflammation and thrombosis, is a leading cause of global mortality. Current therapies are frequently associated with an increased risk of bleeding and do not adequately address the inflammatory component of the disease. The African tree Guibourtia tessmannii represents a promising source of natural anti-inflammatory compounds. This study aimed to synthesize and characterize silver nanoparticles using an aqueous bark extract of G. tessmannii (GT-AgNPs) and to evaluate their anti-inflammatory and anticoagulant properties. Methods: GT-AgNPs were synthesized by reducing silver nitrate with an aqueous extract of G. tessmannii bark. The nanoparticles were comprehensively characterized using UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, and scanning electron microscopy. In vitro anti-inflammatory activity was evaluated through inhibition of bovine serum albumin denaturation, whereas in vivo anti-inflammatory activity was assessed using the carrageenan-induced rat paw edema model. Anticoagulant activity was investigated by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT), corresponding to the intrinsic and extrinsic coagulation pathways, respectively. Results: The synthesis successfully produced GT-AgNPs with an average particle size of approximately 20 nm. Both the aqueous extract and GT-AgNPs exhibited marked anti-inflammatory activity. The nanoparticles achieved 95% inhibition of protein denaturation in vitro and 95% inhibition of carrageenan-induced paw edema in vivo at a dose of 0.4 mg/kg body weight after 5 h. Furthermore, both the extract and GT-AgNPs demonstrated dose-dependent anticoagulant activity. Conclusion: The study demonstrated that GT-AgNPs, synthesized from the bark of G. tessmannii, possess significant anti-inflammatory and anticoagulant properties. These findings highlight the potential of GT-AgNPs as nanotherapeutic candidates for the management of thrombo-inflammatory disorders.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

BackgroundDichapetalin M (Dic M), an active compound extracted from medicinal plants in the Dichapetalum genus, has been previously shown to possess anti-proliferative activity against cancer cell lines. However, the specific mechanism through which it exerts its anticancer effects remains unknown. PurposeThis study focused on elucidating the mechanism of action of dichapetalin M to further explore its potential as a therapeutic agent for resistant and metastatic breast cancer. MethodWe confirmed the Estrogen Receptor (ER) as a target of Dic M, using an in vitro approach. Furthermore, we examined both the apoptotic and migrastatic effects of dichapetalin M by assessing its impact on the expression of key apoptosis-related and cancer cell migration genes. Finally, we evaluated the compounds effect on Multi-drug Resistance Gene MDR1 expression, a gene linked to cancer drug resistance. ResultsOur target validation experiments demonstrated that Dic M exhibited considerably higher cytotoxicity in ER-positive breast cell lines compared to ER-negative cell lines. Furthermore, treatment of MCF-7 cells (which are ER-positive) with Dic M led to a dose-dependent increase in AREG (amphiregulin), a downstream effector of the Estrogen Receptor. Additionally, Dic M inhibited actin polymerization and significantly downregulated genes involved in the turnover of actin monomers. Scratch-wound assay results further demonstrate that Dic M reduces the rate of cell migration, although its impact on EMT-related gene expression was only observed at high doses. Additionally, Dic M treatment in MCF-7 cells resulted in a significant decrease in the expression of pro-apoptotic genes and MDR1 expression. ConclusionsThese findings indicate that Dic M likely interacts with the Estrogen Receptor and employs the apoptotic pathway to exert its cytotoxic and anti-proliferative effects. Dic M exhibits promising potential, such as anti-migrastatic properties and downregulation of a key breast cancer resistance gene, warranting further investigation.

Ovarian and colorectal cancers have the highest incidence and mortality in the world, after breast cancer. Despite the initial response to Pt-drugs or 5-fluorouracil (5-FU), many cancer cells develop resistance to these drugs. For this reason, new therapeutic strategies represent an important medical need, in particular for drugs that are being studied in combination with methods that promote their entry into the cell. Among these strategies, electrochemotherapy (ECT), the combination of drugs with electroporation (EP), a physical method that uses high-frequency electrical pulses to create pores into which chemotherapy drugs can permeate, is gaining interest. In this study, we have evaluated the effect of ECT on the growth of both ovarian (A2780 and A2780/CP) and colorectal (HCT116) cancer cell lines using platinum derivatives (Cisplatin, Carboplatin and Oxaliplatin), as DNA alkylating agents, and human thymidylate synthase (hTS) inhibitors, both traditional (5FU) and novel TS destabilizers (compounds E3 and E7). To this aim, synergism quotient-like analysis to determine whether electroporation gives an advantage in terms of cytotoxicity was applied to the relative IC20 and IC50 concentrations of each drug. Results showed that two of the three Pt-drugs have greater efficacy when combined with EP. 5-FU and the new TS inhibitors E3 and E7 also take advantage of ECT because EP increases drug uptake into the cell, even in resistant cells. In conclusion, ECT appears to be a viable strategy to obviate the problem of resistance in ovarian and colorectal cancers, to deliver compounds inside cells overcoming uptake limits, especially for the low lipophilic compounds whose cytotoxic efficacy is hampered by the obstacle of biological membranes.

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

We have developed a fluorometric assay for detecting reductase activity in biological samples through 4-methoxy-1-naphthalenemethanol (MONOL-41) formation. The enzyme carbonyl reductase 1 (CBR1) and four members of the aldo-keto reductase (AKR) 1 family (AKR1A1, AKR1B1, AKR1B10, AKR1C3) were evaluated for their ability to reduce 4-methoxy-1-naphthaldehyde (MONAL-41). AKR1B1 and CBR1 followed Michaelis-Menten kinetics, whereas AKR1B10, AKR1A1, and AKR1C3 showed substrate inhibition above 10 {micro}M (70 {micro}M for AKR1C3). Among the tested enzymes, AKR1B10 displayed the highest catalytic efficiency in the absence of substrate inhibition. The MONOL-41 assay was compared with the standard NADPH-based method, showing improved sensitivity, robustness, and lower detection limits (0.77 {micro}g/mL vs. 1.49 {micro}g/mL). These results confirm its suitability for monitoring AKR1B10 activity. The assay was then applied to A549 cell extracts, which express multiple reductases. Activity decreased at substrate concentrations above 10 {micro}M, suggesting a predominant role of AKR1B10. Inhibition studies using tolrestat and high MONAL-41 concentrations indicated a limited contribution of CBR1 ([~]7-8%). Considering both catalytic efficiency and expression levels, AKR1B10 appears to be the main contributor to reductase activity in this model. In A549 living cells, MONAL-41 showed no cytotoxicity up to 50 {micro}M and enabled real-time monitoring due to its membrane permeability. However, oxidation by aldehyde dehydrogenases can generate MONOIC-41, which has similar spectral properties but a lower quantum yield, potentially affecting signal interpretation. Overall, this assay represents a sensitive and cost-effective tool for detecting reductase activity and screening inhibitors.

The MDM2-p53 protein-protein interaction is a validated oncology target, yet no food-derived linear peptide has been documented to engage the canonical three-anchor MDM2-p53 interface. We developed a multi-stage computational pipeline (PepVeg) to screen 22 plant and fungal proteomes (337,646 proteins) for MDM2-binding peptides, applying sequential in silico hydrolysis, physicochemical filtering, ESM-2 embedding-based dimensionality reduction, and pharmacophore-driven selection. Twenty-six candidates were evaluated by AlphaFold 3 (AF3) co-folding against MDM2(25-109), yielding 15 binders (iPTM >= 0.75; 58% of evaluated). A 36-peptide benchmark with 29 hard negatives confirmed AF3 discriminative power (Cohens d = 3.41; 95% CI: 1.94-4.88; Hedges g = 3.32; zero overlap). The lead candidate, SPAFESTWDILK -- a tryptic fragment of Zingiber officinale histone deacetylase (UniProt A0A8J5FLH2) -- was evaluated by eight computational assessments: AF3 Server (iPTM 0.83, SD 0.01), Protenix (iPTM 0.923), Chai-1 (iPTM 0.891), EvoEF2 (-55.57 EEU), two GROMACS simulations (no dissociation across two force fields), and two MM-PBSA calculations (-75.30 (SD 4.92) and -55.07 (SD 2.86) kcal/mol). The W8A point mutant produced an iPTM drop of 0.201, closely paralleling the p53 W23A drop of 0.193; we predict W8A substitution will abolish binding. SPAFESTWDILK ranked only #890/2,000 by ESM-2 similarity and was recovered solely through pharmacophore matching, demonstrating that no single pipeline stage alone is sufficient. To our knowledge, this is the first food-database-derived linear peptide with multi-convergent computational evidence supporting engagement of the canonical three-anchor MDM2-p53 interface. Experimental validation by SPR/ITC is warranted.

Room temperature serial crystallography offers advantages over conventional cryo-crystallography, such as simplified crystal handling and the possibility to avoid potential artefacts associated with cryo-trapping. However, to be considered as an alternative for drug discovery, where compound availability may be limited and speed of structure delivery is a key factor, it suffers from several limitations. To address these challenges, we have optimized a serial crystallography workflow for ligand soaking, data collection and data processing, significantly reducing time and reagent consumption to make it a viable option for drug discovery applications, herein exemplified by crystallographic fragment screening. Our approach incorporates the use of dried-in fragment cocktails on fixed target supports, compatible with 96-well plates for crystal soaking, and an efficient data processing pipeline tailored for serial crystallography. To validate our workflow, we conducted an in-crystal fragment screen at room temperature on the protein soluble epoxide hydrolase. The screen comprised 384 compounds and resulted in identification of 40 fragment binders corresponding to a hit rate of 10.4 %. The resulting room-temperature structures are of high quality and reveal opportunities for specific interaction within the highly hydrophobic active site of soluble epoxide hydrolase. Finally, we discuss potential avenues for further workflow optimization, highlighting the future potential of this approach for drug discovery. SynopsisWe have developed a workflow that allowed us to efficiently conduct a fragment screen at room temperature using serial crystallography, of interest for future drug discovery campaigns.

The MYC proto-oncogene encodes a master transcription factor essential for regulating cellular homeostasis, growth, and metabolism. However, its deregulation is involved in up to 70% of human cancers, where it drives uncontrolled cellular proliferation. Structurally, MYC is characterized by an N-terminal transactivation domain and a C-terminal bHLHLZ domain critical for DNA binding and dimerization with MAX. Despite its clinical significance, MYC has remained historically "undruggable" due to its intrinsically disordered structure and the absence of traditional binding pockets. In this study, we combined all-atom Molecular Dynamics and well-tempered Metadynamics simulations to map the conformational landscape of a MYC motif in aqueous ionic solution. The selected energy-minimised structures exhibit stable conformations and drug pockets, providing structural foundations for targetting c-MYC, which is traditionally considered "undruggable". Furthermore, both the electrostatic potential and docking results for the obtained structure and the MYC inhibitor 10074-G5 demonstrated excellent fit, highlighting its potential as a drug target. Remarkably, MYC maintained steady binding to 10074-G5 throughout the 500 ns simulation. These findings provide a previously uncharacterized structural foundation for the design of innovative therapeutic interventions, such as small-molecule inhibitors or peptides aimed at disrupting oncogenic MYC activity.

The blood-sucking ectoparasite Rhipicephalus B. microplus represents one of the greatest economic threats to livestock worldwide. The discovery of novel acaricidal molecules based on environmentally sustainable technologies is therefore required, particularly those suitable for use in dairy and meat-producing livestock without generating contaminant residues. This study aimed to identify and validate plant-derived metabolites with dual activity on acetylcholinesterase and mitochondrial targets as sustainable acaricidal agents against R. microplus, integrating computational screening, biochemical assays and field evaluation. Following in silico screening of 1.300 plant metabolites targeting acetylcholinesterase and mitochondrial function, fourteen compounds were selected for in vitro and in vivo mortality assays using immersion and larval vessel methods. Carvacrol was identified as the most promising metabolite and showed an inhibitory mechanism of action on acetylcholinesterase and mitochondrial complexes I, III, and IV. Its acaricidal efficacy was subsequently confirmed under field conditions using ethanolic and oily formulations (spray and pour-on) applied to cattle (Bos taurus). The spray formulation significantly reduced tick infestation, decreasing tick counts by 20% every 10 days (p < 0.001), whereas the pour-on formulation showed no significant reduction (p = 0.093). These results demonstrate that an integrated discovery pipeline from computational screening to field validation provides a robust strategy for identifying plant-derived acaricidal agents targeting key physiological pathways in R. microplus. HighlightsO_LIStructured and sequential workflow (pipeline) from in silico to Bos taurus C_LIO_LIIdentification and validation of phytometabolites against R. microplus C_LIO_LICarvacrol multitarget activity on acetylcholinesterase and bioenergetic in ticks C_LIO_LIThe spray formulation reduces tick infestation by 20% every 10 days C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/727743v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@11c9931org.highwire.dtl.DTLVardef@8d6088org.highwire.dtl.DTLVardef@1bcc09aorg.highwire.dtl.DTLVardef@108d1d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Avendano, G. (2026) https://BioRender.com/7pm1ffj