Frontiers in Chemistry

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N- and O-glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a true adipocyte profile, and revealed that bisecting GlcNAc type N-glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N-glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de-N-glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de-N-glycosylated CD36 revealed a 41% increase when N-glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de-N-glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/722121v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@189a786org.highwire.dtl.DTLVardef@5563b8org.highwire.dtl.DTLVardef@5cb5borg.highwire.dtl.DTLVardef@69e11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

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

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

Licorice (Glycyrrhiza) is a medicinal plant widely used in approximately 70% of traditional Japanese Kampo formulations and is known to produce a wide array of specialized metabolites with diverse pharmacological properties. Although hundreds of metabolites have been reported, the overall chemical diversity of Glycyrrhiza remains poorly characterized. Here, using mass spectrometry data obtained from fully 13C-labeled leaves and roots of Glycyrrhiza uralensis and Glycyrrhiza glabra, we determined the carbon number, followed by molecular formula and substructure prediction in combination with MS/MS similarity-based molecular networking. After excluding redundant ions, including isotopic peaks, adducts, and in-source fragments, we extracted 3,060 unique metabolite features with assigned carbon numbers. Among these, substructure information was assigned to 1,015 features (33%) across the four plant tissues, revealing the tissue-specific metabolome profiles. Furthermore, we discovered five previously unreported alkaloids, homopipecolic acid-conjugated flavonoids, in the roots of G. uralensis and G. glabra, and Glycine max, another member of the Fabaceae family. Two of these structures were validated using nuclear magnetic resonance spectroscopy. We further proposed a biosynthetic route involving a spontaneous reaction between 1-piperideine and malonyl glycoside substrates and confirmed the formation of the conjugated product using authentic standards.

Prodigiosin is a red pigment produced by various bacteria, including Serratia marcescens. Despite its wide and promising range of biological activities, the large-scale production of prodigiosin is currently limited by its high cost and low yields. Here we propose and optimize an innovative, low-cost, peanut-based solid culture medium that enhances the yield of prodigiosin produced by Serratia marcescens. Colorimetric assays revealed that peanut significantly stimulates prodigiosin synthesis. Further HPLC-MS analysis allowed us to unambiguously identify prodigiosin and shows that our medium specifically improves the yield of prodigiosin. Overall, our innovative culture medium could help lower prodigiosin production costs and, ultimately, open new industrial applications.

Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 {micro}L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples from neonatal intensive care units. For CS quantification, the CS is first enzymatically depolymerised using chondroitinase ABC to release repeating disaccharide units. Matrix complexity is reduced via two rounds of acetonitrile-based protein and lipid precipitation. Disaccharides are separated by hydrophilic interaction liquid chromatography and detected using a Triple Quadrupole Mass Spectrometer, providing robust sensitivity for all CS disaccharides. Method development and validation were performed using pooled mature human milk from term infants. This workflow facilitates detection of all CS disaccharides, with low but reproducible recoveries for total CS. Low- and high-level spike recoveries were 41.3% (RSDr 7.5%, RSDiR 15.9%) and 43.7% (RSDr 24.4%, RSDiR 27.9%), respectively. Despite modest absolute accuracy, precision remained sufficient to make relative comparison of CS concentrations between samples. This method expands the analytical toolkit for human milk glycomics, enabling same day preparation and CS profiling from sample volumes that are 200 times smaller than prior work, supporting future investigations into GAG-mediated functions in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723732v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@176dffborg.highwire.dtl.DTLVardef@16ae4ccorg.highwire.dtl.DTLVardef@d333c2org.highwire.dtl.DTLVardef@1eb3216_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Schematic of sample preparation protocol 25 L of human milk is combined with lyase enzymes and TRIS buffer containing the internal standard prior to incubation. Samples then undergo multiple rounds of centrifugation and refrigeration before analysis via LC-MS/MS. Made using BioRender.com. Glycan nomenclature following Varki et al., 2015. C_FIG

Punicalagin, an ellagic acid polyphenol from pomegranate, has been proposed as an antagonist of protein disulfide isomerase (PDI) and endoplasmic reticulum resident protein 57 (ERp57), thiol oxidoreductases that regulate protein folding and extracellular thrombotic signaling. Here, biochemical oxidase and reductase assays on PDI show that punicalagin inhibits both activities with micromolar potency, thereby extending earlier work that described only disulfide reductase inhibition. In parallel, thiol labeling of catalytic cysteines revealed no change in the redox state, supporting a noncovalent, allosteric of inhibition. Molecular docking and molecular dynamics simulations showed that punicalagin binds stably and preferentially to defined sites on the Nterminal domains of PDI through extensive hydrogen bonding and van der Waals contacts, which is an alternative binding mode to previously reported C-terminal binding. Finally, artificial intelligence-driven network analysis identified PDI as a high-confidence target of punicalagin and related galloylated polyphenols, alongside additional signaling proteins. Together, these findings provide further mechanistic framework for punicalagin-mediated antagonism of PDI and highlight galloylated polyphenols as promising scaffolds for protein disulfide isomerase-targeted therapeutics. HighlightsO_LIPunicalagin, a galloylated polyphenol, antagonizes not only the reductase activity but also the oxidase activity of protein disulfide isomerase C_LIO_LIProtein disulfide isomerase inhibition by punicalagin is through N-terminal binding C_LIO_LIPunicalagin inhibits conformationally rather than catalytic cysteine modification C_LIO_LIArtificial intelligence network analysis reveals pathway inhibition by punicalagin C_LI

While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADCs) are used to treat cancer patients with HER2-amplified tumors, there is a need to improve the efficacy through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2) regulates the internalization of epidermal growth factor receptor (EGFR), leading us to investigate FLOT2 effects on HER2 internalization. Higher FLOT2 expression in nine HER2 amplified cell lines correlated with a higher T-DM1 IC50 in vitro, and breast cancer patients with high FLOT2 expression had worse survival when receiving either T-DXd (16.2 months (m) vs 18.3 m, p=0.04) or T-DM1 (38.0 m vs 41.3 m, p=0.1) in real-world Caris Life Sciences data. FLOT2 interacts with HER2 and positively regulates HER2 activation and downstream signaling, while FLOT2 knockdown reduces the viability of HER2 amplified cancer cells. FLOT2 knockdown results in increased HER2 internalization upon binding of T-DM1, mediated by ubiquitination by the Cbl ubiquitin ligases. We investigated the effects of various small molecules and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances T-DM1 internalization and cytotoxicity. In conclusion, FLOT2 regulates the internalization and cytotoxicity of T-DM1 mediated by Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of T-DM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the HER2-targeting ADC.

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Fungal contamination of food with yeast and moulds is associated with major economic losses due to spoilage and also poses health risks in the form of mycotoxin production. The strain Pantoea agglomerans APC 4211 isolated from leaves of Ilex aquifolium (holly tree) has broad spectrum antifungal activity against a variety of food spoilage fungi. Genomic analysis of the strain confirmed the presence of biosynthetic gene clusters potentially encoding for the enzymatic machinery required for the production of the antifungal lipopeptide herbicolin A. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the cell-free supernatant (CFS) confirmed the presence of molecular masses corresponding to herbicolin A (1300.8 Da), and herbicolin B (1138 Da). Purified herbicolin A has desirable properties for biotechnological applications, including potent antifungal activity against a range of spoilage fungi, thermal stability and resistance to proteases. Herbicolin A has low cytotoxicity against epithelial cell lines and has minimum inhibitory concentrations (MICs) lower than those of some commercial antifungal drugs (0.2 - 2.5 {micro}g/ml). In a model dairy system (10% skim milk), herbicolin A demonstrated excellent solubility and stability, effectively eliminating Aspergillus niger and Penicillium notatum at a concentration of 5 {micro}g/mL. In conclusion, herbicolin A is a potent, naturally occurring antifungal agent with the potential to be applied as a biopreservative in food systems, providing a safe, clean-label, and efficient compound for synthetic preservatives replacement. HighlightsO_LIHerbicolin A has a strong potential as a natural preservative for food protection C_LIO_LIHerbicolin A shows lower MICs than several antifungal agents C_LIO_LIHerbicolin A is stable under heat and resistant to proteolytic degradation C_LIO_LIHerbicolin A has strong solubility and stability in a model dairy system C_LIO_LIHerbicolin A indicates low cytotoxicity against epithelial cell lines C_LI Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Human N-glycoproteins constitute a market worth hundreds of billions of dollars. However, their production in yeast is often limited by misfolding and subsequent degradation, largely due to differences in N-glycan-dependent protein quality control (QC) systems between humans and yeast. Notably, yeast lacks the UGGT-mediated reglucosylation-refolding cycle that rescues misfolded glycoproteins, and its degradation pathway involves fewer rate-limiting steps. To address this, we engineer the glycoprotein QC system in Kluyveromyces marxianus, a promising host for protein production, by introducing key human components and modifying native pathways. Expression of human UGGT1 or UGGT2 enhances the soluble and secretory production of glycoproteins in an activity-dependent manner. This effect is further improved by co-expression of the UGGT cochaperone SEP15 and by reducing native glucosidase II trimming activity. In addition, introduction of human EDEM2, a rate-limiting enzyme in glycoprotein degradation, delays ER-associated degradation and increases secretion. Integration of these engineering strategies substantially enhances the production of several high-value human-derived glycoprotein therapeutics, including etanercept, dulaglutide, and abatacept, with up to a [~]12-fold increase. These findings demonstrate that engineering a human-like glycoprotein QC network in yeast is an effective strategy to improve glycoprotein folding and secretion.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

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.

BackgroundHomozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent genetic alteration in cancer. MTAP, which creates adenine from 5-methylthioadenosine (MTA), is constitutively expressed in all tissues throughout the body. Previously, we described a novel strategy to specifically target MTAP-deleted cancer cells by combining the antipurine prodrug 2-fluoroadenine (2FA) with MTA. In vitro, this combination efficiently killed MTAP- cancer cells, but in vivo the combination was much less effective in vivo. Here, we explored the role of xanthine oxidase (XO) in this process. Materials and MethodsVarious combinations of 2FA, MTA, and the xanthine oxidase inhibitor febuxostat (FX) were tested in various cancer cell lines grown in vitro and in mice. LC-MS/MS was used to examine the levels and ratio of intracellular 2-FA-containing nucleotides compared to adenine-containing nucleotides. Results and conclusionsThe treatment of cells with 2FA+MTA in vitro resulted in much higher 2FANP/ANP ratios than the same treatment in vivo. The addition of XO to culture media in vitro effectively abolished the killing by 2FA, and this effect was fully reversed by the addition of febuxostat (FX), a xanthine oxidase inhibitor. In vivo, the addition of FX to 2FA results in increased cell killing and toxicity and a 1000% increase in the amount of 2FA converted to 2-FA-monophosphate (2FAMP). Xenograft studies using MTAP- HT1080 and MiaPaCa-2 cell lines have shown that a 2FA/MTA/FX cocktail can cause tumor regression in vivo. These studies suggest that the combination of 2FA/MTA/FX should be explored as a treatment for MTAP- cancer.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.