Frontiers in Chemistry

Multiple-drug resistant (MDR) Staphylococcus aureus strains (like methicillin-resistant S. aureus or MRSA) uses an arsenal of antioxidant enzymes to mitigate host-induced oxidative stress. Among them the non-canonical Staphylococcal glutathione peroxidase (SaGpx) plays a crucial role in bacterial redox homeostasis by reducing peroxides via thioredoxin-dependent pathways. Thus, enabling oxidative stress mitigation during host infection. Despite its importance in S. aureus, its role in bacterial pathogenesis remains unexplored. This study aimed to elucidate the possible role of SaGpx in Staphylococcal virulence. First, we determined the high-resolution crystal structure of SaGpx (at 1.65 [A] resolution) using X-ray crystallography. Guided by the catalytic cleft architecture of SaGpx, small-molecule based inhibitors were then rationally designed and synthesized. These inhibitors exhibited good binding affinity to SaGpx and complete enzymatic blockade. These inhibitors exhibited potent anti-S. aureus activity (MICs 6.25-31.25 M) along with no cytotoxicity in L929 fibroblast wound-healing assays. Furthermore, the in vivo antibacterial ability of these inhibitors was evaluated using S. aureus-infected skin wound mouse model, where these compounds show potent antibacterial and wound healing ability supported by subsequent histological as well as immunohistochemical analysis. These findings suggest SaGpx as a possible virulence determinant in S. aureus and position these synthesized inhibitors as promising antivirulence therapeutics. HighlightsO_LIThe high-resolution crystal structure of Staphylococcal glutathione peroxidase is solved. C_LIO_LIBased on the SaGpx catalytic site, ,{beta}-unsaturated ketoesters derivatives are synthesized. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives inhibit SaGpx activity and binds the protein at M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vitro antibacterial activity against S. aureus at low M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vivo antibacterial and wound healing ability S. aureus-infected skin wound mouse model. C_LI

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

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.

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

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

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

The catalytic rate constant (kcat) for product formation is considered a turnover number. Therefore, it is often mistakenly believed that kcat equals the turnover number and the number of substrate molecules changed per unit of time. Therefore, the aim of this study is to show that the rate constant for product synthesis and release is not always the same as the rate constant [Formula] for substrate utilization. To determine the precise substrate concentration at which these two rate constants are identical, it is appropriate to derive equations that allow the computation of [Formula]. In the end, the study will provide the most likely concentration of enzymes that can guarantee minimal or no recycling. An analysis of the literature on invertase (EC 3.2.1.26) and the Bernfeld method of generating Michaelian kinetic parameters for human salivary alpha-amylase (HSAA, EC 3.2.1.1) revealed that all kinetic parameters except [Formula] increased with substrate concentration. Meanwhile, the values for invertase decreased from 0.0697 to 0.0361/min, and the values for HSAA decreased from 5,802.4687 to 3,213.0124/min. The magnitude of [Formula] for each substrate concentration ([ST]) is not always equal, except when [ST] is determined post-assay by computation or extrapolation. The lower [ST] at which [Formula] and kcat for [HSAA] are equal is 3.667540128 g/L (5.682584642 M), which is similar to the molarity of HSAA (5.6101967709 M). The kcat for HSAA was 11,930.9885/min. Future assays should aim to generate large amounts of data for a robust statistical analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/728646v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@c49b65org.highwire.dtl.DTLVardef@1b60655org.highwire.dtl.DTLVardef@159ba67org.highwire.dtl.DTLVardef@1dce0af_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Large-conductance calcium-activated potassium (BKCa) channels are ubiquitously expressed in mammalian cells and regulate electrical activity, intracellular calcium signaling, and cell survival. Although BKCa dysfunction has been linked to multiple diseases, the number of selective channel modulators is limited. In this study, we characterize dibenzoylmethane (DBM), a plant-derived compound isolated from Hottonia palustris, as a novel inhibitor of BKCa channel activity in both plasma membrane and mitochondrial BKCa. Electrophysiological recordings revealed that DBM lowers the open probability of BKCa channels in a concentration-dependent fashion and markedly reduces mean open time, leading to a pronounced flickering behavior - hallmarks of pore-targeted blockade. Competition experiments demonstrated that DBM antagonizes the effect of paxilline, a high-affinity pore-binding inhibitor, suggesting overlapping binding sites. Molecular dynamics simulations further supported this hypothesis, showing that several DBM molecules can block the pore by employing {pi}-{pi} interactions with each other and pore residues. On top of the pore, the carbonyl groups of DBM block the nearest potassium ion in the selectivity filter. The presence of DBM induces the removal of water molecules from the pore. To assess the structural requirements for activity, we tested three DBM analogs: phenyl-1,3-butanedione (PBD), trans-chalcone (T-Ch), and (E)-1,3-diphenylprop-2-en-1-ol (DPE). T-Ch and DPE inhibited BKCa channels with comparable efficacy to DBM, whereas PBD was significantly less potent. These results indicate that diphenyl substitution and structural rigidity are critical determinants of inhibitory activity. Our findings position DBM and its analogs as promising chemical scaffolds for the development of selective BKCa channel modulators with potential pharmacological applications.

Antimycin A (AA) is widely used as an inhibitor of the mitochondrial respiratory chain, targeting the Qi site of cytochrome bc1 (complex III). In photosynthetic organisms, AA is also well known to inhibit the photosynthetic PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron flow around photosystem I (CEF-PSI). Although AA is frequently used as a specific inhibitor of PGR5-dependent CEF-PSI in photosynthetic reactions, we recently clarified that some of the major components of AA, which is typically a mixture of closely related compounds, also exert direct inhibitory effects on photosystem II (PSII). Nevertheless, the binding site and binding mode of AA in PSII remain largely unexplored. Structurally, AA consists of a salicylic acid moiety connected via an amide bond to a hydrophobic dilactone ring moiety. To identify important structural factors of AA for exhibiting inhibitory effects on PSII (assessed by QA- reoxidation measurements), we here investigated the relationship between structure and inhibitory potency using 38 AA-like compounds (AALCs), including commercial compounds and a series of synthetic AA analogs. Some AALCs exhibited substantially stronger impacts on PSII than natural AA. High acidity of the phenolic OH and the presence of a free amide NH of the salicylamide moiety were critical for the effects on PSII. In contrast, while the dilactone ring moiety also affected the inhibitory activity, this was replaceable with certain hydrophobic structures. Based on our results, together with the known structure-activity relationship and binding mode of AA in complex III, we propose tentative binding models for AA in PSII. HighlightsO_LIStructure-activity relationship of AA-like compounds on PSII is examined C_LIO_LISeveral AA-like compounds more potent than AA against PSII are identified C_LIO_LIPhenolic OH acidity and free amide NH of salicylamide moiety are key for AA effects C_LIO_LIThe dilactone ring moiety is replaceable with certain hydrophobic structures C_LIO_LITentative binding models for AA in PSII are proposed C_LI

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.

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.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Raffinose family oligosaccharides (RFOs) are abundant in side streams from food and feed production from legumes, and the transition to plant-based diets increases the volume of such side streams. RFOs in the diet tend to have negative impacts on the consumers gut (e.g., nausea, bloating, diarrhoea), and in many ways, RFOs are comparable to lactose as a side stream from the dairy industry and symptoms associated with lactose intolerance. On the contrary, galactooligosaccharides (GOS) are recognized as prebiotics, and in this study we used a {beta}-galactosidase from Niallia circulans to produce potential prebiotics from RFOs (acceptors) and lactose (donor), which we hypothesized to have a lower fermentability than unmodified RFOs. The transglycosylation reactions resulted in RFO-based -{beta}-GOS, with NMR characterization showing ({beta}1-4) galactosylations on the non-reducing galactose end of RFOs as the major product. In reactions with RFOs, the characteristics were comparable to reactions with lactose alone and the new -{beta}-GOS products made up the largest fraction (by weight). A screening of 11 relevant gut and food microbe strains revealed that the gut commensal Bacteroides ovatus metabolised these modified oligosaccharides for growth whereas other strains grew only after adaption and others did not use them at all. This implies that mixed-linkage -{beta}-GOS are less fermentable by some microbes compared to raffinose, while other (beneficial) bacteria can still ferment them. The enzymatic synthesis established here is an interesting approach to upgrade abundant food side streams towards new prebiotics in a world where functional foods and food waste reduction receive increasing attention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/731070v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@18e0e62org.highwire.dtl.DTLVardef@1525b4borg.highwire.dtl.DTLVardef@1e7be88org.highwire.dtl.DTLVardef@18df278_HPS_FORMAT_FIGEXP M_FIG 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

Chagas disease, caused by Trypanosoma cruzi, demands novel therapeutic strategies that overcome the toxicity and limited efficacy of current treatments. To address this need, herein we report an integrative, target-centric strategy that combines parasite proteome mining, structural modeling, and experimental validation. Functional enrichment and druggability analyses identified phosphopyruvate hydratase (PPH) as a promising candidate due to its essential metabolic role and limited similarity to human homologs. Notably, proteome mining revealed the presence and conservation of PPH across kinetoplastid parasites, including Leishmania donovani, supporting its evaluation beyond T. cruzi. For the selected PPH sequences, AlphaFold-derived three-dimensional models underwent extensive molecular dynamics refinement, yielding stable conformational ensembles suitable for structure-based studies. Using this validated model, virtual screening of the Latin American Natural Products Database - LANaPDB - identified aptosimon as a top-ranked compound candidate. Molecular dynamics simulations further showed ligand-dependent binding behavior, suggesting alternative binding modes distinct from the canonical substrate configuration. In vitro assays demonstrated consistent antiparasitic activity against intracellular T. cruzi amastigotes (IC = 3.52 {+/-} 0.023 {micro}g/mL) and Leishmania donovani promastigotes (IC = 13.06 {+/-} 0.018 {micro}g/mL), supporting the biological relevance of the aptosimon-related lignan chemotype, hinokinin, across two kinetoplastid parasite models. Together, these results support PPH as a structurally tractable and biologically relevant candidate target, while identifying an aptosimon-related lignan chemotype, represented experimentally by hinokinin, as a cross-species antiparasitic scaffold that warrants further biochemical target-validation studies.

Vodobatinib (VBN) is a weakly basic (pKa {approx} 2.3), anticancer treatment with poor enteric solubility and low oral bioavailability. This study demonstrates how an emerging polymeric amorphization technique, slurry conversion, can yield amorphous drug-polymer salts with enhanced dissolution rates. The technique had not previously been applied to a weakly basic drug, so design rules for this class of active were unknown. Two acidic polymers, poly(styrene sulfonic acid) (PSSA) and poly(acrylic acid) (PAA), were individually evaluated for salt formation with VBN. Formulation involved blending the drug and polymer in a 1:2 (v/v) ratio of a protic liquid to solvent and a 1:9 (w/w) ratio of solid to solvent. Design rules for effective combinations of solvents and protic liquids were developed and optimized to thread the needle between dissolution of all species and acid-base interactions, both of which were required to form amorphous salts. Drug loadings of 10%, 20%, and 40% by mass were tested. X-ray photoelectron spectroscopy was employed to evaluate protonation of the quinoline nitrogen atoms on VBN, a key indicator of successful salt formation. Powder X-ray diffraction was used to confirm that the resulting slurry contained amorphous VBN, and 1H NMR spectroscopy indicated residual solvent remained after drying, which remains an area for improvement. In dissolution kinetics tests in FeSSIF, the lead drug-polymer salt formulation achieved a concentration of dissolved VBN up to 140 {micro}g/mL, an improvement of >35-fold compared to <4 {micro}g/mL (LLD) for crystalline VBN. These results demonstrate that slurry conversion is a viable polymeric amorphization technique even for weakly basic drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/734800v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1812ceforg.highwire.dtl.DTLVardef@1ad06dcorg.highwire.dtl.DTLVardef@9d8bb7org.highwire.dtl.DTLVardef@13fcbe8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Isoflavone hydroxylases (IFHs, CYP81E) convert isoflavone aglycones into their respective hydroxylated intermediates, which direct legume isoflavones into specialized defense pathways. In soybean, their functions have been studied mostly in the context of the daidzein-derived glyceollin biosynthesis. Here we combine metabolomics-guided feature mining, phylogenetic analysis, heterologous enzymology, structural elucidation, and in planta metabolite validation to determine the functional landscape of the soybean IFH family. Analysis of a soybean isoflavonoid-enriched metabolomic dataset revealed unidentified hydroxyisoflavone features that co-accumulated with glyceollins, indicating branch chemistry that is not well-recognized. The systematic characterization of the repertoire of soybean CYP81E has demonstrated that 9 out of 11 GmIFHs are catalytically active and collectively span both 2'- and 3'- hydroxylation of the major soybean isoflavone aglycones. Among them, GmIFH9A showed broad substrate scope and regioselectivity, yielding canonical and previously unknown hydroxylated isoflavone products. NMR and LC-MS/MS were used to identify and validate the hydroxylated isoflavone products as 2'-hydroxyglycitein and 2'-hydroxyformononetin, whose presence was also confirmed in soybean roots, thus confirming two of the hidden soybean isoflavonoid network metabolites. Kinetic studies also indicated that, although the majority of GmIFHs prefer daidzein and genistein as substrates, a few isoforms are active towards methoxylated isoflavones as well, indicating functional divergence in this expanded family. Our findings collectively redefine soybean IFHs as a multi-functional enzyme module that expands the hydroxyisoflavone chemical space and reveals new biosynthetic entry points beyond canonical glyceollin pathway.

Pyrrolizidine alkaloids (PA) are well-known and widespread natural products from plants, which have also been identified in several different bacteria. In the latter case, the core structure is constructed by a non-ribosomal peptide synthetase (NRPS), which then undergoes oxidative ring contraction catalyzed by a Baeyer-Villiger monooxygenase. By deploying various NRPS engineering strategies, we have successfully generated five novel peptides carrying the unusual PA moiety at their C-terminus. Nonetheless, efforts to obtain a larger library of PAs were unsuccessful. Combined computational modelling and docking experiments suggest that this failure stems from the strict specificity of the thioesterase (TE) domain at the end of the NRPS, which discriminates against peptides carrying more than two amino acids. Our work thus suggests protein design strategies by which this intrinsic limitation to NRPS engineering may be overcome in future.