Active Protease Research Articles

Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47.

CD47 is a cell surface glycoprotein that is expressed on normal human tissues and has a key role as a marker of self. Tumor cells have coopted CD47 overexpression to evade immune surveillance and thus blockade of CD47 is a highly active area of clinical exploration in oncology. However, clinical development of CD47-targeted agents has been complicated by its robust expression in normal tissues and the toxicities that arise from blocking this inhibitory signal. Further, pro-phagocytic signals are not uniformly expressed in tumors and antibody blockade alone is often not sufficient to drive antitumor activity. The inclusion of an IgG1 antibody backbone into therapeutic design has been shown to serve as an additional pro-phagocytic signal but also exacerbates toxicities in normal tissues. Therefore, a need persists for more selective therapeutic modalities targeting CD47. To address these challenges, we developed SGN-CD47M, a humanized anti-CD47 IgG1 monoclonal antibody linked to novel masking peptides through linkers designed to be cleaved by active proteases enriched in the tumor microenvironment. Masking technology has the potential to increase the amount of drug that reaches the tumor microenvironment, while concomitantly reducing systemic toxicities. We demonstrate that SGN-CD47M is well tolerated in cynomolgus monkeys and displays a 20-fold improvement in tolerability to hematologic toxicities when compared to the unmasked antibody. SGN-CD47M also displays preferential activation in the tumor microenvironment that leads to robust single-agent antitumor activity. For these reasons, SGN-CD47M may have enhanced antitumor activity and improved tolerability relative to existing therapies that target the CD47-SIRPα interaction.

Cathepsin B- and L-like Protease Activities Are Induced During Developmental Barley Leaf Senescence

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC50 values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC50 in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves.

Role of Na+-K+ ATPase Alterations in the Development of Heart Failure.

Na+-K+ ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α1, α2, and α3), β-subunit with two isoforms (β1 and β2) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na+-K+ ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na+-K+ ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na+-K+ ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na+-K+ ATPase activity in association with a depressed level of intracellular Na+ in failing hearts is considered to decrease intracellular Ca2+ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na+-K+ ATPase by cardiac glycosides in association with an increased level of Na+ in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na+-K+ ATPase activity associated with an increased level of intracellular Na+ in failing hearts has been demonstrated to result in an intracellular Ca2+ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na+-K+ ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca2+-overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca2+ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na+-K+ ATPase activity due to a shift in its isoform subunits in association with intracellular Ca2+-overload, cardiac energy depletion, increased membrane permeability, Ca2+-handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure.

L-histidine makes Ni2+ ‘visible’ for plant signalling systems: Shading the light on Ni2+-induced Ca2+ and redox signalling in plants.

Nickel is both an important nutrient and an ecotoxicant for plants. Organic ligands, such as L-histidine (His), play a key role in Ni2+ detoxification. Here, we show that His (added together with 0.01-10 mM Ni2+) decreases Ni2+ toxicity to Arabidopsis thaliana roots not only as a result of a decrease in Ni2+ activity, but also via the induction of signalling phenomena important for adaptation such as the generation of reactive oxygen species (ROS) and cytosolic Ca2+ transients. With the use of EPR spectroscopy, we demonstrate that Ni-His complexes generate hydroxyl radicals that is not detected by the addition of Ni2+ or His separately. Similarly, Ni-His complexes, but not Ni2+, activate Ca2+ influx and K+ efflux currents in patch-clamped root protoplasts resulting in distinct cytosolic Ca2+ signals and a transient K+ release. His prevented programmed cell death symptoms (cytoplasm shrinkage, protease and endonuclease activation) induced by Ni2+ and inhibited Ni2+ accumulation at [Ni2+]>0.3 mM. Intriguingly, priming of roots with Ni-His stimulated plant resistance to Ni2+. Overall, these data show that His triggers ROS-Ca2+-mediated reactions making Ni2+ ‘visible’ for plant signalling machinery and facilitating adaptation to the excess Ni2+.

ADAMTS12 promotes fibrosis by restructuring extracellular matrix to enable activation of injury-responsive fibroblasts.

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.

Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases)

An uncontrolled activity of neutrophil serine proteases (NSPs) contributes to inflammatory diseases. Cathepsin C (CatC) is known to activate NSPs during neutrophilic differentiation and represents a promising pharmacological target in NSP-mediated diseases. In humans, Papillon-Lefèvre syndrome (PLS) patients have mutations in theirCTSC gene, resulting in the complete absence of CatC activity. Despite this, low residual NSP activities are detected in PLS neutrophils (<10% vs healthy individuals), suggesting the involvement of CatC-independent proteolytic pathway(s) in the activation of proNSPs. This prompted us to characterize CatC-independent NSP activation pathways by blocking proCatC maturation. In this study, we show that inhibition of intracellular CatS almost completely blocked CatC maturation in human promyeloid HL-60 cells. Despite this, NSP activation was not significantly reduced, confirming the presence of a CatC-independent activation pathway involving a CatC-like protease that we termed NSPs-AAP-1. Similarly, when human CD34+ progenitor cells were treated with CatS inhibitors during neutrophilic differentiation in vitro, CatC activity was nearly abrogated but ∼30% NSP activities remained, further supporting the existence of NSPs-AAP-1. Our data indicate that NSPs-AAP-1 is a cysteine protease that is inhibited by reversible nitrile compounds designed for CatC inhibition. We further established a proof of concept for the indirect, although incomplete, inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP maturation using a CatS inhibitor, alone or in combination with a CatC/NSPs-AAP-1 inhibitor, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases.

Factor XII signaling via uPAR-integrin β1 axis promotes tubular senescence in diabetic kidney disease

Coagulation factor XII (FXII) conveys various functions as an active protease that promotes thrombosis and inflammation, and as a zymogen via surface receptors like urokinase-type plasminogen activator receptor (uPAR). While plasma levels of FXII are increased in diabetes mellitus and diabetic kidney disease (DKD), a pathogenic role of FXII in DKD remains unknown. Here we show that FXII is locally expressed in kidney tubular cells and that urinary FXII correlates with kidney dysfunction in DKD patients. F12-deficient mice (F12-/-) are protected from hyperglycemia-induced kidney injury. Mechanistically, FXII interacts with uPAR on tubular cells promoting integrin β1-dependent signaling. This signaling axis induces oxidative stress, persistent DNA damage and senescence. Blocking uPAR or integrin β1 ameliorates FXII-induced tubular cell injury. Our findings demonstrate that FXII-uPAR-integrin β1 signaling on tubular cells drives senescence. These findings imply previously undescribed diagnostic and therapeutic approaches to detect or treat DKD and possibly other senescence-associated diseases.

Intrinsic Proteolytic Activities from Cancer Cells Are Sufficient to Activate Alkoxyamine Prodrugs and Induce Cell Death.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

LRG1-Targeted Nintedanib Delivery for Enhanced Renal Fibrosis Mitigation.

Renal fibrosis lacks effective nephroprotective drugs in clinical settings due to poor accumulation of therapeutic agents in damaged kidneys, underscoring the urgent need for advanced renal-targeted delivery systems. Herein, we exploited the significantly increased expression of the leucine-rich α-2 glycoprotein 1 (LRG1) protein during renal fibrosis to develop a novel drug delivery system. Our engineered nanocarrier, DENNM, preferentially targets fibrotic kidneys via the decorated ET peptide's high affinity for LRG1. Once internalized by damaged renal cells, DENNM releases its encapsulated nintedanib, triggered by the active caspase-3 protease, disrupting the nanomedicine's structural integrity. The released nintedanib effectively reduces the level of expression of the extracellular matrix and impedes the progression of renal fibrosis by inhibiting the transforming growth factor-β (TGF-β)-Smad2/3 pathway. Our comprehensive in vitro and in vivo studies validate DENNM's antifibrotic efficacy, emphasizing LRG1's potential in renal targeted drug delivery and introducing an innovative approach to nanomedicine for treating renal fibrosis.

A breath-based in vitro diagnostic assay for the detection of lower respiratory tract infections.

An accurate diagnosis is critical to reducing mortality in people with lower respiratory tract infections (LRTIs). Current microbiological culture is time-consuming, and nucleic acid amplification-based molecular technologies cannot distinguish between colonization and infection. Previously, we described developing a sampling system for effectively capturing biomolecules from human breath. We identified a new class of proteoform markers of protease activation, termed proteolytic products of infection, for detecting LRTIs in people with mechanical ventilation. Here, we further developed an in vitro assay by designing a specific substrate sensor for human neutrophil elastase (HNE) to detect LRTIs in breath samples. In the proof-of-concept study, we then applied this in vitro assay to breath samples collected from intubated patients and healthy volunteers. The findings revealed that the LRTI group demonstrated a significant mean differential, showing a 9.8-fold elevation in measured HNE activity compared with the non-LRTI group and a 9.2-fold compared with healthy volunteers. The in vitro assay's diagnostic potential was assessed by constructing a receiver operating characteristic curve, resulting in an area under the curve of 0.987. Using an optimal threshold for HNE at 0.2 pM, the sensitivity was determined to be 1.0 and the specificity to be 0.867. Further correlation analysis revealed a strong positive relationship between the measured HNE activity and the protein concentration in the breath samples. Our results demonstrate that this breath-based in vitro assay provides high diagnostic performance for LRTIs, suggesting that the technology may be useful in the near term for the accurate diagnosis of LRTIs.

Assessing the impact of different Urushiol primer solvents on dentin remineralization and bond strength.

To investigate urushiol's potential as a dentin cross-linking agent, promoting remineralization of etched dentin and preventing activation of endogenous proteases causing collagen degradation within the hybrid layer. The goal is to improve bond strength and durability at the resin-dentin interface. Urushiol primers with varying concentrations were prepared using ethanol and dimethyl sulfoxide (DMSO) as solvents. Dentin from healthy molars underwent grinding and acid etching for 15s, followed by a 1min application of urushiol primer. After 14 and 28days of remineralization incubation and remineralization were usedtoassessby Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR), Micro-Raman spectroscopy, X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), Vickers Hardness, Scanning Electron Microscopy (SEM), and Energy X-ray dispersive spectroscopy (EDS). The overall performance of urushiol primers as dentin adhesives was observed by microtensile bond strength (μTBS) testing and nanoleakage assessment. Investigated the inhibitory properties of the urushiol primers on endogenous metalloproteinases (MMPs) utilizing in situ zymography, and the cytotoxicity of the primers was tested. Based on ATR-FTIR, Raman, XRD, EM-EDS and Vickers hardness analyses, the 0.7%-Ethanol group significantly enhanced dentin mineral content and improved mechanical properties the most. Pretreatment notably increased the μTBS of restorations, promoted the stability of the mixed layer, and reduced nanoleakage and MMPs activity after 28days. The urushiol primer facilitates remineralization in demineralized dentin, enhancing remineralization in etched dentin, effectively improving the bonding interface stability, with optimal performance observed at a 0.7 wt% concentration of the urushiol primer.

Brensocatib in Patients With Bronchiectasis: Subgroup Analyses From the WILLOW Trial

Bronchiectasis is a chronic inflammatory airway disease. Brensocatib, an oral, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), reduces pulmonary inflammation by preventing the activation of neutrophil serine proteases. In the phase 2 WILLOW trial, brensocatib prolonged time to first exacerbation in patients with bronchiectasis. In this post-hoc analysis we compare clinical outcomes in patients from WILLOW according to baseline disease characteristics.Adults with bronchiectasis treated with brensocatib (10 or 25 mg) or placebo once-daily were analysed by baseline Bronchiectasis Severity Index (BSI) score (≤4 [mild], 5–8 [moderate], or ≥9 [severe]), exacerbation history (2 or ≥3 in the prior year), blood eosinophil count (&lt;300 cells·µL−1or ≥300 cells·µL−1), long-term macrolide use (≥6 months; no or yes), andPseudomonas aeruginosaculture at screening (negative or positive). End points were time to first exacerbation, annualised exacerbation rate, change in lung function from baseline, and safety. All patients who received brensocatib were pooled and compared with placebo.Treatment with brensocatibversusplacebo was associated with a longer time to first exacerbation (hazard ratio [95% confidence interval], BSI: ≤4, 0.28 [0.08–0.96]; 5–8, 0.75 [0.35–1.60]; ≥9, 0.61 [0.35–1.04]; prior exacerbations: 2, 0.56 [0.34–0.90]; ≥3, 0.71 [0.32–1.59]; blood eosinophils/µL: &lt;300, 0.66 [0.42–1.06]; ≥300, 0.49 [0.20–1.20]; long-term macrolide use: no, 0.60 [0.38–0.94]; yes, 0.60 [0.25–1.45];Pseudomonas aeruginosaculture: negative, 0.54 [0.32–0.92]; positive, 0.68 [0.37–1.27]). Safety results were similar across subgroups.Patients treated with brensocatib had a numerically longer time to first exacerbation and reduced annualised rate of exacerbationversusplacebo across all key baseline disease characteristics.

Novel cathepsin C inhibitor, BI 1291583, intended for treatment of bronchiectasis: Phase I characterization in healthy volunteers.

Novel treatments are needed to reduce inflammation, improve symptoms, address exacerbations, and slow disease progression in bronchiectasis. Cathepsin C (CatC) inhibition promises to achieve this through reduction of neutrophil-derived serine protease (including neutrophil elastase [NE] and proteinase 3 [PR3]) activation. Here, we present the phase I characterization of the novel CatC inhibitor, BI 1291583. Five phase I trials of BI 1291583 in healthy subjects are presented: a single-rising-dose study (NCT03414008) and two multiple-rising-dose studies (NCT03868540 and NCT04866160) assessing the safety, tolerability, pharmacodynamics, and pharmacokinetics of BI 1291583; a food effect study (NCT03837964); and a drug-drug interaction study (NCT03890887) of BI 1291583 and itraconazole. BI 1291583 was safe and well tolerated across the doses tested in these trials. Most adverse events (AEs) were mild or moderate in intensity, with no serious AEs, AEs of special interest or deaths reported in any trial. Drug-related skin exfoliation was not reported more frequently in subjects treated with BI 1291583 compared with placebo. BI 1291583 was readily absorbed, and pharmacokinetics were supra-proportional over the dose ranges assessed. Additionally, BI 1291583 inhibited CatC in a dose-dependent manner, inhibited downstream NE activity, and decreased PR3 levels. No food effect was observed. Co-administration of multiple doses of itraconazole increased BI 1291583 exposure approximately twofold. Due to these promising phase I results, a multinational phase II program of BI 1291583 in adults with bronchiectasis is ongoing (Airleaf™ [NCT05238675], Clairafly™ [NCT05865886], and Clairleaf™ [NCT05846230]).

Treatment with ensitrelvir for COVID-19 in hospitalized patients of very advanced age: Case series.

Ensitrelvir fumaric acid (ensitrelvir) is an orally active 3C-like protease inhibitor used to treat severe acute respiratory syndrome coronavirus 2 infection. Ensitrelvir was granted an emergency use authorization in Japan in 2022, but reports on the effectiveness and safety of ensitrelvir in actual clinical settings are limited. Here, we report a case series of 9 patients with laboratory-confirmed symptomatic coronavirus disease of 2019 (COVID-19) who completed a 5-day course of ensitrelvir at Tomita Hospital from November 2022 to April 2023. Data on clinical symptoms, oxygen saturation, and food intake were collected for 14 days, beginning on the first day of ensitrelvir administration. The outcome of COVID-19 in each patient was also evaluated during this period. All patients were female, 80 years old or older, and the mean age was 90.2 ± 5.5 years. All patients received ensitrelvir within 2 days after the onset of COVID-19. At baseline, 7 among the 9 patients had their body temperature above 37.5 °C and all of them had oxygen saturation levels of 94% or higher. All patients recovered without worsening of COVID-19, and none received oxygen or additional antiviral drugs during the observation period; no deaths were reported within 14 days. After receiving ensitrelvir for 5 days, all patients had resolution of fever (<37 °C). There was no significant decrease in food intake of patients due to COVID-19. All patients maintained oxygen saturation above 93%. Our results provide information on the real-world usage of ensitrelvir in elderly, hospitalized patients with COVID-19, and suggests that ensitrelvir is an option for treatment of COVID-19 in these population.

Elucidating the biotechnological potential of the genera Parageobacillus and Saccharococcus through comparative genomic and pan-genome analysis

BackgroundThe genus Geobacillus and its associated taxa have been the focal point of numerous thermophilic biotechnological investigations, both at the whole cell and enzyme level. By contrast, comparatively little research has been done on its recently delineated sister genus, Parageobacillus. Here we performed pan-genomic analyses on a subset of publicly available Parageobacillus and Saccharococcus genomes to elucidate their biotechnological potential.ResultsPhylogenomic analysis delineated the compared taxa into two distinct genera, Parageobacillus and Saccharococcus, with P. caldoxylosilyticus isolates clustering with S. thermophilus in the latter genus. Both genera present open pan-genomes, with the species P. toebii being characterized with the highest novel gene accrual. Diversification of the two genera is driven through the variable presence of plasmids, bacteriophages and transposable elements. Both genera present a range of potentially biotechnologically relevant features, including a source of novel antimicrobials, thermostable enzymes including DNA-active enzymes, carbohydrate active enzymes, proteases, lipases and carboxylesterases. Furthermore, they present a number of metabolic pathways pertinent to degradation of complex hydrocarbons and xenobiotics and for green energy production.ConclusionsComparative genomic analyses of Parageobacillus and Saccharococcus suggest that taxa in both of these genera can serve as a rich source of biotechnologically and industrially relevant secondary metabolites, thermostable enzymes and metabolic pathways that warrant further investigation.

The Establishment of Complement System Is from Gene Duplication and Domain Shuffling.

The mammalian complement system constitutes a highly sophisticated body defense machinery. The evolutionary origin of the complement system can be traced to Coelenterata as the presence of the central component C3 and two activation proteases BF and MASP. In the present study, the main complement components were screened and analyzed from the genomes of different species in metazoan subphyla/phyla. C1q with classical domains can be traced to Annelida, and ficolin and MBL to Urochordata. C1r and C1s are only found in Chondrichthyes and even higher species, and MASP is traced to Coelenterata. In the evolutionary tree, C1r from Vertebrates is close to MASP1/2/3 from Deuterostomia and Coelenterata, and C1s from Vertebrates is close to MASP-like protease (MASPL) from Arthropoda, Mollusca, and Annelida. C2, BF, and DF can be traced to Mollusca, Coelenterata, and Porifera, respectively. There are no clear C2 and BF branches in the evolutionary tree. C3 can be traced to Coelenterata, and C4 and C5 are only in Chondrichthyes and even higher species. There are three clear C3, C4, and C5 branches in the evolutionary tree. C6-like (C6L) and C8 can be traced to Urochordata, and C7-like (C7L) can be traced to Cephalochordara. C6L, C7L, and C8 from Urochordata and Cephalochordara provide the structural conditions for the formation of Vertebrate MAC components. The findings unveil the evolutionary principles of the complement system and provide insight into its sophistication.

Rapid screening and isolation of potential xanthine oxidase inhibitors in Ganoderma lucidum

Given the abundance and intricate nature of the chemical components found in natural food ingredients, functional food research has long grappled with two significant technical challenges: rapid and precise screening of active substances and targeted preparation. To enhance the efficiency of fungal food preparation, novel methodologies for screening and the production of xanthine oxidase inhibitors from G. lucidum have been developed. The xanthine oxidase inhibitors were rapidly screened using AUF-LC-MS. Molecular docking and molecular dynamics simulation were used to verify the anti-gout activity of active compounds. Subsequently, with activity screening as the guide, HSCCC and SPLC were employed to separate the active ingredients. Simultaneously, the mechanism of action of xanthine oxidase inhibitors was analyzed by enzymatic reaction kinetics. Finally, the anti-gout activity of chemical components in G. lucidum was verified by network pharmacology, and its mechanism of action was discussed. Four potential xanthine oxidase inhibitors, namely ganoderic acid C2, B, A, and D2, were screened from G. lucidum. Their docking binding energies with xanthine oxidase were −6.29, −6.65, −4.29, and −5.96 kcal/mol, respectively. The purity of the four triterpenoid active ingredients isolated from G. lucidum was more than 90 %. These triterpenoid active ingredients showed a reversible inhibitory effect on xanthine oxidase. Network pharmacological studies revealed 71 common targets of active ingredients and gout, obtained by screening and intersecting the two targets, and identified the main metabolic pathways. The results indicate that G. lucidum extract can be employed for the prevention and treatment of gout disease. We enhance the efficiency and purity of active compounds preparation by incorporating enzyme inhibition assays and computerized virtual screening with compound chromatography. With this approach, we seamlessly incorporate fast discovery, screening, and focused preparation of active ingredients, as well as delving into the mechanisms of action of active monomers and proteases, and the molecular-level interactions between active ingredients and proteases. This study providing more opportunities to discover and develop new potential therapeutics from health food resources.

In silico exploration of 4(α-l-rhamnosyloxy)-benzyl isothiocyanate: A promising phytochemical-based drug discovery approach for combating multi-drug resistant Staphylococcus aureus

Multidrug-resistant (MDR) Staphylococcus aureus infections significantly threaten global health. With rising resistance to current antibiotics and limited solutions, the urgent discovery of new, effective, and affordable antibacterials with low toxicity is imperative to combat diverse MDR S. aureus strains. Hence, in this study, we introduce an in silico phytochemical-based approach for discovering novel antibacterial agents, underscoring the potential of computational approaches in therapeutic discovery. Glucomoringin Isothiocyanate (GMG-ITC) from Moringa oleifera Lam. is one of the phytochemical compounds with several biological activities, including antimicrobial, anti-inflammatory, and antioxidant activities, and is also effective against S. aureus. This study focuses on screening GMG-ITC as a potential drug candidate to combat MDR S. aureus infections through a molecular docking approach. Moreover, interaction amino acid analysis, in silico pharmacokinetics, compound target prediction, pathway enrichment analysis and molecular dynamics (MD) simulations were conducted for further investigation. Molecular docking and interaction analysis showed strong binding affinity towards S. aureus lipase, dihydrofolate reductase, and other MDR S. aureus proteins, including penicillin-binding protein 2a, MepR, D-Ala:D-Ala ligase, and RPP TetM, through hydrophilic and hydrophobic interactions. GMG-ITC also showed a strong binding affinity to cyclooxygenase-2 and FAD-dependent NAD(P)H oxidase, suggesting that it is a potential anti-inflammatory and antioxidant candidate that may eliminate inflammation and oxidative stress associated with S. aureus infections. MD simulations validated the stability of the GMG-ITC molecular interactions determined by molecular docking. In silico pharmacokinetic analysis highlights its potency as a drug candidate, showing strong absorption, distribution, and excretion properties in combination with low toxicity. It acts as an active protease and enzyme inhibitor with moderate activity against GPCR ligands, ion channels, nuclear receptor ligands, and kinases. Enrichment analysis further elucidated its involvement in important biological, molecular, and cellular functions with potential therapeutic applications in diseases like cancer, hepatitis B, and influenza. Results suggest that GMG-ITC is an effective antibacterial agent that could treat MDR S. aureus-associated infections.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Neutrophil PAD4 Expression and Its Pivotal Role in Assessment of Alcohol-Related Liver Disease.

Neutrophils release neutrophil extracellular traps (NETs) as a defense strategy in response to broad-spectrum infections and sterile triggers. NETs consist of a DNA scaffold decorated with antimicrobial peptides (AMPs) and enzymatically active proteases, including peptidyl arginine deiminase type 4 (PAD4). Susceptibility to infections and inflammatory dysregulation are hallmarks of alcohol-related liver disease (ALD). Sixty-two patients with ALD were prospectively recruited, and they were followed for 90 days. Twenty-four healthy volunteers served as the control group. PAD4 concentrations were quantified using immunoenzymatic ELISAs. Correlation coefficients between PAD4 blood concentrations and markers of systemic inflammation; liver dysfunction severity scores; and ALD complications were calculated. The receiver operating curves (ROCs) and their areas under the curve (AUCs) were checked in order to assess the accuracy of PAD4 expression in predicting the degree of liver failure and the development of ALD complications. Systemic concentrations of PAD4 were significantly increased in the patients with ALD in comparison with controls. PAD4 levels correlated with the standard markers of inflammation and revealed a good predictive AUC (0.76) for survival in the whole ALD group. PAD4 seems to be an inflammatory mediator and may be potentially applied as a predictor of patient survival in ALD.