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Aberrant activation of NLRP3 inflammasome is associated with a variety of inflammatory diseases. Advances in understanding the molecular mechanisms of NLRP3 inflammasome have revealed that NEK7 is an essential component for its activation, but the development of drugs specifically targeting NEK7 remains challenging. Here we identify rociletinib (ROC), an anticancer drug in phase III clinical trial with high safety profile, as a highly potent and specific small-molecule antagonists of NEK7. Mechanistically, ROC covalent binds to the cysteine 79 of NEK7 through its reactive α, β-unsaturated carbonyl group, thereby inhibiting the interaction between NLRP3 and NEK7, and the subsequent assembly and activation of NLRP3 inflammasome. Furthermore, ROC alleviates the pathological features of metainflammation on the mouse model of type 2 diabetes (T2D). In summary, our results identify ROC as a covalent inhibitor of NEK7 and demonstrates that targeting NEK7 provides a potential and promising strategy for the treatment of NLRP3 inflammasome-driven T2D. [ABSTRACT FROM AUTHOR]

Electrochemical devices, using renewable energy sources, for CO2 reduction (CO2R) coupled with water oxidation is an attractive strategy for the carbon‐neutral generation of solar fuels and chemical feedstocks. Highly reduced multi‐carbon (C2+) products (e.g., ethylene, ethanol, and propanol) are among the most attractive CO2R chemicals because of their commercial value and high energy densities. Here a new molecular hybrid material is reported that combines the capacity of molecular CO2R catalyst to generate CO with the capacity of Cu(0) materials to achieve C2+ products and further tune their reactivity with organic modifiers. A Fe‐A2B2 porphyrin containing two quaternary ammonium groups that set the right CO2R onset potential for a synergistic tandem performance with Cu2O nanocubes as the precursors is prepared. Furthermore, Fe‐A2B2 porphyrin is functionalized with two thiolate substituents in order to covalently anchor the molecular catalyst onto Cu providing an intimate interaction and great stability. The structural design enables a substantially enriched CO species adsorbed on Cu facilitated by the iron porphyrin catalyst that in turn facilitates the evolution of C2+ products, as demonstrated by in situ Raman spectroscopy. In addition, the whole electrode is further coated with phenyl modifiers that regulate the proton content and hydrophilicity in the neighborhood of the active centers. This approach affords Faradaic efficiencies in the range of 50% for ethylene and 77% for C2+ products at an applied potential of −1.05 V versus RHE. [ABSTRACT FROM AUTHOR]

Abstract Aberrant activation of NLRP3 inflammasome is associated with a variety of inflammatory diseases. Advances in understanding the molecular mechanisms of NLRP3 inflammasome have revealed that NEK7 is an essential component for its activation, but the development of drugs specifically targeting NEK7 remains challenging. Here we identify rociletinib (ROC), an anticancer drug in phase III clinical trial with high safety profile, as a highly potent and specific small-molecule antagonists of NEK7. Mechanistically, ROC covalent binds to the cysteine 79 of NEK7 through its reactive α, β-unsaturated carbonyl group, thereby inhibiting the interaction between NLRP3 and NEK7, and the subsequent assembly and activation of NLRP3 inflammasome. Furthermore, ROC alleviates the pathological features of metainflammation on the mouse model of type 2 diabetes (T2D). In summary, our results identify ROC as a covalent inhibitor of NEK7 and demonstrates that targeting NEK7 provides a potential and promising strategy for the treatment of NLRP3 inflammasome-driven T2D.

Background: The structure of proanthocyanidins (PC) contains a large number of active phenolic hydroxyl groups, which makes it have strong antioxidant capacity. This study investigated the structural and functional properties of ovalbumin (OVA) modified by its interaction with PC. Results: It was found that on increasing the concentration ratio of PC to OVA from 10:1 to 40:1, the free amino and total sulfhydryl contents of OVA decreased from 470.59 ± 38.77 and 29.81 ± 0.31 nmol mg−1 to 96.61 ± 4.55 and 21.22 ± 0.78 nmol mg−1, respectively, and the free sulfhydryl content increased from 7.65 ± 0.41 to 9.48 ± 0.58 nmol mg−1. These results indicated that CN and CS bonds were formed and PC was covalently linked with OVA. The PC content in the OVA–PC conjugates increased from 281.93 ± 12.92 to 828.81 ± 46.09 nmol mg−1 on increasing the concentration ratio of PC to OVA from 10:1 to 40:1. The contents of α‐helix and β‐turn of OVA decreased, and the contents of β‐sheet and random coil increased, confirmed by circular dichroism. The tertiary structure of OVA was also altered according to the results of fluorescence and ultraviolet absorption spectra. The surface hydrophobicity of OVA–PC conjugates decreased with increasing bound polyphenol content. The conjugation of OVA to PC significantly improved its emulsification and antioxidant activity and denaturation temperature. Conclusion: This study may provide valuable information for improving OVA's functional properties and its PC conjugates for applications in the food industry. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin‐3‐glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography‐electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events. [ABSTRACT FROM AUTHOR]

Objective: The marked success of prostate-specific membrane antigen (PSMA)-targeting radioligands with albumin binder (ALB) is attributed to the improvement of blood retention and tumor accumulation. [111In]In-PNT-DA1, our PSMA-targeting radioligand with ALB, also achieved improved tumor accumulation due to its prolonged blood retention. Although the advantage of ALBs is related to their reversible binding to albumin, the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands remains unclear because of the lack of information about radioligands with stronger albumin-binding than ALBs. In this study, we designed and synthesized [111In]In-PNT-DM-HSA, a new radioligand that consists of a PSMA-targeting radioligand covalently bound to albumin. The pharmacokinetics of [111In]In-PNT-DM-HSA was compared with those of [111In]In-PNT-DA1 and [111In]In-PSMA-617, a non-ALB-conjugated radioligand, to evaluate the relationship between albumin-binding and tumor accumulation. Method: The [111In]In-PNT-DM-HSA was prepared by incubation of [111In]In-PNT-DM, a PSMA-targeting radioligand including a maleimide group, and human serum albumin (HSA). The ability of [111In]In-PNT-DM-HSA was evaluated by in vitro assays. A biodistribution study using LNCaP tumor-bearing mice was conducted to compare the pharmacokinetics of [111In]In-PNT-DM-HSA, [111In]In-PNT-DA1, and [111In]In-PSMA-617. Results: The [111In]In-PNT-DM-HSA was obtained at a favorable radiochemical yield and high radiochemical purity. In vitro assays revealed that [111In]In-PNT-DM-HSA had fundamental characteristics as a PSMA-targeting radioligand interacting with albumin covalently. In a biodistribution study, [111In]In-PNT-DM-HSA and [111In]In-PNT-DA1 showed higher blood retention than [111In]In-PSMA-617. On the other hand, the tumor accumulation of [111In]In-PNT-DA1 was much higher than [111In]In-PNT-DM-HSA and [111In]In-PSMA-617. Conclusions: These results indicate that the moderate reversible binding of ALB with albumin, not covalent binding, may play a critical role in enhancing the tumor accumulation of PSMA-targeting radioligands. [ABSTRACT FROM AUTHOR]

This study systematically investigated the impact of covalent binding of tannic acid (TA) to different proteins on protein structure and the stability of a nanoemulsion of Perilla essential oil (PEO). Additionally, the antimicrobial activity of the nanoemulsion was assessed. The interactions between TA and various proteins were explored using fluorescence spectroscopy and Fourier transform infrared (FTIR) spectroscopy, revealing that TA exhibited the most binding sites (n = 1.65) to whey protein isolate (WPI), forming a stable protein-TA complex. The emulsion prepared using TA had high stability. One-factor-at-a-time experiments showed that the emulsion prepared under the conditions of 675 W ultrasonic power and an oil-to-water ratio of 1:9 had the smallest droplet size of (292.3 ± 1.96) nm, a relatively high absolute zeta potential value of (–62.5 ± 0.29) mV, and the highest PEO encapsulation efficiency of (74.51 ± 0.35)%. The interaction between WPI-TA complex and PEO could stabilize the structure of the emulsion, and the emulsion maintained high stability at 4 ℃ for 28 days. Furthermore, emulsion encapsulation significantly reduced the loss of PEO, with the loss of PEO in the emulsion being only one-third of that of the pure essential oil under the same conditions. This study provides new ideas for future research on the synthesis of protein-polyphenol complexes and the encapsulation of volatile substances, laying a foundation for expanding the application of nanoemulsion in the food industry.

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Abstract The near‐infrared (NIR)‐II bioimaging technique is highly important for both diagnosing and treating life‐threatening diseases due to its exceptional imaging capabilities. However, the lack of suitable NIR‐II fluorescent probes has hindered their widespread clinical application. To address this issue, the binding of albumin to cyanine dyes has emerged as a practical and efficient method for developing high‐performance NIR‐II probes. Cyanine dyes can bind with exogenous and endogenous albumin through either covalent or noncovalent interactions, serving various purposes. The resulting cyanine@albumin (or albumin@cyanine) fluorophores offer significant advantages, including strong brightness, excellent photostability, good biosafety, and a long‐term, high‐resolution imaging window. Cyanine dye in situ binding with endogenous albumin can also enhance the targeting imaging capability. This review provides a summary of the interaction mechanism, performance enhancement, tumor‐targeting feature, and in vivo imaging applications of the cyanine@albumin fluorophores. These advancements not only highlight the unique characteristics of cyanine@albumin fluorophores in preclinical research but also emphasize their potential for clinical diagnosis.

BACKGROUND: It is well known that hemp proteins have the disadvantages of poor solubility and poor emulsification. To improve these shortcomings, an alkali covalent cross‐linking method was used to prepare hemp protein isolate‐epigallocatechin‐3‐gallate biopolymer (HPI‐EGCG) and the effects of different heat treatment conditions on the structure and emulsifying properties of the HPI‐EGCG covalent complex were studied. RESULTS: The secondary and tertiary structures, solubility, and emulsification ability of the HPI‐EGCG complexes were evaluated using particle size, zeta potential, circular dichroism (CD), and fluorescence spectroscopy indices. The results showed that the absolute value of zeta potential of HPI‐EGCG covalent complex was the largest, 18.6 mV, and the maximum binding amount of HPI to EGCG was 29.18 μmol g−1. Under heat treatment at 25–35 °C, the α‐helix content was reduced from 1.87% to 0%, and the β‐helix content was reduced from 82.79% to 0% after the covalent binding of HPI and EGCG. The solubility and emulsification properties of the HPI‐EGCG covalent complexes were improved significantly, and the emulsification activity index (EAI) and emulsion stability index (ESI) were increased by 2.77‐fold and 1.21‐fold, respectively. CONCLUSION: A new HPI‐EGCG covalent complex was developed in this study to provide a theoretical basis for the application of HPI‐EGCG in food industry. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

A series of nanoscale-level metal complexes with bidentate (N2MCl 2) and tetradentate (MN) chromophores have been employed as efficient cisplatin analogues and intercalating agents towards DNA and studied their anticancer activities against A549 cancer cells. Among them, complexes 1–6 exist with two labile chlorides similar to the chemotherapeutic inorganic drug cisplatin, which have been found to bind covalently with herring sperm DNA. Complexes 7–12 may undergo an intercalative mode of binding due to their structural differences relative to 1–6. The intrinsic binding constants obtained for complexes 7–12 were also significantly varied due to their structure and binding mode (0. 9 5 − 1. 6 × 1 0 5 and 1. 5 − 2. 4 × 1 0 4 M − 1 for complexes, 1–3 and 4–6, respectively). However, complexes 7–9 have shown much stronger binding constants (0. 9 8 − 1. 6 4 × 1 0 6 M − 1 ) due to their existing planarity which leads them to strong intercalation than their counterparts, 10–12 (3. 1 9 − 4. 1 4 × 1 0 4 M − 1 ). The significantly higher binding constant values for complexes 1–3 may due to their planar structure and also may possibly lead to intercalation. Though all these complexes (1–12) have shown almost similar binding constants (1.26–2.0) in the electrochemical analysis, it implies that these complexes interact towards DNA with equal strength at their reduced oxidation (M + I ) states. The obtained results in various spectral, electrochemical, relative viscosity changes, DNA cleavage studies revealed that these complexes 1–6 mimic the functional properties of cisplatin. Therefore, these nanostructured complexes could be substituted for cisplatin as a new family of non-platinum-based anticancer drugs in the future, after a sequence of in-vivo investigation. Their potential activity against A549 cancer cells has also been recorded using Hoechst staining and Propidium iodide images. [ABSTRACT FROM AUTHOR]

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed. [ABSTRACT FROM AUTHOR]

The ongoing evolution of influenza strains and the limited effectiveness of existing anti-influenza drugs underscore the imperative for novel therapeutics. In addressing this, we developed and synthesized several sialic acid (SA) analogs, designed as potential covalent inhibitors by targeting lysine residues (Lys133 and Lys222) proximal to the substrate binding site of hemagglutinin (HA). The affinities of these compounds for HA were meticulously assessed using surface plasmon resonance (SPR). Intriguingly, all target compounds exhibited superior affinities for HA compared to the parent compound SA. Notably, compound 1d demonstrated a binding potency similar to that of the positive control (2,6-SLN), with KD values of 38.2 µM and 39.6 µM, respectively. Importantly, all compounds demonstrated non-cytotoxicity to MDCK cells, and compound 1d displayed robust inhibitory effects on the influenza virus in vitro. The development of covalent inhibitors targeting HA, as outlined in this study, presented a rational and promising strategy for the creation of potent HA inhibitors. [ABSTRACT FROM AUTHOR]

Metal ion mordants are commonly used in the process of dyeing natural fabrics, such as cotton, wool, and silk, to improve their color fastness, but the use of metal salts results in toxic metal wastes which can harm the environment and are costly to process. This study investigated the dyeing and functionalization of wool fabrics with Alizarin Red S (ARS), catalyzed by horseradish peroxidase (HRP) and H2O2, as well as the polymerization mechanism of the ARS/HRP-H2O2 system and possible covalent conjugation mechanisms with wool fiber proteins. The dyeing process was optimized by determining the effects of HRP and ARS dosages, reaction temperature, and reaction time on ARS dyeing performance, as well as the color fastness and antibacterial properties of the resulting dyed fabrics. The HRP and H2O2 catalyzed polymerization of ARS and covalent attachment of the polymers to the wool fibers resulted in dyed fabrics with high color fastness; the rubbing and washing fastness were both 4–5. The antibacterial properties of dyed wool fabrics were excellent, achieving 92.3% growth inhibition of Escherichia coli. Therefore, the HRP-H2O2 system has great potential to replace metal mordants in the dyeing process of natural fibers and reduce the production of toxic wastes by this industry. [ABSTRACT FROM AUTHOR]

Background: A noninvasive method to track implanted biomaterials is desirable for real‐time monitoring of material interactions with host tissues and assessment of efficacy and safety. Purpose: To explore quantitative in vivo tracking of polyurethane implants using a manganese porphyrin (MnP) contrast agent containing a covalent binding site for pairing to polymers. Study Type: Prospective, longitudinal. Animal Model: Rodent model of dorsal subcutaneous implants (10 female Sprague Dawley rats). Field Strength/Sequence: A 3‐T; two‐dimensional (2D) T1‐weighted spin‐echo (SE), T2‐weighted turbo SE, three‐dimensional (3D) spoiled gradient‐echo T1 mapping with variable flip angles. Assessment: A new MnP‐vinyl contrast agent to covalently label polyurethane hydrogels was synthesized and chemically characterized. Stability of binding was assessed in vitro. MRI was performed in vitro on unlabeled hydrogels and hydrogels labeled at different concentrations, and in vivo on rats with unlabeled and labeled hydrogels implanted dorsally. In vivo MRI was performed at 1, 3, 5, and 7 weeks postimplantation. Implants were easily identified on T1‐weighted SE, and fluid accumulation from inflammation was distinguished on T2‐weighted turbo SE. Implants were segmented on contiguous T1‐weighted SPGR slices using a threshold of 1.8 times the background muscle signal intensity; implant volume and mean T1 values were then calculated at each timepoint. Histopathology was performed on implants in the same plane as MRI and compared to imaging results. Statistical Tests: Unpaired t‐tests and one‐way analysis of variance (ANOVA) were used for comparisons. A P value <0.05 was considered to be statistically significant. Results: Hydrogel labeling with MnP resulted in a significant T1 reduction in vitro (T1 = 517 ± 36 msec vs. 879 ± 147 msec unlabeled). Mean T1 values of labeled implants in rats increased significantly by 23% over time, from 1 to 7 weeks postimplantation (651 ± 49 msec to 801 ± 72 msec), indicating decreasing implant density. Data Conclusion: Polymer‐binding MnP enables in vivo tracking of vinyl‐group coupling polymers. Evidence Level: 1. Technical Efficacy: Stage 1. [ABSTRACT FROM AUTHOR]

G‐quadruplexes (G4s) have been revived as promising therapeutic targets with the development of immunotherapy, but the G4‐mediated immune response remains unclear. We designed a novel class of G4‐binding organic‐platinum hybrids, L1‐cispt and L1‐transpt, with spatial matching for G4 binding and G4 DNA reactivity for binding site locking. The solution structure of L1‐transpt‐MYT1L G4 demonstrated the effectiveness of the covalent binding and revealed the covalent binding‐guided dynamic balance, accompanied by the destruction of the A5‐T17 base pairs to achieve the covalent binding of the platinum unit to N7 of the G6 residue. Furthermore, L1‐cispt‐ and L1‐transpt‐mediated genomic dysfunction could activate the retinoic acid‐induced gene I (RIG‐I) pathway and induce immunogenic cell death (ICD). The use of L1‐cispt/L1‐transpt‐treated dying cells as therapeutic vaccines stimulated a robust immune response and effectively inhibited tumor growth in vivo. Our findings highlight the importance of the rational combination of specific spatial recognition and covalent locking in G4‐trageting drug design and their potential in immunotherapy. [ABSTRACT FROM AUTHOR]

Blood glucose monitoring (BGM) using disposable electrodes is commonly used in healthcare diagnosis. The BGM method is not suitable for people with diabetes requiring real-time monitoring who might experience sudden hypoglycemia or hyperglycemia owing to a single measurement at a specific moment. This study aimed to achieve an enhanced stability of glucose diagnosis for continuous glucose measurement systems (CGMs). A representative mediator of a second-generation glucose sensor was synthesized and coordinated with a polymer for immobilization on an indium tin oxide (ITO) electrode. For electrode immobilization, an electrode for enhanced stability was fabricated using the silanization method. The morphological properties of the electrodes were confirmed via cyclic voltammetry (CV), impedance spectroscopy, and SEM. The loss rate of the current density was only 10.11% of the initial current after 8 d. The electrode exhibited a coefficient of determination of R2 = 0.9924, sensitivity of 1.5454 μA/cm2·mM, limit of quantitation (LOQ) of 7.604 μM, and limit of detection (LOD) of 2.509 μM for glucose concentrations between 0.1 and 20.0 mM. The electrode system developed in this study is applicable to the CGM healthcare industry and is expected to be applicable to biofuel cells. [ABSTRACT FROM AUTHOR]

Several carboxylic acid-containing drugs have been withdrawn from clinical use due to adverse drug reactions including idiosyncratic hepatotoxicity and anaphylaxis. Carboxylic acid drugs are increasingly being shown to be metabolized to chemically-reactive acyl-CoA thioester-linked intermediary metabolites that are proposed to potentially mediate these toxicities. Acyl-CoA thioesters possess an electrophilic carbonyl-carbon and are able to undergo non-enzyme catalyzed transacylation reactions with biological nucleophiles leading to covalent binding to protein and drug-protein adduct formation. Such drug-protein adducts are proposed to mediate idiosyncratic drug toxicity reactions to carboxylic acid drugs, similar to the more studied mechanism of chemically-reactive acyl glucuronides potentially mediating carboxylic acid drug toxicity through the formation of immunogenic covalent protein adducts. Carboxylic acid drug acyl-CoA formation is catalyzed by acyl-CoA synthetase enzymes located with highest activity in liver tissue and in mitochondrial, peroxisomal, microsomal, and cytosolic cellular fractions. As is also true for unstable and reactive acyl glucuronide metabolites, the role of reactive acyl-CoA thioester metabolites in mediating idiosyncratic drug toxicity is difficult to assess. Importantly, in comparison to acyl glucuronide formation, results from mechanistic studies have revealed that acyl-CoA thioester formation can be the predominant pathway leading to covalent protein adduct formation in vitro and in vivo. This review includes an examination of the metabolism of carboxylic acid-containing drugs to reactive acyl-CoA thioesters leading to covalent binding to protein and potential implications with regard to carboxylic acid drug toxicity. [ABSTRACT FROM AUTHOR]

Definition of the relationship between drug protein adduct formation (haptenation) and development of immunological adverse drug reactions (drug hypersensitivity) has been an area of active research for over 80 years. The hapten hypothesis which states that "immunogenicity of low molecular weight organic chemicals is dependent on modification of self-proteins," evolved from Landsteiner and Jacob's discovery of a correlation between the reactivity of dinitro-halogenated benzenes and their sensitization potential. The hypothesis rapidly evolved to encompass drugs that often require metabolic activation to generate electrophilic, protein-reactive intermediates. As tissue culture methods advanced, the importance of drug hapten-specific T-cells in the disease pathogenesis was defined. This led to a plethora of studies describing the uptake and processing of drug(metabolite) protein adducts by antigen presenting cells, and the subsequent surface display of hapten-modified peptides in the context of MHC molecules. Although the pathway of hapten-specific T-cell activation is now well established, several questions need to be addressed: first, what is the nature of the hapten-modified peptides displayed by MHC? Second, how many of these peptides stimulate T-cells?; third, what are the critical protein modifications involved in T-cell activation; and finally, what is the role of hapten-specific T-cells in the iatrogenic disease? These questions will become increasingly important as more and more targeted covalent binding inhibitor drugs are approved for human use. In this review, we provide a brief synopsis of hapten research and then describe the approaches used by Pharma and academia to study hapten covalent binding and the role of drug protein adducts in the activation of human T-cells. [ABSTRACT FROM AUTHOR]

The influence of ultrasound-assisted free radical modification on the structure and functional properties of ovalbumin-epigallocatechin gallate (OVA-EGCG) conjugates was investigated by experimental measurements and computer simulations. Compared with the traditional free radical condition, the ultrasonic-assisted processing significantly increased the conjugating efficiency of OVA and EGCG and shortened the conjugating from 24 h to 1 h without affecting the equivalent amount of EGCG conjugating. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and multi-spectroscopy analysis (Fourier transform infrared spectroscopy, intrinsic fluorescence spectroscopy, and UV spectroscopy) indicated that the covalent conjugates could be formed between OVA and EGCG. And modification in the conformation of OVA was induced by EGCG. Furthermore, molecular docking results demonstrated the possession of high-affinity EGCG binding location on OVA, supporting and clarifying the experimental results. In addition, the functional properties of OVA including emulsification (emulsifying activity and emulsion stability) and antioxidant properties (DPPH scavenging capacity and ABTS scavenging capacity) were significantly improved after conjugation with EGCG, especially in ultrasound-assisted conditions. Overall, OVA-EGCG conjugates produced by ultrasound-assisted free radical treatment could be applied as a potential emulsifier and antioxidant, thereby expanding the application of OVA as a dual-functional ingredient.

Flucloxacillin is a β-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581. [ABSTRACT FROM AUTHOR]

The use of agricultural residue as a substrate and implementation of nanomaterials for cellulase immobilization can improve the efficiency at higher temperature and will lead to reduce the cost of cellulose-assisted biofuel production. The immobilization utilizing cellulase with amino, chitosan, or polymeric functionalities enhanced stability, activity, reusability, inhibition reduction, purification, and selectivity during enzymatic hydrolysis. Covalent interaction between the substrate and the cellulose marks in greater enzymatic immobilization, which pilots to higher biofuel production. Among the various techniques available for immobilization of cellulase on activated and functionalized magnetic nanoparticles, glutaraldehyde-based covalent binding are the most efficient process for cellulase immobilization. This review provides an overview of different cellulase immobilization strategies, factors, and its kinetics for enhanced biofuel production. The expanding need for low-cost immobilized cellulase, as well as its diverse applications in a variety of industries, is propelling research in this field. [ABSTRACT FROM AUTHOR]

Structured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity‐based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min−1 g−1 (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF "Amano" IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct. Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties. [ABSTRACT FROM AUTHOR]

Metal nanoclusters (NCs) with precise structure and ultrasmall size have attracted great interests in catalysis. However, the poor stability has limited its large-scale use. Herein, we proposed the "covalence bridge" strategy to effectively connect atomically precise metal NCs and metal-organic frameworks. Benefiting from the covalent linkage, the synthesized UiO-66-NH2-Au25(L-Cys)18 showed outstanding stability after 16 h photocatalysis. Moreover, the covalence bridge created a strong metal-support interaction between the two components and provided an effective charge transport channel and thereby enhanced photocatalytic activity. UiO-66-NH2-Au25(L-Cys)18 displayed an exceptional photocatalytic H2 production rate, which is 21 and 90 times higher than that of UiO-66-NH2/Au25(PET)18 (made by physically combination) and bare UiO-66-NH2, respectively. Thermodynamic and kinetic studies demonstrated that UiO-66-NH2-Au25(L-Cys)18 exhibited higher charge transfer efficiency, lower overpotential of water reduction and activation energy barrier compared with its counterparts. [ABSTRACT FROM AUTHOR]

BTK plays a pivotal role in the B cell signaling, abnormal activation of which is associated with the progression of SLE or other diseases. Targeting BTK has emerged as a promising therapeutic strategy for SLE. [Display omitted] • BTK is a key kinase downstream of BCR, FcR and multiple pathways. • Overactivated BTK contributes to SLE development by driving B cell abnormalities. • Inhibitors targeting BTK are a promising and effective option for SLE treatment. Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disorder involving multiple organs and systems. There is growing evidence that autoreactive B cells occupy a central role in the occurrence and progression of SLE due to their ability to generate pathogenic autoantibodies. Small molecule inhibitors targeting Bruton's tyrosine kinase (BTK), a crucial intracellular kinase regulating B cell development and function, emerge as a new strategy to treat SLE in recent years and are superior to biologic agents depleting B cells in many aspects. Supportive data obtained from lupus-prone mice preliminarily demonstrated the promising therapeutic potential of BTK inhibition. However, these BTK inhibitors, including elsubrutinib, evobrutinib, etc., mostly face with unsatisfactory efficacy and certain safety issues during clinical use, driving the quest for new-generation inhibitors with improved potency and higher selectivity. This paper elaborates the importance of BTK involvement in SLE pathogenesis, reviews the clinical research progress of BTK inhibitors for SLE and discusses limitations and challenges the drugs met in development, in order to contribute to a deeper understanding of disease mechanism and provide a reference for new-generation BTK inhibitor research. [ABSTRACT FROM AUTHOR]

The oil-water separation phenomenon in sesame paste poses a challenge for the storage of sauce products. In this study, soy protein isolate (SPI), peanut protein isolate (PPI) and flaxseed gum (FG) conjugates were prepared to stabilize the solid-liquid phases of sesame paste. The results indicated that the highest degree of grafting (25.07%) was observed in a ratio of PPI:FG = 4:1 (PF2). The interaction between proteins and FG generated the extension of the protein molecule and increased structural disorder. PPI-FG conjugates exhibited the lower surface hydrophobicity and higher thermal stability. The addition of 4% PF2 to sesame paste led to a reduction of 5.06% oil separation rate and showed higher storage modulus (G′), loss modulus (G″), apparent viscosity, and hardness. A stable and continuous three-dimensional network was created to physically entrap the oil, resulting in a more uniform distribution of oil droplets and a reduced particle interaction of sesame paste. • Peanut protein isolate-flaxseed gum conjugate possessing network structure was prepared. • The three-dimensional network structure of conjugates improved the oil absorption capacity of sesame paste. • Conjugate incorporation reduced particle interaction and notably decreased oil separation rate. • Conjugate incorporation improved the stability and commodity value of sesame paste. [ABSTRACT FROM AUTHOR]

Background: The potential applications of protein-engineered functional materials are so wide and exciting that the interest in these eco-friendly advanced materials will further expand in the future. Tag-mediated protein purification/immobilization technologies have emerged as green and cost-effective approaches for the fabrication of such materials. Strategies that combine the purification and immobilization of recombinant proteins/peptides onto/into natural, synthetic or hybrid materials in a single-step are arising and attracting increasing interest. Aim of Review: This review highlights the most significant advances of the last 5 years within the scope of tag-mediated protein purification/immobilization and elucidates their contributions for the development of efficient single-step purification and immobilization strategies. Recent progresses in the field of protein-engineered materials created using innovative protein-tag combinations and future opportunities created by these new technologies are also summarized and identified herein. Key Scientific Concepts of Review: Protein purification/immobilization tags present a remarkable ability to establish specific non-covalent/covalent interactions between solid materials and biological elements, which prompted the creation of tailor-made and advanced functional materials, and of next-generation hybrid materials. Affinity tags can bind to a wide range of materials (of synthetic, natural or hybrid nature), being most suitable for protein purification. Covalently binding tags are most suitable for long-term protein immobilization, but can only bind naturally to protein-based materials. Hybrid affinity-covalently binding tags have allowed efficient one-step purification and immobilization of proteins onto different materials, as well as the development of innovative protein-engineered materials. Self-aggregating tags have been particularly useful in combination with other tags for generating protein-engineered materials with self-assembling, flexible and/or responsive properties. While these tags have been mainly explored for independent protein purification, immobilization or functionalization purposes, efficient strategies that combine tag-mediated purification and immobilization/functionalization in a single-step will be essential to guarantee the sustainable manufacturing of advanced protein-engineered materials.

We report the refinement of a high-throughput, liquid chromatography/mass spectrometry (LC/MS)-based screening method for the identification of covalent small-molecule binders to proteins. Using a custom library of 1600 disulfide-capped fragments targeting surface cysteine residues, we optimize sample preparation, chromatography, and ionization conditions to maximize the reliability and flexibility of the approach. Data collection at a rate of 84 s per sample balances speed with reliability for sustained screening over multiple, diverse projects run over a 24-month period. The method is applicable to protein targets of various classes and a range of molecular masses. Data are processed in a custom pipeline that calculates a percent bound value for each compound and identifies false positives by calculating significance of detected masses (signal significance). An example pipeline is available through Biovia's ScienceCloud Protocol Exchange. Data collection and analysis methods for the screening of covalent adducts of intact proteins are now fast enough to screen the largest covalent compound libraries in 1 to 2 days.

Lipid oxidation is still a major problem complicating the development of food emulsions. In this study, an antioxidant Pickering emulsion stabilized by resveratrol-grafted zein (Z-R) conjugates and pectin (P) complex particles was prepared. The hydrophilic pectin successfully adjusted the wettability of Z-R; when the mass ratio of Z-R to P was 2:1 (Z-R/P2:1), the three-phase contact angle was 90.68°, and the wettability of the particles was close to neutral. Rheological analysis showed that the emulsion formed an elastic gel structure. FTIR spectra indicated that there was a hydrogen bond and electrostatic interaction between Z-R and P. The disappearance of characteristic infrared peaks of corn oil was due to a dense protective film formed on the surface of oil drops by Z-R/P2:1 particles, which was confirmed by confocal laser scanning microscopy. The emulsion stabilized by Z-R/P2:1 had excellent physical stability at a wide range of pH values (4–9), salt ion concentrations (0.04–0.15 mol·L−1) and storage times (0–30 days). The anti-lipid oxidation ability of the emulsion was outstanding; after storage for 14 days at room temperature, the MDA content in the emulsion was only 123.85 μmol/kg oil. In conclusion, the Z-R/P2:1 particles prepared in this study can effectively stabilize a Pickering emulsion and expand the usability of the method for constructing antioxidant Pickering emulsions. [ABSTRACT FROM AUTHOR]

An extracellular pectinase from a mixed consortium of Bacillus sp. (BSP) was immobilized onto graphene oxide/chitosan composite (GO/CS) through covalent binding to enhance its recycling and operational stability features. Different parameters were optimized, including cross-linker concentration (%), time, pH, and GO/CS-pectinase ratios. GO/CS-pectinase was further characterized by FT-IR and XRD. The activity of GO/CS-pectinase was reached up to 804 μmolmin−1 with an immobilization efficiency of 80.64 ± 1.15 % under optimum conditions. GO/CS-pectinase exhibited a 3.0-folds higher half-life (t 1/2) than free pectinase at 50, 55, and 60 °C, respectively. The V max and K M values of GO/CS-pectinase were found to be nearly equal to the free pectinase indicating that conformational flexibility was retained. K d , t 1/2 , ∆ G *, ∆ H *, and ∆ S * of both free pectinase and GO/CS-pectinase was 0.0339 & 0.0721 min−1, 9.62 and 40.44 min, 81.35, 90.72 kJmol−1, 47.098 & 63.635 kJmol−1, −102.86 & −81.340 Jmole−1 K−1. SEM morphological analysis further confirmed the successful binding of pectinase with GO/CS, which retained about 92 % of its original catalytic activity after ten consecutive reaction cycles. Finally, GO/CS-pectinase was employed for guava juice clarification which exhibited the turbidity reduction up to 81 % after 75 min of treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

In the present study, the non-blue laccase Melac13220 from Methylobacterium extorquens was immobilized using three methods to overcome problems related to the stability and reusability of the free enzyme: entrapment of the enzyme with sodium alginate, crosslinking of the enzyme with glutaraldehyde and chitosan-, and site-specific covalent immobilization of the enzyme on Fe3O4 nanoparticles by an aldehyde tag. The site-specific covalent immobilization method showed the highest immobilization efficiency and vitality recovery. The optimum temperature of Melac13220 was increased from 65 °C to 80 °C. Immobilized Melac13220 showed significant tolerance to some organic solvents and maintained approximately 80% activity after 10 cycles of use. Differential scanning calorimetry (DSC) indicated that the melting temperature of the enzyme was increased (from 57 °C to 79 °C). Immobilization of Melac13220 also led to improvement in dye decolorization such that Congo Red was completely decolorized within 10 h. The immobilized enzyme can be easily prepared without purification, demonstrating the advantages of using the aldehyde tag strategy and providing a reference for the practical application of different immobilized laccase methods in the industrial field. [ABSTRACT FROM AUTHOR]

Protein modification can improve the bioavailability of polyphenols, while polyphenols can also improve the functional properties of pea protein. Four representative polyphenols (gallic acid, ferulic acid, catechin and kaempferol) were covalently combined with pea protein by ultrasonic assisted grafting (free radical grafting, laccase method and alkali treatment), and the structure and functional properties of the conjugated compounds were characterized. The interaction was evaluated by surface hydrophobicity, free sulfhydryl content determination and SDS-PAGE. Fluorescence spectra and Fourier transform infrared spectra revealed that polyphenol binding reduced the α-helix content of pea protein and changed its structure. In addition, the antioxidant activity of the conjugate was significantly improved compared to natural pea protein (DPPH by 7.5 to 20.2 times, ABTS + by 2.8 to 4.3 times, FRAP by 1.4 to 4.1 times). At the same time, the introduction of polyphenols also optimized the thermal stability of pea protein, and increased the denaturation temperature of pea protein (113.1 °C) by 0.19-6.63 °C. The emulsifying properties were also enhanced by covalent modification, and the emulsifying properties of the ultrasound-assisted conjugates were better. In summary, the pea protein-polyphenol conjugate provides a new solution for improving the bioavailability of polyphenols and preparing polyphenol delivery systems for complex emulsions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tracking chemical reactions by measuring incurred mass shifts upon successful binding is a direct and attractive alternative to existing assays based on chemical tags. Traditional methods use liquid chromatography-mass spectrometry (LC-MS), and because the required buffers are not amenable to direct MS injection, sample pre-treatment is needed to desalt. This leads to analysis times from ten seconds to minutes per sample, limiting throughput and preventing widespread application. Combining an acoustic ejection (AE) interface with a time-of-flight mass spectrometer (MS) removes this bottleneck, as samples can be directly introduced at rates of up to one second per sample. This article describes a complete workflow for measuring the covalent binding of compounds to proteins in real-time, from assay to data evaluation. It is noteworthy that this is the first instance of using SCIEX Echo® MS+ system with ZenoTOF 7600 system to study the kinetic regimes of covalent binding., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christian Opitz reports equipment, drugs, or supplies was provided by Sciex. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Covalent binding with bioactive substances is one of effective and promising strategies to improve bioactivities of artificial materials. However, there is still a lack of systematic understanding of how the microstructure of carriers affect performances of the covalently bound bioactive substances. In this study, we firstly introduced four chemical functional groups into two kinds of differently microstructured carriers, multi-walled carbon nanotubes (MWCNTs) and graphene (GP), with equal grafting rate. Then two types of osteogenic-related peptides were respectively covalently bound with the eight kinds of carriers with the same molar amount. Subsequently, performances of the MWCNTs/Peptides and GP/Peptides composites were evaluated using ectopic osteogenesis model in rats and calvarial defect model in rabbits respectively. The results showed that the ability of inducing osteogenesis and repairing bone defect of the peptides covalently bound with the MWCNTs was better than that of the peptides covalently bound with the GP, and that the positive effect order of the four covalent binding modes on bioactivities of the peptides was for the first time announced. Furthermore, the possible mechanism was proposed and discussed. Our study could provide important guidance for the research and development of high-performance tissue repair materials covalently bound with related bioactive substances.

Purpose: Michael receptor molecules derived from plants are biologically active due to electrophilic groups in their structure. They can target nucleophilic residues on disease-related proteins, with significant therapeutic effects and low toxicity for many diseases. They provide a good option for relevant disease treatment. The aim of this study is to summarize the existing MAMs and their applications, and lay a foundation for the application of Michael receptor molecules in life science in the future.Methods: This review summarizes the published studies on Michael receptor molecules isolated from plants in literature databases such as CNKI, Wanfang Data, PubMed, Web of Science, ScienceDirect, and Wiley. Latin names of plants were verified through https://www.iplant.cn/. All relevant compound structures were verified through PubChem and literature, and illustrated with ChemDraw 20.0.Result: A total of 50 Michael receptor molecules derived from various plants were discussed. It was found that these compounds have similar pharmacological potential, most of them play a role through the Keap1-Nrf2-ARE pathway and the NF-κB pathway, and have biological activities such as antioxidant and anti-inflammatory. They can be used to treat inflammatory diseases and tumors.Conclusion: The Michael receptor molecule has electrophilicity due to its unsaturated aldehyde ketone structure, which can combine with nucleophilic residues on the protein to form complexes and activate or inhibit the protein pathway to play a physiological role. Michael receptor molecules can regulate the Keap1-Nrf2-ARE pathway and the NF-κB pathway. Michael receptor molecules can be used to treat diseases such as inflammation, cancer, oxidative stress, etc.

Blood glucose monitoring (BGM) using disposable electrodes is commonly used in healthcare diagnosis. The BGM method is not suitable for people with diabetes requiring real-time monitoring who might experience sudden hypoglycemia or hyperglycemia owing to a single measurement at a specific moment. This study aimed to achieve an enhanced stability of glucose diagnosis for continuous glucose measurement systems (CGMs). A representative mediator of a second-generation glucose sensor was synthesized and coordinated with a polymer for immobilization on an indium tin oxide (ITO) electrode. For electrode immobilization, an electrode for enhanced stability was fabricated using the silanization method. The morphological properties of the electrodes were confirmed via cyclic voltammetry (CV), impedance spectroscopy, and SEM. The loss rate of the current density was only 10.11% of the initial current after 8 d. The electrode exhibited a coefficient of determination of R2 = 0.9924, sensitivity of 1.5454 μA/cm2·mM, limit of quantitation (LOQ) of 7.604 μM, and limit of detection (LOD) of 2.509 μM for glucose concentrations between 0.1 and 20.0 mM. The electrode system developed in this study is applicable to the CGM healthcare industry and is expected to be applicable to biofuel cells.

Engineering electrode surfaces through the electrodeposition of gold may provide a range of advantages in the context of biosensor development, such as greatly enhanced surface area, improved conductivity and versatile functionalization. In this work we report on the development of an electrochemical biosensor for the laccase-catalyzed assay of two catecholamines—dopamine and L-epinephrine. Variety of electrochemical techniques—cyclic voltammetry, differential pulse voltammetry, electrochemical impedance spectroscopy and constant potential amperometry have been used in its characterization. It has been demonstrated that the laccase electrode is capable of sensing dopamine using two distinct techniques—differential pulse voltammetry and constant potential amperometry, the latter being suitable for the assay of L-epinephrine as well. The biosensor response to both catecholamines, examined by constant potential chronoamperometry over the potential range from 0.2 to −0.1 V (vs. Ag|AgCl, sat KCl) showed the highest electrode sensitivity at 0 and −0.1 V. The dependencies of the current density on either catecholamine's concentration was found to follow the Michaelis—Menten kinetics with apparent constants KMapp = 0.116 ± 0.015 mM for dopamine and KMapp = 0.245 ± 0.031 mM for L-epinephrine and linear dynamic ranges spanning up to 0.10 mM and 0.20 mM, respectively. Calculated limits of detection for both analytes were found to be within the sub-micromolar concentration range. The biosensor applicability to the assay of dopamine concentration in a pharmaceutical product was demonstrated (with recovery rates between 99% and 106%, n = 3). [ABSTRACT FROM AUTHOR]

The aberrant activation of FGFRs plays a critical role in various cancers, leading to the development of several FGFR inhibitors in clinic. However, the emergence of drug resistance, primarily due to gatekeeper mutations in FGFRs, has limited their clinical efficacy. To address the unmet medical need, a series of 5-amino-1 H -pyrazole-4-carboxamide derivatives were designed and synthesized as novel pan-FGFR covalent inhibitors targeting both wild-type and the gatekeeper mutants. The representative compound 10h demonstrated nanomolar activities against FGFR1, FGFR2, FGFR3 and FGFR2 V564F gatekeeper mutant in biochemical assays (IC 50 = 46, 41, 99, and 62 nM). Moreover, 10h also strongly suppressed the proliferation of NCI–H520 lung cancer cells, SNU-16 and KATO III gastric cancer cells with IC 50 values of 19, 59, and 73 nM, respectively. Further X-ray co-crystal structure revealed that 10h irreversibly binds to FGFR1. The study provides a new promising point for anticancer drug development medicated by FGFRs. The emergence of drug resistance caused by gatekeeper mutations in FGFRs has limited the current drug clinical efficacy, representing a significant unmet medical need. In this study, a series of 5-amino-1 H -pyrazole-4-carboxamide derivatives were designed and synthesized as novel irreversible pan-FGFR inhibitors. The representative compound 10h exhibited high potency against FGFR1, FGFR2, FGFR3, and FGFR2 V564F with IC 50 values of 46, 41, 99, and 62 nM, respectively. Moreover, 10h also strongly suppressed the proliferation of FGFR1-amplified NCI–H520 lung cancer cells, FGFR2-amplified SNU-16 and KATO III gastric cancer cells with IC50 values of 19, 59, and 73 nM, respectively. Further X-ray crystal structures revealed that 10h irreversibly binds to FGFR1. [Display omitted] • A series of 5-amino-1 H -pyrazole-4-carboxamide derivatives were designed as FGFR covalent inhibitors. • 10h can overcome drug resistant FGFR gatekeeper mutants. • X-ray co-crystal structure revealed 10h irreversibly binds to FGFR1. [ABSTRACT FROM AUTHOR]

The characteristics of the crosslinking between rice protein (RP) and ferulic acid (FA), gallic acid (GA), or tannin acid (TA) by covalent binding of Laccase and non-covalent binding were evaluated. The RP-polyphenol complexes greatly improved the functionality of RP. The covalent effect with higher polyphenol binding equivalence showed higher emulsion activity than the non-covalent effect. The solubility, and antioxidant activity of covalent binding were higher than that of non-covalent binding in the RP-FA group, but there was a contrasting behavior in the RP-GA group. The RP-FA was most soluble in conjugates, while the RP-GA had the highest solubility in mixtures. It was found that the covalent complexes were more stable in the intestinal tract. The content of polyphenols in the RP-TA group was rapidly increased at the later intestinal digestion, which indicated the high polyphenol-protective effect in this group. Meanwhile, the RP-TA group showed high reducing power but low digestibility. [Display omitted] • Polyphenols greatly improve the functionality of the protein. • The RP-FA was most soluble in conjugates, while RP-GA had the highest solubility in mixtures. • The RP-TA complexes showed higher reducing power but lower digestibility. • The TA group has a high polyphenol-protective effect in vitro digestion. [ABSTRACT FROM AUTHOR]

The unique structural of anthocyanins makes them vulnerable to degradation within food processing environments. Proteins commonly coexist with polyphenols in natural food matrices on a non-covalent and covalent basis, serving to stabilize protection for polyphenols. Here, three natural pea-extracted proteins, including albumin, vicilin, and legumin were bound to cyanidin-3-O-glucoside (C3G), and binding modes, conjugate structures and functions were characterized. Isothermal titration calorimetry showed a strongly exothermic interaction between C3G and proteins. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fourier transform infrared spectroscopy revealed that the C3G-quinone (C3GQ) formed by C3G oxidation established covalent cross-links with C–S and C–N in the amino acid residues of proteins, and could form more large molecular weight aggregates at an addition amount of 1 mM. The free sulfhydryl and amino contents of the three protein conjugates decreased with the addition of C3GQ. Due to the presence of 4–5 times more sulfhydryl groups in albumin as compared to globulin, albumin showed a 20% higher binding capacity for C3GQ than globulin. This consequently led to the highest antioxidant activity in the C3GQ-albumin conjugate. In contrast to native pea proteins, the introduction of covalent modifications induces changes in the secondary structure of the pea proteins. Specifically, the structure of albumin tends to exhibit increased orderliness, while the molecular flexibility of globular proteins was augmented. Thus, the conjugates have enhanced thermal stability. Our results affirmed that the stability of C3G can be more effectively improved through designing it to covalently interact with proteins relative to non-covalent interactions. This study aimed at offering novel theoretical underpinnings for the functional utilization of C3G and pea proteins in the food industry. [Display omitted] • Quinone-mediated covalent 2 S/7 S/11 S-cyanidin-3-O-glucoside (C3GQ) were prepared. • Main covalent bonds that contribute to the conjugate were C–N and C–S. • The binding preference order for C3GQ was 2 S > 11 S > 7 S. • Covalent binding generated new polyphenol binding sites on pea proteins. • Antioxidant and thermal stability of conjugates were superior to natural pea protein. [ABSTRACT FROM AUTHOR]

[Display omitted] • Tannic acid (TA) and soybean protein isolated (SPI) form TA-SPI complex. • Magnesium ion (M) aggregate TA-SPI to form stronger M-TA-SPI structure. • M retards gastric digestion of M-TA-SPI, so as to protect TA during gastric digestion. • M-TA-SPI is a fine carrier and is beneficial for TA release in intestinal digestion. A ternary complex composed of soybean protein isolated (SPI), tannic acid (TA) and magnesium ion (M) was established to enhance the capability of protein carriers for TA delivery. SPI was firstly covalently bind with TA (TA-SPI) and then M was employed to form the ternary complex (M-TA-SPI). Their structures, gel and digestion properties were further investigated. TA was observed to covalently bind with SPI. TA-SPI and M-TA-SPI complexes showed different molecule size and spatial structures after binding with M and TA. The increasing of TA amount changed the intramolecular interactions, microstructure and texture properties of M-TA-SPI gels. Compared with TA-SPI, M retarded the gastric digestion of M-TA-SPI and caused higher TA release amount in intestinal tract. In this study, M-TA-SPI was determined to be a good carrier to protect and release TA in gastrointestinal digestion. This kind of complex may have potential applications for loading polyphenols in nutraceuticals. [ABSTRACT FROM AUTHOR]

Blockade of the adenosine A2B receptor (A2BAR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A2BAR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα15 protein activation assays by performing ligand wash-out and kinetic experiments. p-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (6a, PSB-21500) was the most potent and selective irreversible A2BAR antagonist of the present series with an apparent Ki value of 10.6 nM at the human A2BAR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative 6c (PSB-21502) was similarly potent, but was non-selective versus A1- and A2AARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (12, Ki 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K2697.32 was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A2BAR, indicating that 6a and related irreversible A2BAR antagonists do not interact with K2697.32. The new irreversible A2BAR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs. [ABSTRACT FROM AUTHOR]

A series of colchicine and thiocolchicine derivatives bearing a Michael acceptor moiety in ring A were synthesized. Some of these compounds exhibit cytotoxic activity in the nanomolar range, efficiently disrupt the mitotic spindle, and cause the accumulation of G2/M-phase cells resulting in the development of apoptosis. These derivatives can be covalently bound to the cysteine residues at the colchicine-binding site of tubulin. [ABSTRACT FROM AUTHOR]

[Display omitted] • Recent advances in tag-mediated protein purification/immobilization are highlited. • Their contribution for single-step protein purification & immobilization is clarified. • Innovative protein-engineered materials developed using protein tags are outlined. • The impact of these technologies on the fabrication of hybrid materials is discussed. • Future opportunities created by these new technologies are identified. The potential applications of protein-engineered functional materials are so wide and exciting that the interest in these eco-friendly advanced materials will further expand in the future. Tag-mediated protein purification/immobilization technologies have emerged as green and cost-effective approaches for the fabrication of such materials. Strategies that combine the purification and immobilization of recombinant proteins/peptides onto/into natural, synthetic or hybrid materials in a single-step are arising and attracting increasing interest. This review highlights the most significant advances of the last 5 years within the scope of tag-mediated protein purification/immobilization and elucidates their contributions for the development of efficient single-step purification and immobilization strategies. Recent progresses in the field of protein-engineered materials created using innovative protein-tag combinations and future opportunities created by these new technologies are also summarized and identified herein. Protein purification/immobilization tags present a remarkable ability to establish specific non-covalent/covalent interactions between solid materials and biological elements, which prompted the creation of tailor-made and advanced functional materials, and of next-generation hybrid materials. Affinity tags can bind to a wide range of materials (of synthetic, natural or hybrid nature), being most suitable for protein purification. Covalently binding tags are most suitable for long-term protein immobilization, but can only bind naturally to protein-based materials. Hybrid affinity-covalently binding tags have allowed efficient one-step purification and immobilization of proteins onto different materials, as well as the development of innovative protein-engineered materials. Self-aggregating tags have been particularly useful in combination with other tags for generating protein-engineered materials with self-assembling, flexible and/or responsive properties. While these tags have been mainly explored for independent protein purification, immobilization or functionalization purposes, efficient strategies that combine tag-mediated purification and immobilization/functionalization in a single-step will be essential to guarantee the sustainable manufacturing of advanced protein-engineered materials. [ABSTRACT FROM AUTHOR]

As a series of metal organic framework (MOFs), materials of institute lavoisier frameworks (MILs) are expected to be excellent supports for enzyme immobilization due to their good water-stability and acid tolerance. However, enzyme loading on MIL-125-NH2 is very low due to small pore size and few amino groups on the surface of the MIL-125-NH2. In this work, catalase (CAT) was immobilized on the MIL-125-NH2 and amino functionalized MIL-125-NH2 by adsorption (CAT@MIL-125-NH2) and covalent binding (CAT@Amino MIL-125-NH2), respectively. Compared with the CAT@MIL-125-NH2 and free CAT, the CAT@Amino MIL-125-NH2 displayed high activity recovery, good pH stability, stability against denaturants, and thermostability. Furthermore, activity recovery of CAT@Amino MIL-125-NH2 was 56% higher than CAT@MIL-125-NH2. The CAT@Amino MIL-125-NH2 still retained 50% residual activity for 14 days at room temperature, whereas free CAT lost activity after storage for 1 day at the same storage conditions. Furthermore, the CAT@Amino MIL-125-NH2 maintained 62% of its initial activity after 4 consecutive uses, showing good reusability. The results showed that the amino functionalized MIL-125-NH2 is an excellent carrier of enzyme immobilization. [ABSTRACT FROM AUTHOR]

Some drugs with carboxylic acid moieties can potentially cause rare but severe hepatotoxicity. The reactive chemical species generated by drug metabolism are thought to be one reason for this event. Although the phase II conjugation metabolism of carboxylic acids generally renders a compound more polar and inactive, it is also responsible for the formation of reactive metabolites. This study aimed to provide a new approach towards the risk assessment of carboxylic acids in the aspect of reactive acyl CoA metabolites. Although acyl CoA metabolites have been concerned, it is difficult to detect them because of their instability. We investigated the trapping agents for acyl CoA metabolites. We found that cysteine is a good trapping agent and developed an assay method for the reactivity of acyl CoA metabolites. We evaluated 17 drugs with carboxylic acid moieties, all drugs concerned with hepatotoxicity displayed reactive potential. With consideration of the exposure of each parent drug, the correlation between drug labels and the calculated risk of carboxylic drugs was improved. These evaluations can be conducted without radiochemical reagents or the authentic standards of metabolites. We believe that the method will be beneficial for drug discovery. [ABSTRACT FROM AUTHOR]