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The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4 days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture. [ABSTRACT FROM AUTHOR]

AbstractContext The effect of the active ingredients in traditional Chinese medicines on the activity of cytochrome P450 enzymes (CYP450s) is a critical factor that should be considered in TCM prescriptions. Physcion, the major active ingredient of Rheum spp. (Polygonaceae), possesses wide pharmacological activities.Objectives The effect of physcion on CYP450 activity was investigated to provide a theoretical basis for use.Materials and methods The experiments were conducted in pooled human liver microsomes (HLMs). The activity of CYP450 isoforms was evaluated with corresponding substrates and probe reactions. Blank HLMs were set as negative controls, and typical inhibitors were employed as positive controls. The inhibition model was fitted with Lineweaver Burk plots. The concentration (0, 2.5, 5, 10, 25, 50 and 100 μM physcion) and time-dependent (0, 5, 10, 15 and 30 min) effects of physcion were also assessed.Results Physcion suppressed CYP2C9, 2D6 and 3A4 in a concentration-dependent manner with IC50 values of 7.44, 17.84 and 13.50 μM, respectively. The inhibition of CYP2C9 and 2D6 was competitive with the Ki values of 3.69 and 8.66 μM, respectively. The inhibition of CYP3A4 was non-competitive with a Ki value of 6.70 μM. Additionally, only the inhibition of CYP3A4 was time-dependent with the KI and Kinact parameters of 3.10 μM−1 and 0.049 min−1, respectively.Conclusions The inhibition of CYP450s by physcion should be considered in its clinical prescription, and the study design can be employed to evaluate the interaction of CYP450s with other herbs.

The emergence of drug resistance in cancer cells eventually causing relapse is a serious threat that demands new advances. Upregulation of the ATP‐dependent binding cassette (ABC) transporters, such as ABCB1, significantly contributes to the emergence of drug resistance in cancer. Despite more than 30 years of therapeutic discovery, and several generations of inhibitors against P‐gp, the search for effective agents that minimize toxicity to human cells, while maintaining efflux pump inhibition is still underway. Leads derived from natural product scaffolds are well‐known to be effective in various therapeutic approaches. Inspired by the biosynthetic pathway to Nocardioazine A, a marine alkaloid known to inhibit the P‐gp efflux pump in cancer cells, we devised a regioselective pathway to create structurally unique indole‐C3‐benzyl cyclo‐L‐Trp‐L‐Trp diketopiperazines (DKPs). Using bat cells as a model to derive effective ABCB1 inhibitors for targeting human P‐gp efflux pumps, we have recently identified exo‐C3‐N‐Dbn‐Trp2 (13) as a lead ABCB1 inhibitor. This C3‐benzylated lead inhibited ABCB1 better than Verapamil.[21] Additionally, C3‐N‐Dbn‐Trp2 restored chemotherapy sensitivity in drug‐resistant human cancer cells and had no adverse effect on cell proliferation in cell cultures. For a clearer structure‐activity relationship, we developed a broader screen to test C3‐functionalized pyrroloindolines as ABCB1 inhibitors and observed that C3‐benzylation is outperforming respective isoprenylated derivatives. Results arising from the molecular docking studies indicate that the interactions at the access tunnel between ABCB1 and the inhibitor result in a powerful predictor for the efficacy of the inhibitor. Based on fluorescence‐based assays, we conclude that the most efficacious inhibitor is the p‐cyano‐derived exo‐C3‐N‐Dbn‐Trp2 (33 a), closely followed by the p‐nitro substituted analogue. By combining assay results with molecular docking studies, we further correlate that the predictions based on the inhibitor interactions at the access tunnel provide clues about the design of improved ABCB1 inhibitors. As it has been well documented that ABCB1 itself is powerfully engaged in multi‐drug resistance, this work lays the foundation for the design of a new class of inhibitors based on the endogenous amino acid‐derived cyclo‐L‐Trp‐L‐Trp DKP scaffold. [ABSTRACT FROM AUTHOR]

Novel inhibitors of GlcNAc-Ts have been reported based on benzoxazolone appended amino acids/esters which showed a competitive inhibition of the enzyme with IC50 values in lower micromolar range. The compounds 1b–5b showed a better inhibition profile against GlcNAc-Ts as compared to 1a–5a as evidenced by the in vitro 96 well plate ELISA experiment and the in vitro cytotoxicity studies on the cancer cell lines PDAC, Caco-2, MDA-MB-231, HT-29, PC-3, and A549. The execution of ELISA and cytotoxicity experiments on the compounds 1a–5a in the presence of esterase enzyme along with the appraisal of the lipophilicity index, and calculation of Ki and Ka values for the enzyme-inhibitor complex further indicated that the compounds 1a–5a serve as prodrugs of the compounds 1b–5b owing to a better lipophilicity index of the former. Double reciprocal plots were constructed for 2 different concentrations of the test compounds 1b–5b and that of the natural substrate of OGT. A competitive mode of inhibition was observed for the test molecules which was further confirmed by molecular docking analysis of these molecules in the active site of OGT in the presence of its natural inhibitor. Mechanistic evaluation of the inhibition of OGT by test molecules was performed by NMR and HPLC experiments where hydrolysis of exocyclic urea linkage in the test molecules was observed on their incubation with the enzyme over a period. [ABSTRACT FROM AUTHOR]

The H 2 O 2 -assisted oxidation by a peroxidase enzyme takes place to help plants maintain the concentrations of organic compounds at physiological levels. Cells regulate the oxidation rate by inhibiting the action of this enzyme. The cells use two inhibitory processes to regulate the enzyme: a noncompetitive substrate inhibitory process and a competitive substrate inhibitory process. Numerous applications of peroxidase have been developed in clinical biochemistry, enzyme immunoassays, the treatment of waste water containing phenolic compounds, the synthesis of various aromatic chemicals, and the removal of peroxide from industrial wastes. The kinetic mechanism of the Spanish broom peroxidase enzyme is a Ping Pong Bi Bi mechanism with the presence of competitive inhibition by substrates. A mathematical model may help in identifying the key mechanism from amongst a set of competing mechanisms. In this study, we developed a fractional mathematical model to describe the H 2 O 2 -supported oxidation by the enzyme Spanish broom peroxidase. Numerical simulations of the model produced results that are consistent with the known behaviour of Spanish broom peroxidase. Finally, some future investigations of the study are briefly indicated as well. [ABSTRACT FROM AUTHOR]

To assess the effect of Rehmannioside A on CYP450s activity and to estimate its inhibitory properties. The effect of Rehmannioside A on the activity of major CYP450s in human liver microsomes (HLMs) was assessed with the corresponding substrates and marker reactions, and compared with a blank control and the respective inhibitors. Suppression of CYP3A4, 2C9 and 2D6 was assessed by the dose-dependent assay and fitted with non-competitive or competitive inhibition models. The inhibition of CYP3A4 was determined in a time-dependent manner. Rehmannioside A suppressed the activity of CYP3A4, 2C9, and 2D6 with IC50 values of 10.08, 12.62, and 16.43 μM, respectively. Suppression of CYP3A4 was fitted to a non-competitive model with Ki value of 5.08 μM, whereas CYP2C9 and 2D6 were fitted to a competitive model with Ki values of 6.25 and 8.14 μM. Additionally, the inhibitory effect on CYP3A4 was time-dependent with KI value of 8.47 μM−1 and a Kinact of 0.048 min−1. In vitro suppression of CYP3A, 2C9 and 2D6 by Rehmannioside A indicated that Rehmannioside A or its source herbs may interact with drugs metabolised by these CYP450s, which could guide the clinical application. [ABSTRACT FROM AUTHOR]

Trichoderma parestonica is a novel source of manganese peroxidase (MnP), [E.C.1.11.1.13] enzyme. The following was the sequence in which T. parestonica induced the production of MnP in the liquid culture medium: rice husk, corncob, sawdust, coir-dust and dried luffa on days 3, 4, 5, 2 and 3, respectively. The molecular weight of the enzyme was found to be 45kDa, determined from SDS-PAGE analysis which was further confirmed by mass analysis and was found to be 51.9kDa. Enzyme characterization parameters like optimum pH, temperature, Km, Kcat and catalytic efficiency are 5, 20ᵒC, 0.014mM (for Mn2+) and 0.028mM (for H2O2), 23.9 S-1 and 1.7 μM-1 S-1 respectively. Effect of various metal ions, such as bivalent Copper(Cu), Cobalt(Co), Mercury(Hg), Nickel(Ni), Cadmium(Cd), trivalent Chromium(Cr), Iron(Fe), Tetra-valent cerium(Ce), Penta-valent vanadium(V) and hexavalent molybdenum(Mo) the enzyme-catalyzed steady-state velocity was studied. The inhibition constant and nature of inhibition by these metal ions were determined. In the current investigation, MnP catalytic activities demonstrated that it could effectively catalyze the oxidation of phenols, aromatic amines and wastewater treatment. This communication suggested potential pathways for aromatic amine bioconversion mediated by MnP [ABSTRACT FROM AUTHOR]

The development of new substances with the ability to interact with a biological target is only the first stage in the process of the creation of new drugs. The 5-nitroisatin derivatives considered in this study are new inhibitors of cyclin-dependent kinase 2 (CDK2) intended for anticancer therapy. The research, carried out based on the ADMET (absorption, distribution, metabolism, excretion, toxicity) methods, allowed a basic assessment of the physicochemical parameters of the tested drugs to be made. The collected data clearly showed the good oral absorption, membrane permeability, and bioavailability of the tested substances. The analysis of the metabolite activity and toxicity of the tested drugs did not show any critical hazards in terms of the toxicity of the tested substances. The substances' low solubility in water meant that extended studies tested compounds were required, which helped to select solvents with a high dissolving capacity of the examined substances, such as DMSO or NMP. The use of aqueous binary mixtures based on these two solvents allowed a relatively high solubility with significantly reduced toxicity and environmental index compared to pure solvents to be maintained, which is important in the context of the search for green solvents for pharmaceutical use. [ABSTRACT FROM AUTHOR]

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0–50 μM) were incubated with isoform‐specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2‐catalyzed phenacetin O‐deethylation, exhibiting IC50 values of 9.39 ± 2.05 and 3.07 ± 0.45 μM with and without preincubation in the presence of β‐NADPH, respectively. Moreover, suberosin showed concentration‐dependent, but not time‐dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver‐Burk plot, we found that suberosin competitively inhibited CYP1A2‐catalyzed phenacetin O‐deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb–drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates. [ABSTRACT FROM AUTHOR]

The interactions of the classic phytohormones gibberellic acid (gibberellin A3, GA3) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S‐transferase P1‐1 (hpGSTP1‐1), an enzyme that plays a role in endo‐ or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1‐1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1‐1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f–[GSH]v and at [GSH]f–[CDNB]v, Vm, Km, and Ki values were statistically estimated to be 205 ± 16 μmol/min‐mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 μmol/min‐mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm, Km, and Ki obtained with GA3 at [CDNB]f–[GSH]v and at [GSH]f–[CDNB]v were found to be 303 ± 14 μmol/min‐mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 μmol/min‐mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen‐bonding and electrostatic interactions with the key residues that line the G‐ and H‐sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1‐1 may guide medicinal chemists through the structure‐based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics. [ABSTRACT FROM AUTHOR]

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

The H2O2-assisted oxidation by a peroxidase enzyme takes place to help plants maintain the concentrations of organic compounds at physiological levels. Cells regulate the oxidation rate by inhibiting the action of this enzyme. The cells use two inhibitory processes to regulate the enzyme: a noncompetitive substrate inhibitory process and a competitive substrate inhibitory process. Numerous applications of peroxidase have been developed in clinical biochemistry, enzyme immunoassays, the treatment of waste water containing phenolic compounds, the synthesis of various aromatic chemicals, and the removal of peroxide from industrial wastes. The kinetic mechanism of the Spanish broom peroxidase enzyme is a Ping Pong Bi Bi mechanism with the presence of competitive inhibition by substrates. A mathematical model may help in identifying the key mechanism from amongst a set of competing mechanisms. In this study, we developed a fractional mathematical model to describe the H2O2-supported oxidation by the enzyme Spanish broom peroxidase. Numerical simulations of the model produced results that are consistent with the known behaviour of Spanish broom peroxidase. Finally, some future investigations of the study are briefly indicated as well.

Objectives: To better understand the unique inhibitory behavior of a non-natural cofactor preferred formaldehyde dehydrogenase (FalDH) mutant 9B2. Results: We described our serendipitous observation that 9B2 was reversibly inhibited by residual imidazole introduced during protein preparation, while the wild-type enzyme was not sensitive to imidazole. Kinetic analysis showed that imidazole was a competitive inhibitor of formaldehyde with a Ki of 16 μM and an uncompetitive inhibitor of Nicotinamide Cytosine Dinucleotide for 9B2, indicating that formaldehyde and imidazole were combined in the same position. Molecular docking results of 9B2 showed that imidazole could favorably bind very close to the nicotinamide moiety of the cofactor, where formaldehyde was expected to reside for catalysis, which was in line with a competitive inhibition. Conclusion: The mutant 9B2 can be competitively inhibited by imidazole, suggesting that cautions should be taken to evaluate activities as protein mutants might attain unexpected sensitivity to a component in buffers for purification or activity assays. [ABSTRACT FROM AUTHOR]

Context Alpinetin, the major active constitutes of Alpinia katsumata Hayata (Zingiberaceae), has been demonstrated to possess the activity of anti-breast cancer. Cytochrome P450 enzymes (CYP450s) plays vital roles in the biotransformation of various drugs.Objective To assess the effect of alpinetin on the activity of CYP450s and estimate the inhibition characteristics.Materials and methods The activity of CYP450s was evaluated in pooled human liver microsomes with corresponding substrates and marker reactions. The effect of alpinetin was compared with blank control (negative control) and corresponding inhibitors (positive control). The dose-dependent and time-dependent experiments were conducted in the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM alpinetin and incubated for 0, 5, 10, 15, and 30 min.Results Alpinetin suppressed CYP3A4, 2C9, and 2E1 activity. All the inhibitions were significantly influenced by alpinetin contration with the IC50 values of 8.23 μM (CYP3A4), 12.64 μM (CYP2C9), and 10.97 μM (CYP2E1), respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with a Ki value of 4.09 μM and was time-dependent with KI and Kinact values of 4.67 min and 0.041 μM−1, respectively. While CYP2C9 and 2E1 were inhibited by alpinetin competitively with Ki values of 6.42 (CYP2C9) and 5.40 μM (CYP2E1), respectively, in a time-independent manner.Discussion and conclusion The in vitro inhibitory effect of alpineticn on CYP3A, 2C9, and 2E1 implied the potential interaction of alpinetin or its origin herbs with the drugs metabolised by those CYP450s, which needs further in vivo validation.

Medicinal plants belonging to the genus Berberis may be considered an interesting source of drugs to counteract the problem of antimicrobial multiresistance. The important properties associated with this genus are mainly due to the presence of berberine, an alkaloid with a benzyltetrahydroisoquinoline structure. Berberine is active against both Gram-negative and Gram-positive bacteria, influencing DNA duplication, RNA transcription, protein synthesis, and the integrity of the cell surface structure. Countless studies have shown the enhancement of these beneficial effects following the synthesis of different berberine analogues. Recently, a possible interaction between berberine derivatives and the FtsZ protein was predicted through molecular docking simulations. FtsZ is a highly conserved protein essential for the first step of cell division in bacteria. The importance of FtsZ for the growth of numerous bacterial species and its high conservation make it a perfect candidate for the development of broad-spectrum inhibitors. In this work, we investigate the inhibition mechanisms of the recombinant FtsZ of Escherichia coli by different N-arylmethyl benzodioxolethylamines as berberine simplified analogues appropriately designed to evaluate the effect of structural changes on the interaction with the enzyme. All the compounds determine the inhibition of FtsZ GTPase activity by different mechanisms. The tertiary amine 1c proved to be the best competitive inhibitor, as it causes a remarkable increase in FtsZ Km (at 40 μM) and a drastic reduction in its assembly capabilities. Moreover, a fluorescence spectroscopic analysis carried out on 1c demonstrated its strong interaction with FtsZ (Kd = 26.6 nM). The in vitro results were in agreement with docking simulation studies. [ABSTRACT FROM AUTHOR]

The soluble and fermentable carbohydrate contents was detected over 47% of glucose and fructose in Chinese Elaeagnus angustifolia fruit powder (EAF), being over 47 wt% sugar content more than that of grape. Ethanol was therefore fermented directly from EAF, and different submerged fermentation modes were comparatively employed to optimize ethanol harvest. The results indicated that glucose has certain competitive inhibition on fructose bio-utilization, as well as the EAF solid residue involved fermentation mode also hindered the fermented-ethanol titer. Pectinase addition and in situ hydrolysis seemed to assist somewhat the fermentation. The water-solute fermentation mode is preferable, and glucose and fructose components were completely consumed and converted to 80.96 g/L ethanol at 87.6% ethanol yield even under tannin and pectin inhibition. The fermentation result could provide some experimental data and an approach to not only new biomass resource explores of bioethanol and alcohol beverage production, but also the technological development on valorization commercials of EAF in global draught areas. [ABSTRACT FROM AUTHOR]

Athrixia phylicoides DC. (Bush tea) is a shrub harvested in the north-eastern mountain regions of South Africa and belongs to the Asteraceae family. Generally, A. phylicoides is consumed as a hot tea beverage for its associated health benefits. The use of bush tea extracts for beauty enhancement has not been investigated even though several ethnobotanical reports have indicated its usage against skin imperfections. Therefore, the aim of the study was to assess plant metabolites of A. phylicoides for their inhibition of tyrosinase from mushroom and determine their inhibitory mechanism. Methanolic extracts (80% v/v) of A. phylicoides were evaluated using a tyrosinase-based TLC (thin-layer chromatography) autography technique. The inhibitory mechanism of active metabolites against the enzyme was determined using Lineweaver–Burk plots. Quercetin and an unknown metabolite with a retention factor (Rf) value of 0.73 inhibited melanogenesis. However, the IC50 value for Quercetin was reported as 51.07 ± 2.43 µg/mL higher than that of kojic acid of 5.22 ± 1.44 µg/mL. Chlorogenic acid was reported to have a similar Rf value as kojic acid, suggesting similarities in polarity and affinity towards the adsorbent material. However, chlorogenic acid with an IC50 value of 15.25 ± 1.18 µg/mL and an inhibitory strength of 2.92 could not inhibit melanogenesis with a similar intensity as kojic acid. The inhibitory mechanism for A. phylicoides extract was reported to be mixed inhibition (competitive/uncompetitive). The IC50 value for A. phylicoides was recorded as 20.65 ± 0.14 µg/mL with an inhibitory strength of 3.96. These results suggest that A. phylicoides extracts could be used against dark spots associated with scarring and ageing through modulation of tyrosinase activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cryptic sites potentially expand the space of druggable proteins. • Cryptic sites formed solely by side chain motion don't provide nanomolar ligand binding affinity. • However, cryptic sites formed by loop and domain motion can be valuable drug targets. • The difference in affinity is due to the much shorter time scale of side chain motion. • The different behavior is explained in terms of inhibition models and the kinetics of binding. Cryptic sites can expand the space of druggable proteins, but the potential usefulness of such sites needs to be investigated before any major effort. Given that the binding pockets are not formed, the druggability of such sites is not well understood. The analysis of proteins and their ligands shows that cryptic sites that are formed primarily by the motion of side chains moving out of the pocket to enable ligand binding generally do not bind drug-sized molecules with sufficient potency. By contrast, sites that are formed by loop or hinge motion are potentially valuable drug targets. Arguments are provided to explain the underlying causes in terms of classical enzyme inhibition theory and the kinetics of side chain motion and ligand binding. [ABSTRACT FROM AUTHOR]

The development of new substances with the ability to interact with a biological target is only the first stage in the process of the creation of new drugs. The 5-nitroisatin derivatives considered in this study are new inhibitors of cyclin-dependent kinase 2 (CDK2) intended for anticancer therapy. The research, carried out based on the ADMET (absorption, distribution, metabolism, excretion, toxicity) methods, allowed a basic assessment of the physicochemical parameters of the tested drugs to be made. The collected data clearly showed the good oral absorption, membrane permeability, and bioavailability of the tested substances. The analysis of the metabolite activity and toxicity of the tested drugs did not show any critical hazards in terms of the toxicity of the tested substances. The substances’ low solubility in water meant that extended studies tested compounds were required, which helped to select solvents with a high dissolving capacity of the examined substances, such as DMSO or NMP. The use of aqueous binary mixtures based on these two solvents allowed a relatively high solubility with significantly reduced toxicity and environmental index compared to pure solvents to be maintained, which is important in the context of the search for green solvents for pharmaceutical use.

BackgroudLeptospira interrogans is the major causative agent of leptospirosis, a worldwide zoonotic disease. Hemorrhage is a typical pathological feature of leptospirosis. Binding of von Willebrand factor (vWF) to platelet glycoprotein-Ibα (GPIbα) is a crucial step in initiation of platelet aggregation. The products of L. interrogans vwa-I and vwa-II genes contain vWF-A domains, but their ability to induce hemorrhage has not been determined.MethodsHuman (Hu)-platelet- and Hu-GPIbα-binding abilities of the recombinant proteins expressed by L. interrogans strain Lai vwa-I and vwa-II genes (rLep-vWA-I and rLep-vWA-II) were detected by flowcytometry, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Hu-platelet aggregation and its signaling kinases and active components were detected by lumiaggregometry, Western analysis, spectrophotometry and confocal microscopy. Hu-GPIbα-binding sites in rLep-vWA-I and rLep-vWA-II were identified by SPR/ITC measurements.FindingsBoth rLep-vWA-I and rLep-vWA-II were able to bind to Hu-platelets and inhibit rHu-vWF/ristocetin-induced Hu-platelet aggregation, but Hu-GPIbα-IgG, rLep-vWA-I-IgG and rLep-vWA-II-IgG blocked this binding or inhibition. SPR and ITC revealed a tight interaction between Hu-GPIbα and rLep-vWA-I/rLep-vWA-II with KD values of 3.87 × 10-7-8.65 × 10-8 M. Hu-GPIbα-binding of rL-vWA-I/rL-vWA-II neither activated the PI3K/AKT-ERK and PLC/PKC kinases nor affected the NO, cGMP, ADP, Ca2+ and TXA2 levels in Hu-platelets. G13/R36/G47 in Lep-vWA-I and G76/Q126 in Lep-vWA-II were confirmed as the Hu-GPIbα-binding sites. Injection of rLep-vWA-I or rLep-vWA-II in mice resulted in diffuse pulmonary and focal renal hemorrhage but this hemorrhage was blocked by rLep-vWA-I-IgG or rLep-vWA-II-IgG.InterpretationThe products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation.

Macroautophagy/autophagy is a finely-regulated process in which cytoplasm encapsulated within transient organelles termed autophagosomes is delivered to lysosomes or vacuoles for degradation. Phospholipids, particularly phosphatidic acid (PA) that functions as a second messenger, play crucial and differential roles in autophagosome formation; however, the underlying mechanism remains largely unknown. Here we demonstrated that PA inhibits autophagy through competitive inhibition of the formation of ATG3 (autophagy-related)-ATG8e and ATG6-VPS34 (vacuolar protein sorting 34) complexes. PA bound to GAPC (glyceraldehyde-3-phosphate dehydrogenase) or PGK (phosphoglycerate kinase) and promoted their interaction with ATG3 or ATG6, which further attenuated the interactions of ATG3-ATG8e or ATG6-VPS34, respectively. Structural and mutational analyses revealed the mechanism of PA binding with GAPCs and PGK3, and that GAPCs or ATG8e competitively interacted with ATG3, and PGK3 or VPS34 competitively interacted with ATG6, at the same binding interface. These results elucidate the molecular mechanism of how PA inhibits autophagy through binding GAPC or PGK3 proteins and expand the understanding of the functional mode of PA, demonstrating the importance of phospholipids in plant autophagy and providing a new perspective for autophagy regulation by phospholipids. Abbreviation: ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; Con A: concanamycin A; ER: endoplasmic reticulum; EZ: elongation zone; FRET-FLIM: fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; MDC: monodansylcadaverine; MZ: meristem zone; PA: phosphatidic acid; PAS: phagophore assembly site; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PGK3: phosphoglycerate kinase; PtdIns3K: phosphatidylinositol 3-kinase; PLD: phospholipase D; TEM: transmission electron microscopy; TOR: target of rapamycin; VPS34: vacuolar protein sorting 34; WT: wild type; Y2H: yeast two-hybrid. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin type A (BoNT/A) is an exceptionally potent neurotoxin of great therapeutic value; however, it is also considered a weapon of mass destruction, as it is one of the most poisonous biological substances known to man. The etiology behind BoNT/A is its action as a zinc-dependent protease, which can cause extended paralysis through the cleavage of SNARE proteins. Thiosemicarbazones, known zinc chelators, provide a privileged scaffold that can be leveraged for the development of BoNT/A LC inhibitors. Through a combination of biochemical and kinetic assays, it was demonstrated that the thiosemicarbazone ZMC1, an antitumor agent, is an effective competitive inhibitor of the BoNT/A LC. Based on these results, a series of thiosemicarbazones were designed/synthesized using structure-based analysis and examined in enzyme activity and cell-based assays. From this screen, two analogues presented noteworthy cellular activity. The most potent inhibitors were then tested in a BoNT/A mouse lethality assay, providing statistically significant prolonged survival.

The endo-polygalacturonase D (PgaD) from Aspergillus niger JL15 was recombinantly expressed in Escherichia coli BL21, exhibiting an optimal activity at 55 °C and pH 4.0. Hydrolysis products of citrus pectin by recombinant PgaD included galacturonic acid (GalA), digalacturonic acid (GalA2), trigalacturonic acid (GalA3), and tetragalacturonic acid (GalA4). The hydrolysates exhibited significant antioxidant capacity and dose-dependent competitive inhibition of α-glucosidase. GalA2 and GalA3 acted as competitive inhibitors of α-glucosidase, with inhibition constant of 0.0589 mmol.L -1 and 0.6732 mmol.L -1 , respectively. Molecular dynamics (MD) simulations revealed that both GalA2 and GalA3 penetrated the catalytic pocket of α-glucosidase and formed stable hydrogen bonds with key catalytic residues D352 and D215. The binding free energies of GalA2-α-glucosidase and GalA3-α-glucosidase complexes were - 10.3 ± 0.6 kcal·mol -1 and -10.8 ± 0.7 kcal·mol -1 , respectively. These findings might offer new ideas for the development of α-glucosidase inhibitors sourced from citrus pectin, as well as enhance utilization of the renewable plant polysaccharide resources., 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 Ltd. All rights reserved.)

Tumors are still one of the main causes of death; therefore, the search for new therapeutic agents that will enable the implementation of effective treatment is a significant challenge for modern pharmacy. One of the important factors contributing to the development of neoplastic diseases is the overexpression of enzymes responsible for the regulation of cell division processes such as cyclin-dependent kinases. Numerous studies and examples of already-developed drugs confirm that isatin is a convenient basis for the development of new groups of inhibitors for this class of enzyme. Therefore, in this work, a new group of potential inhibitors of the CDK2 enzyme, utilizing isatin derivatives and substituted benzoylhydrazines, has been designed based on the application of computational chemistry methods, such as docking and molecular dynamics, and their inhibiting ability was assessed. In the cases of the selected compounds, a synthesis method was developed, and the selected physicochemical properties of the newly synthesized derivatives were estimated. As part of the completed project, new compounds are developed which are potential inhibitors of the CDK2 enzyme. [ABSTRACT FROM AUTHOR]

Camellia sinensis (tea) seeds have been identified as potential sources of nutraceutical compounds. In this study, caffeine and theaflavanoside IV were annotated as the most abundant phytochemicals in the seed shells of C. sinensis. Both compound displayed potent inhibitions against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 37.9 ± 3.5 and 8.7 ± 1.1 µM, respectively. In the kinetic study, caffeine inhibited PTP1B with mixed type I mode, which prefers to bind to free enzyme. Theaflavanoside IV showed competitive and reversible simple slow-binding inhibition [k3 = 0.1 µM−1·min−1, k4 = 0.002 min−1, Kiapp = 0.0002 µM]. This is the first report on PTP1B-inhibitory activity of these compounds and their action mechanisms. These results suggest their potential in the development of antidiabetic agents. [ABSTRACT FROM AUTHOR]

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyse the same reaction, oxidative cleavage of L-tryptophan (L-Trp) to N -formyl-kynurenine. In both enzymes, the ferric form is inactive and ascorbate (Asc) is frequently used as a reductant in in vitro assays to activate the enzymes by reducing the heme iron. Recently, it has been reported that Asc activates IDO2 by acting as a reductant; however, it is also a competitive inhibitor of the enzyme. Here, the effect of Asc on human TDO (hTDO) is investigated. Similar to its interaction with IDO2, Asc acts as both a reductant and a competitive inhibitor of hTDO in the absence of catalase, and its inhibitory effect was enhanced by the addition of H2O2. Interestingly, however, no inhibitory effect of Asc was observed in the presence of catalase. TDO is known to be activated by H2O2 and a ferryl-oxo (FeIV=O) intermediate (Compound II) is generated during the activation process. The observation that Asc acts as a competitive inhibitor of hTDO only in the absence of catalase can be explained by assuming that the target of Asc is Compound II. Asc seems to compete with L-Trp in an unusual manner. [ABSTRACT FROM AUTHOR]

Angiotensin I-converting enzyme (ACE) regulates blood pressure through the renin-angiotensin system. Douchi, a traditional fermented soybean condiment, may have antihypertensive effects, but research on ACE inhibitory peptides from Douchi hydrolysates is limited. We hypothesized that enzymatic treatment could enhance ACE inhibitory peptide diversity and efficacy. We tested ten single enzymes and four combinations, finding pepsin-trypsin-chymotrypsin most effective. Hydrolysates were purified using Sephadex G-15 and reversed-phase HPLC, and peptides were identified via LC-MS/MS. Five peptides (LF, VVF, VGAW, GLFG, NGK) were identified, with VGAW as the most potent ACE inhibitor (IC 50 46.6 ± 5.2 μM) showing excellent thermal and pH stability. Lineweaver-Burk plots confirmed competitive inhibition, and molecular docking revealed eight hydrogen bonds between VGAW and ACE. In hypertensive rats, VGAW significantly reduced blood pressure at 12.5, 25, and 50 mg/kg. These findings highlight Douchi as a source of ACE inhibitory peptides and suggest VGAW as a promising functional food ingredient. [Display omitted] • Douchi hydrolysate was identified as a source of ACE inhibitory peptides. • VGAW was a competitive ACE inhibitor with an IC 50 of 46.6 ± 5.2 μM. • Oral administration of VGAW reduced systolic and diastolic blood pressure in SHRs. • VGAW formed eight hydrogen bonds with amino acids in ACE S1 and S2 pockets. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the regulatory mechanism of ferulic acid (FA) on proso millet starch (PMS) digestion. When the FA addition amount 0.2 g per g of starch, the relative crystallinity values of nonwaxy PMS (NS)-FA complexes and waxy PMS (WS)-FA complexes were higher by 15% and 6%, and the specific surface area decreased by 0.10 and 0.03 m2/g, respectively. The PMS-FA complexes gradually formed non-V-type inclusion aggregates with increased FA addition. FA could also inhibit digestive enzyme activity by binding to the active sites of α-amylase and amyloglucosidase. Compared with WS-FA, NS-FA complexes were more suitable for the preparation of low-glycemic index foods. The steric hindrance of enzymes by the complex structure and inhibitory effect of FA on the enzymes were the major reasons for the reduced digestibility of starch in complexes. These findings provided evidence for the use of FA to reduce the postprandial glycemic response of starchy foods. [Display omitted] • Ferulic acid altered starch structure forming steric hindrance to enzymes. • The particle size of complexes gradually increased with ferulic acid added. • The starch in vitro digestibility gradually reduced with ferulic acid added. • Ferulic acid bound to the active sites of enzyme by competitive inhibition. • Nonwaxy proso millet starch is more suitable for preparing low-GI foods. [ABSTRACT FROM AUTHOR]

Improving substrate utilization is crucial for industrial conversion of starch. The introduction of pullulanase in cyclodextrin (CD) production has improved substrate utilization. However, complete utilization of debranched starches has not been achieved, mainly due to the retrogradation of debranched starches and the inhibitory effect of CDs on pullulanase during the catalytic reaction. This study aimed to establish an efficient CD preparation system by regulating the substrate concentration and reaction temperature. A Bacillus thermoleovorans pullulanase (BtPul) was selected for investigation of its interaction with CDs through inhibition pattern assays, fluorescence spectroscopy, and molecular docking. Results clarified that CDs inhibited BtPul, with α-CD showing the highest inhibition, followed by β-CD and γ-CD. γ-CD inhibited BtPul through a mixed mode of competitive and noncompetitive inhibition, while the other CDs inhibited BtPul competitively. When pullulan was used as the substrate, increasing the substrate concentration weakened the inhibitory effect of CDs to some degree, while changes in reaction temperature only had a minor contribution. When BtPul and β-CGTase collaboratively converted 30% (w/v) starch substrate, an increase in reaction temperature improved the conversion rate of β-CD from 67.6% to 86.8%. Optimization of substrate concentration showed that high concentration starch substrate had an adverse effect on CDs yield due to retrogradation. The optimal yields of α-CD and β-CD produced by BtPul and α-/β-CGTases were 55.7% and 90.3%, respectively. This study provides a reliable method and reference for the efficient conversion of starch into CDs. [ABSTRACT FROM AUTHOR]

Medicinal plants belonging to the genus Berberis may be considered an interesting source of drugs to counteract the problem of antimicrobial multiresistance. The important properties associated with this genus are mainly due to the presence of berberine, an alkaloid with a benzyltetrahydroisoquinoline structure. Berberine is active against both Gram-negative and Gram-positive bacteria, influencing DNA duplication, RNA transcription, protein synthesis, and the integrity of the cell surface structure. Countless studies have shown the enhancement of these beneficial effects following the synthesis of different berberine analogues. Recently, a possible interaction between berberine derivatives and the FtsZ protein was predicted through molecular docking simulations. FtsZ is a highly conserved protein essential for the first step of cell division in bacteria. The importance of FtsZ for the growth of numerous bacterial species and its high conservation make it a perfect candidate for the development of broad-spectrum inhibitors. In this work, we investigate the inhibition mechanisms of the recombinant FtsZ of Escherichia coli by different N-arylmethyl benzodioxolethylamines as berberine simplified analogues appropriately designed to evaluate the effect of structural changes on the interaction with the enzyme. All the compounds determine the inhibition of FtsZ GTPase activity by different mechanisms. The tertiary amine 1c proved to be the best competitive inhibitor, as it causes a remarkable increase in FtsZ Km (at 40 μM) and a drastic reduction in its assembly capabilities. Moreover, a fluorescence spectroscopic analysis carried out on 1c demonstrated its strong interaction with FtsZ (Kd = 26.6 nM). The in vitro results were in agreement with docking simulation studies.

This article discusses the importance of D-xylose for fighting viruses (especially SARS-CoV-2) that use core proteins as receptors at the cell surface, by providing additional supporting facts that these viruses probably bind at HS/CS attachment sites (i.e., the hydroxyl groups of Ser/Thr residues of the core proteins intended to receive the D-xylose molecules to initiate the HS/CS chains). Essentially, the additional supporting facts, are: some anterior studies on the binding sites of exogenous heparin and soluble HS on the core proteins, the inhibition of the viral entry by pre-incubation of cells with heparin, and additionally, corroborating studies about the mechanism leading to type 2 diabetes during viral infection. We then discuss the mechanism by which serine protease inhibitors inhibit SARS-CoV-2 entry. The biosynthesis of heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (Hep) is initiated not only by D-xylose derived from uridine diphosphate (UDP)-xylose, but also bioactive D-xylose molecules, even in situations where cells were previously treated with GAG inhibitors. This property of D-xylose shown by previous anterior studies helped in the explanation of the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This explanation is completed here by a preliminary estimation of xyloside GAGs (HS/CS/DS/Hep) in the body, and with other previous studies helping to corroborate the mechanism by which the D-xylose exhibits its antiglycaemic properties and the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This paper also discusses the confirmatory studies of regarding the correlation between D-xylose and COVID-19 severity. [ABSTRACT FROM AUTHOR]

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18β-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

Context: The effect of the active ingredients in traditional Chinese medicines on the activity of cytochrome P450 enzymes (CYP450s) is a critical factor that should be considered in TCM prescriptions. Physcion, the major active ingredient of Rheum spp. (Polygonaceae), possesses wide pharmacological activities., Objectives: The effect of physcion on CYP450 activity was investigated to provide a theoretical basis for use., Materials and Methods: The experiments were conducted in pooled human liver microsomes (HLMs). The activity of CYP450 isoforms was evaluated with corresponding substrates and probe reactions. Blank HLMs were set as negative controls, and typical inhibitors were employed as positive controls. The inhibition model was fitted with Lineweaver Burk plots. The concentration (0, 2.5, 5, 10, 25, 50 and 100 μM physcion) and time-dependent (0, 5, 10, 15 and 30 min) effects of physcion were also assessed., Results: Physcion suppressed CYP2C9, 2D6 and 3A4 in a concentration-dependent manner with IC 50 values of 7.44, 17.84 and 13.50 μM, respectively. The inhibition of CYP2C9 and 2D6 was competitive with the K i values of 3.69 and 8.66 μM, respectively. The inhibition of CYP3A4 was non-competitive with a K i value of 6.70 μM. Additionally, only the inhibition of CYP3A4 was time-dependent with the K I and K inact parameters of 3.10 μM -1 and 0.049 min -1 , respectively., Conclusions: The inhibition of CYP450s by physcion should be considered in its clinical prescription, and the study design can be employed to evaluate the interaction of CYP450s with other herbs.

Copyright of China Surfactant Detergent & Cosmetics (1001-1803) is the property of China Surfactant Detergent & Cosmetics Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Most plant primary transcripts undergo alternative splicing (AS), and its impact on protein diversity is a subject of intensive investigation. Several studies have uncovered various mechanisms of how particular protein splice isoforms operate. However, the common principles behind the AS effects on protein function in plants have rarely been surveyed. Here, on the selected examples, we highlight diverse tissue expression patterns, subcellular localization, enzymatic activities, abilities to bind other molecules and other relevant features. We describe how the protein isoforms mutually interact to underline their intriguing roles in altering the functionality of protein complexes. Moreover, we also discuss the known caseswhen these interactions have been placed inside the autoregulatory loops. This review is particularly intended for plant cell and developmental biologists who would like to gain inspiration on how the splice variants encoded by their genes of interest may coordinately work. [ABSTRACT FROM AUTHOR]

1, 5-Anhydro-D-fructose (1, 5-AF), which is produced from starch by the action of alpha-1, 4-glucan lyase, competitively inhibited Agaricus tyrosinase (L-tyrosine, L-dopa: oxygen oxidoreductase, EC 1.14.18.1) with a Ki value of ~20 mM. In silico modelling of the catalytic subunit of the enzyme docked with a 1, 5-AF molecule showed that only the keto form of the 1, 5-AF molecule could bind to the bottom of the active site of tyrosinase. The binding mode seemed to be very similar to that of tropolone, a strong, competitive inhibitor. 1, 5-AF also inhibited an enzyme partially purified from wheat flour and retarded the browning of commercial avocado paste. Thus, its colour protection activity, in conjunction with its bacteriostatic effect, appears applicable to food processing. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The rich chemical, physical, electronic, and surface properties of two-dimensional (2D) materials have witnessed an explosion of research leading to enrichment in the 2D family of materials. Cobalt sulfide (CoxSy), a transition metal chalcogenide, has attracted significant interest as its different stoichiometric and non-stoichiometric phases offer diverse properties. To date, the Co3S4 phase has seen limited developments with recent reports suggesting good catalytic properties of this material. In the current study, we show the facile synthesis of phase pure 2D Co3S4 nanosheets using a hydrothermal approach and demonstrate the importance of the chemical reactivity of the surface atoms in achieving target selectivity during sensing. First, the catalytic activity of Co3S4 nanosheets was observed to follow Michaelis–Menten kinetics, suggesting that the nanosheets mimic the catalytic activity of the natural peroxidase enzymes. The presence of dangling sulfur atoms on the surface of the Co3S4 nanosheets allowed them to specifically interact with l-cysteine amino acid that resulted in the temporary loss of its catalytic activity. This interaction allowed us to develop a "turn-off" colorimetric sensor to detect l-cysteine even in complex mixtures of amino acids and other sulfur containing compounds. In-depth understanding of the sensor mechanism revealed a reversible competition between l-cysteine and peroxidase substrates for binding to the Co3S4 nanosheets. Due to the higher affinity of l-cysteine to the nanosheets, it first binds to the Co3S4 nanosheets blocking its nanozyme activity. Subsequently, the outstanding catalytic activity of 2D Co3S4 nanosheets assists in the oxidation of l-cysteine, and as oxidized products leave the nanozyme surface, its catalytic activity resumes. The findings of our study are likely to instigate further research into the exploration of the chemical reactivity of 2D surfaces for diverse applications. [ABSTRACT FROM AUTHOR]

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (K i) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was −30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239 OsXIP stably interacted with the catalytic site E140 AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104 AfXylA10 /V99 AfXylA10 -Q5 OsXIP , R256 AfXylA10 -E235 OsXIP , D155 AfXylA10 -Y243 OsXIP and D145 AfXylA10 -R194 OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP. • Family GH10 AfXylA10 (reAfXylA10) was recombinantly expressed in P. pastoris X33. • reAfXylA10 was inhibited by rePOsXIP via competitive inhibition. • α-7 helix of OsXIP anchored in catalytic cleft of AfXylA10 based on MD simulations. • Hydrogen bonding played an essential role in AfXylA10-OsXIP interaction. [ABSTRACT FROM AUTHOR]

Most plant primary transcripts undergo alternative splicing (AS), and its impact on protein diversity is a subject of intensive investigation. Several studies have uncovered various mechanisms of how particular protein splice isoforms operate. However, the common principles behind the AS effects on protein function in plants have rarely been surveyed. Here, on the selected examples, we highlight diverse tissue expression patterns, subcellular localization, enzymatic activities, abilities to bind other molecules and other relevant features. We describe how the protein isoforms mutually interact to underline their intriguing roles in altering the functionality of protein complexes. Moreover, we also discuss the known cases when these interactions have been placed inside the autoregulatory loops. This review is particularly intended for plant cell and developmental biologists who would like to gain inspiration on how the splice variants encoded by their genes of interest may coordinately work.

Aims: Allergic airway disease manifestation is induced by lysophosphatidylcholine (LPC) through CD1d-restricted Natural killer T (NKT) cells. Choline chloride (ChCl) and LPC both have the “choline” moiety in their structure and this may interplay the effect in allergic airway disease pathway. Main methods: To test the hypothesis, mice were sensitized with cockroach extract (CE); challenged with CE or exposed to LPC and were given ChCl 1hr later. Key findings: A significant increase in Airway hyperresponsiveness (AHR), total and differential cell count, Th2 cytokines, 8-isoprostanes level in bronchoalveolar lavage fluid (BALF) and inflammation score based on lung histology were observed on challenge with CE or exposure to LPC (p ​

Fermentable sugar for bioethanol production can be produced from molasses due to its high sucrose content but Ca2+ ions found in the molasses may affect the hydrolysis. Therefore, this paper was focused to study the effect of Ca2+ ions as CaO on sucrose hydrolysis using invertase and to obtain the kinetic parameters. The kinetic parameters (KM and Vmax) were obtained using a Lineweaver-Burk plot. The value of KM and Vmax parameters were 36.181 g/L and 21.322 g/L.h, respectively. The Ca2+ ions act as competitive inhibitor in sucrose hydrolysis using invertase. Therefore, the inhibition mechanism was followed the competitive inhibition mechanism. The value of inhibition constant was 0.833 g/g. These parameters were obtained from the non-substrate inhibition process because this study used the low substrate concentrations which means the fermentable sugar production was low. Hence, there were still more challenges to studying the simultaneous effect of substrate and Ca2+ on sucrose hydrolysis to produce high fermentable sugar.

Athrixia phylicoides DC. (Bush tea) is a shrub harvested in the north-eastern mountain regions of South Africa and belongs to the Asteraceae family. Generally, A. phylicoides is consumed as a hot tea beverage for its associated health benefits. The use of bush tea extracts for beauty enhancement has not been investigated even though several ethnobotanical reports have indicated its usage against skin imperfections. Therefore, the aim of the study was to assess plant metabolites of A. phylicoides for their inhibition of tyrosinase from mushroom and determine their inhibitory mechanism. Methanolic extracts (80% v/v) of A. phylicoides were evaluated using a tyrosinase-based TLC (thin-layer chromatography) autography technique. The inhibitory mechanism of active metabolites against the enzyme was determined using Lineweaver–Burk plots. Quercetin and an unknown metabolite with a retention factor (Rf) value of 0.73 inhibited melanogenesis. However, the IC50 value for Quercetin was reported as 51.07 ± 2.43 µg/mL higher than that of kojic acid of 5.22 ± 1.44 µg/mL. Chlorogenic acid was reported to have a similar Rf value as kojic acid, suggesting similarities in polarity and affinity towards the adsorbent material. However, chlorogenic acid with an IC50 value of 15.25 ± 1.18 µg/mL and an inhibitory strength of 2.92 could not inhibit melanogenesis with a similar intensity as kojic acid. The inhibitory mechanism for A. phylicoides extract was reported to be mixed inhibition (competitive/uncompetitive). The IC50 value for A. phylicoides was recorded as 20.65 ± 0.14 µg/mL with an inhibitory strength of 3.96. These results suggest that A. phylicoides extracts could be used against dark spots associated with scarring and ageing through modulation of tyrosinase activity.

Enzyme inhibition is an important area of medicinal research. Enzyme inhibitor possess a wide application in agriculture, clinical science and used as drug, antibiotics and preservatives. Benzoic acid is used as preservative in many food materials. Hence inhibition of Jack bean urease catalysed hydrolysis of urea by p-nitro benzoic acid and p-chloro benzoic acid has been studied on the basis of kinetic study of hydrolysis of urea. During the experimental study the concentration of substrate was varied in the range of 1.00 x 10-2 to 2.65 x 10-2 and that of inhibitor was maintained at 2.5 x 10-3. The results of the inhibition effect are quantitatively expressed in terms of Apparent Michaelis constants Kmapp and enzymeinhibitor constant, Ki at pH 6.80 and temperature 37.0°C. Both the inhibitor shows competitive type of inhibition where both substrate and inhibitor compete with each other to react with active site of enzyme. [ABSTRACT FROM AUTHOR]

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