Action Of Proteases Research Articles

Nutritional implications of exogenous proteases in fish feeding

The increase in demand for fish production drives the search for food strategies to maximize productivity. In this review, the nutritional implications of the use of exogenous proteases on fish growth were described. Exogenous proteases help in digestive processes, acting in the hydrolysis of macromolecules and in the release of smaller particles, such as amino acids and peptides. Dietary supplementation improves fish growth, mainly due to the availability and greater use of nutrients. The action of proteases is directly linked to better intake, feed efficiency and protein synthesis. On the other hand, enzyme activity depends on substrate and dietary composition as there are limitations on digestibility. Furthermore, in feed processing, thermal stability and the form of inclusion of the protease influence its efficiency. However, the increase in fish weight gain can offset the cost of including the enzyme in the diet. However, there are still gaps regarding the effects of protease in fish feeding, for example, enzyme: specific substrate and enzyme: digestive system ratio; and, stabilization technologies (mainly extruded diets), so further studies are needed.

Brain mast cell accumulation and activation in an asymptomatic mouse model of cow’s milk allergy

Abstract Mast cells (MCs) are best known as the effector cells of allergy. Due to their presence in the brain and production of potent inflammatory mediators, MCs have been implicated in the pathogenesis of neuroinflammatory disorders. Previously, we showed that acutely challenging allergically sensitized mice increased the numbers of brain MCs. However, the consequences and mechanism of MC recruitment and activation within the brain are still unclear. We hypothesized that MCs increase in the brain due to allergen infiltration into the central nervous system, which subsequently activates MCs that release histamine, increasing blood-brain barrier (BBB) permeability and neuroinflammation. C57BL6/J mice were sensitized to a bovine whey allergen β-lactoglobulin (BLG) and placed on a whey-containing diet for two weeks to simulate prolonged allergen consumption. Although no overt allergic symptoms were observed, BLG-sensitized mice exhibited depression-like behavior after the allergen consumption period. Serum BLG-specific IgE and histamine were also significantly elevated, validating their hypersensitivity status. In the brains of sensitized mice, MC numbers and histamine levels were increased. IgG extravasation and perivascular astrogliosis were also detected, indicating BBB impairment and neuroinflammation, respectively. Furthermore, myelin staining revealed demyelination in select cortical regions of BLG-sensitized mice, suggesting a potential action of MC-derived proteases. Together, our findings support the involvement of MCs in peripheral-to-central communication during a chronic allergic response. MC stabilization or allergen avoidance may be useful to treat neuroinflammation in susceptible individuals. Supported by grants from the NIH/NIGMS (P20GM103442), UND Host-Pathogen CoBRE Pilot Grant (P20GM113123) and funds from the University of North Dakota SMHS Department of Biomedical Sciences

Design of variants of human fibroblast growth factor 1 with enhanced stability and cell proliferation activity

Human Fibroblast growth factor 1 (hFGF1) is a member of FGF1 subfamily which play significant biological roles, including cell proliferation, cell differentiation, and wound healing. Under physiological temperatures, hFGF1 has low thermodynamic stability and susceptible to the action of proteases. Circular dichroism, fluorescence spectroscopy, and other related studies reveal that reversal of charge on R136 by acidic amino acids (Glu) drastically increases the enzymatic resistance to thrombin, thermal stability, and cell proliferation activity of the growth factor. Interestingly, introduction of R136D mutation shows that the protein stability and cell proliferation activity is similar to the wild‐type protein. Microsecond molecular dynamics studies suggest that length of the side chain of acidic amino acid at position 136 is critical to confer stability to the protein. Detailed analysis of the results will be presented.

Plant Kunitz Inhibitors and Their Interaction with Proteases: Current and Potential Pharmacological Targets

The action of proteases can be controlled by several mechanisms, including regulation through gene expression; post-translational modifications, such as glycosylation; zymogen activation; targeting specific compartments, such as lysosomes and mitochondria; and blocking proteolysis using endogenous inhibitors. Protease inhibitors are important molecules to be explored for the control of proteolytic processes in organisms because of their ability to act on several proteases. In this context, plants synthesize numerous proteins that contribute to protection against attacks by microorganisms (fungi and bacteria) and/or invertebrates (insects and nematodes) through the inhibition of proteases in these organisms. These proteins are widely distributed in the plant kingdom, and are present in higher concentrations in legume seeds (compared to other organs and other botanical families), motivating studies on their inhibitory effects in various organisms, including humans. In most cases, the biological roles of these proteins have been assigned based mostly on their in vitro action, as is the case with enzyme inhibitors. This review highlights the structural evolution, function, and wide variety of effects of plant Kunitz protease inhibitors, and their potential for pharmaceutical application based on their interactions with different proteases.

LIMITED PROTEOLYSIS OF FIBRINOGEN αC-REGION REVEALS ITS STRUCTURE

Aim. The purpose of our study was to compare hydrolytic action of proteases from Gloydius halys halys, Agkistrodon contortrix contortrix and Calloselasma rhodostoma rhodostoma snake venoms and from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium on αC-regions of fibrinogen molecule. Methods. Products of hydrolysis were characterized by SDS-PAGE under reducing conditions with following Western-Blot using the mouse monoclonal 1-5А (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen hydrolysis products was performed using a Voyager-DE. Results. Combination of SDS-PAGE, FPLC and MALDI-TOF analysis enabled to detect the peptide bonds cleaved by studied proteases. In particular proteases from Gloydius halys halys and Agkistrodon contortrix contortrix snake venoms cleaved peptide bond Aα413-414. Action of protease from Calloselasma rhodostoma rhodostoma on fibrinogen led to the formation of hydrolytic product generated from C-terminal portion of Aα-chain that corresponded to fragments generated by enzymes from two other snakes. On the other hand protease from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium cleaved peptide bond Aα504-505. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.

Interaction between protease from Staphylococcus epidermidis and pork myofibrillar protein: Flavor and molecular simulation

This study investigated the influence of a protease from Staphylococcus (S.) epidermidis on the hydrolysis and flavor development in pork myofibrillar protein (MP). The surface hydrophobicity, fluorescence, Fourier transform infrared spectra, and atomic force microscopy analysis indicated that hydrolysis significantly changed surface hydrophobicity and secondary structure of MP (p < 0.05), and improved the stability of MP in water. The contents of free amino acid from MP, especially glutamic and alanine, significantly increased (p < 0.05), and the production of volatile compound such as aldehydes, alcohols and acid were promoted under the action of protease. MP treated with S. epidermidis protease is non-cytotoxic to the HEK-293 cells. Molecular docking analysis suggested that the interaction between the protease and actin was spontaneous and mainly involved hydrogen bonding forces. In summary, this study provides a theoretical basis for the future application of S. epidermidis protease in fermented meat products.

Novel Design of Neuropeptide-Based Drugs with β-Sheet Breaking Potential in Amyloid-Beta Cascade: Molecular and Structural Deciphers.

Our work discusses the investigation of 75 peptide-based drugs with the potential ability to break the β-sheet structures of amyloid-beta peptides from senile plaques. Hence, this study offers a unique insight into the design of neuropeptide-based drugs with β-sheet breaker potential in the amyloid-beta cascade for Alzheimer’s disease (AD). We started with five peptides (15QKLVFF20, 16KLVFF20, 17LVFF20, 16KLVF19 and 15QKLV18), to which 14 different organic acids were attached at the N-terminal. It was necessary to evaluate the physiochemical features of these sequences due to the biological correlation with our proposal. Hence, the preliminary analysis of different pharmacological features provided the necessary data to select the peptides with the best biocompatibility for administration purposes. Our approaches demonstrated that the peptides 17LVFF20, NA-17LVFF20, 16KLVF19 and NA-16KLVF19 (NA-nicotinic acid) have the ability to interfere with fibril formation and hence improve the neuro and cognitive functions. Moreover, the peptide conjugate NA-16KLVF19 possesses attractive pharmacological properties, demonstrated by in silico and in vitro studies. Tandem mass spectrometry showed no fragmentation for the spectra of 16KLVF19. Such important results suggest that under the action of protease, the peptide cleavage does not occur at all. Additionally, circular dichroism confirmed docking simulations and showed that NA-16KLVF19 may improve the β-sheet breaker mechanism, and thus the entanglement process of amyloid-beta peptides can be more effective.

Dipeptidyl Peptidase IV Inhibitory Peptides Generated in Dry-Cured Pork Loin during Aging and Gastrointestinal Digestion.

The ability of peptides from an aqueous and salt-soluble protein extract of dry-cured pork loins to inhibit the action of dipeptidyl peptidase IV was determined. This activity was assessed at different times of the production process, i.e., 28, 90, 180, 270 and 360 days. The resistance of the biological property during the simulated digestive process was also assessed. For this, the extracts were hydrolyzed with pepsin and pancreatin as a simulated digestion step of the gastrointestinal tract and fractionated (>7 kDa) as an intestinal absorption step. The results indicate that dried-pork-loin peptides may have potential as functional food ingredients in the prevention and treatment of type 2 diabetes mellitus. In particular, the APPPPAEV, APPPPAEVH, KLPPLPL, RLPLLP, VATPPPPPPK, VPIPVPLPM and VPLPVPVPI sequences show promise as natural food compounds helpful in maintaining good health.

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity.

Laccases are enzymes produced by plants and white rot fungi, such as Pleurotus ostreatus, with industrial applications. Fungal laccases have been widely studied, and investigations, such as those involving recombinant DNA technology or adding inducers, have been made to increase laccase production. On the other hand, it has been proposed that extracellular proteases could decrease laccase activity when both types of enzymes are produced by P. ostreatus. The aim of this work was to evaluate the effects of proteases on the activity of extracellular laccases produced by P. ostreatus PoB in submerged culture. Results showed that P. ostreatus PoB produced alkaline, acidic, and neutral proteases. Protease activity was quantified, and the highest activity at alkaline pH (9.0) was 5.63 IU/L (192 h), that at acidic pH (2.0) was 3.38 IU/L (192 h), and that at neutral pH (7.0) was 6.20 IU/L (312 h). The protease activity decreased in the presence of different protease inhibitors, as phenylmethylsulfonyl fluoride (PMSF), EDTA, pepstatin A, and a cocktail of protease inhibitors. Laccase activity was determined in cultures with and without protease inhibitors. In the control culture (without inhibitor), the highest laccase specific activity was 99.88 IU/mg protein. In cultures with PMSF, pepstatin A, or a cocktail of protease inhibitors, laccase activity increased by approximately 1.35-fold (138 IU/mg protein) with respect to the control culture. The inhibitor EDTA did not produce a positive effect on extracellular laccase activity. These results suggest that laccase activity is affected by the actions of acidic and neutral extracellular proteases.

Peptidomics as a useful tool in the follow-up of food bioactive peptides.

There is an intense research activity on bioactive peptides derived from food proteins in view of their health benefits for consumers. However, their identification is quite challenging as a consequence of their small size and low abundance in complex matrices such as foods or hydrolyzates. Recent advances in peptidomics and bioinformatics are getting improved sensitivity and accuracy and therefore such tools are contributing to the development of sophisticated methodologies for the identification and quantification of peptides. These developments are very useful for the follow-up of peptides released through proteolysis either in the food itself through the action of endogenous peptidases during processing stages like fermentation, drying or ripening, or from food proteins hydrolyzed by commercial peptidases or microorganisms with proteolytic activity. This chapter is presenting the latest advances in peptidomics and its use for the identification and quantification of peptides, and as a useful tool for controlling the proteolysis phenomena in foods and protein hydrolyzates.

The Study of Sensitivity to Proteolysis of Full-length and Truncated Forms of Recombinant Human Renalase Expressed in the Prokaryotic System

Renalase (RNLS) is a flavoprotein; its N-terminal peptide (amino acid residues 1-17) performs various important functions. Inside cells, it is involved in the Rossmann fold formation (residues 2-35), which is necessary for the binding of the FAD cofactor and the manifestation of the enzymatic activity of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). When RNLS is secreted into the extracellular space, this peptide is cleaved off, and the resulting truncated extracellular RNLS can no longer bind FAD and, therefore, numerous effects described in the literature are carried out by non-catalytic mechanisms. In this work, we have investigated the sensitivity to trypsinolysis of two recombinant forms of human RNLS expressed in prokaryotic cells: (a) full-length RNLS containing the FAD cofactor; (b) a truncated RNLS lacking the 1-17 N-terminal peptide (truncatedRNLS, tRNLS) unable to bind the FAD cofactor. Trypsin (1 unit/20 μL of medium) effectively cleaved both forms of renalase (RNLS and tRNLS). When exposed to a lower concentration of trypsin (0.1 U/20 μL of medium), full length RNLS was more trypsin resistant than tRNLS. We suggest that the different sensitivity of RNLS and tRNLS is apparently determined by the presence of the FAD cofactor in the full-length recombinant protein, which contributes to the formation of a spatial structure that is more resistant to the action of certain proteases.

Zona pellucida in the process of fertilization and mammal embryonal development

In mammals, oocytes, fertilized eggs and pre-implantation embryos are surrounded by an acellular zona pellucida (zona pellucida – ZP). This structure has a fibro-spongy character but it undergoes constant modifications throughout its existence depending on many internal and external factors. ZP consists of glycoproteins marked as ZP1, ZP2, ZP3 and ZP4, the presence of which is species different. ZP1 and probably ZP4 molecules stabilize the fibrillary skeleton of the zona pellucida formed of ZP2 and ZP3 protein polymers which are ligands for specific spermatozoid receptors. The oligosaccharide chains of ZP3 are responsible for the primary attachment of the male gamete which induces the acrosomal reaction. ZP2 enhances this connection by secondary binding to an acrosome-free spermatozoid. Additionally, oviductal specific glycoprotein 1 which plays a role in interspecific oocyte-sperm interactions, appears around the postovulatory oocyte surrounded by ZP. In addition, this protein modifies the resistance of ZP to the action of proteases released as a result of the cortical reaction during polyspermia block. After fertilization, ZP not only protects the egg and then the embryo until implantation, but also has an embryotrophic effect. Understanding the molecular basics of the structure and properties of ZP can significantly improve animal fertility as well as reproductive rates.

LIMITED PROTEOLYSIS OF FIBRINOGEN BY PROTEASE OF Gloydius halys halys SNAKE VENOM

Aim. One of the approaches for studying structure and functions of proteins is their limited proteolysis. Proteolytic fragments of macromolecules can preserve the biological activity and can be used for the study of their structural and functional peculiarities. Thus, the characterization of new proteolytic enzymes and determination of the specificity of their action can be of interest for exploration. In the present work, we focused on the action of protease from the venom of Gloydius halys halys on fibrinogen, the crucial protein of blood coagulation system. Methods. Products of fibrinogen hydrolysis by protease from the venom of G. halys halys were studied by SDS-PAGE electrophoresis and western-blot analysis using monoclonal antibodies ІІ-5 Сand 1-5A targeted to 20‒78 and 549‒610 fragments of fibrinogen Aα-chain. Molecular weights of hydrolytic products were determined using MALDI-TOF analysis on Voyager DE PRO (USA). Sequence of hydrolytic products were predicted by «Peptide Mass Calculator» soft ware. Results. SDS-PAGE showed that protease from the venom of Gloydius halys halys initially cleaved Аα-chain of fibrinogen molecule. Western-blot analysis confirmed that this protease specifically cleaves off fragment of C-terminal parts of Аα-chain with apparent molecular weight of 22 kDa. Cleaved fragment was identified by MALDI-TOF analysis as the 21.1 kDa polypeptide. «Peptide Mass Calculator» predicted that such a fragment corresponded to Аα414-610 residue of fibrinogen molecule. Thus, we showed that studied protease cleaved peptide bond AαK413-L414 with the formation of stable partly hydrolyzed fibrinogen desAα414-610. Conclusions. The use of protease from the venom of Gloydius halys halys would allow obtaining the unique partly hydrolyzed fibrinogen des Aα 414‒610 that is suitable for the study of structure and functions of fibrinogen αС-regions.

Fate of undigested proteins in the pig large intestine: What impact on the colon epithelium?

Apart from its obvious agronomic interest in feeding billions of people worldwide, the porcine species represents an irreplaceable experimental model for intestinal physiologists and nutritionists. In this review, we give an overview on the fate of proteins that are not fully digested in the pig small intestine, and thus are transferred into the large intestine. In the large intestine, dietary and endogenous proteins are converted to peptides and amino acids (AA) by the action of bacterial proteases and peptidases. AA, which cannot, except in the neonatal period, be absorbed to any significant level by the colonocytes, are used by the intestinal microbes for protein synthesis and for the production of numerous metabolites. Of note, the production of the AA-derived metabolites greatly depends on the amount of undigested polysaccharides in the pig's diet. The effects of these AA-derived bacterial metabolites on the pig colonic epithelium have not yet been largely studied. However, the available data, performed on colonic mucosa, isolated colonic crypts and colonocytes, indicate that some of them, like ammonia, butyrate, acetate, hydrogen sulfide (H2S), and p-cresol are active either directly or indirectly on energy metabolism in colonic epithelial cells. Further studies in that area will certainly gain from the utilization of the pig colonic organoid model, which allows for disposal of functional epithelial unities. Such studies will contribute to a better understanding of the potential causal links between diet-induced changes in the luminal concentrations of these AA-derived bacterial metabolites and effects on the colon epithelial barrier function and water/electrolyte absorption.

Health Implications of Bt Cotton Seeds in Animals and Man

Cotton (Gossypium spp.) is an arborous plant from the Malvaceae family. Cottonseed meal is a by-product of cotton that is used for animal feeding because it is rich in oil and proteins. However, gossypol toxicity limits cottonseed use in animal feed. Gossypol is a phenolic compound produced by pigment glands in cotton stems, leaves, seeds and flower buds. Cotton is a long duration crop and is attacked by a number of insect pests. Earlier, insecticide quantity applied on cotton was highest but with the advent of Bt cotton, there is a great reduction in insecticide and pesticide usage as majority of the bollworms are controlled by it. Bt cotton refers to transgenic cotton which contains endotoxin protein inducing gene from soil bacterium Bacillus thuringiensis. It was discovered by a Japanese scientist Ishiwata in 1901. B.thuringiensis strains produces 3 types of insecticidal toxins that are Crystal toxins (Cry), Cytolytic toxins (Cyt) and vegetatively expressed insecticidal proteins (vip). Bt-delta-endotoxins which functions as oral toxins are ingested by insect and protoxins are proteolytically activated to trypsin-resistant active core δ- endotoxin in alkaline mid-gut. The active toxin binds to cadherin receptors present on the brush border membrane of the insect midgut. Cadherins process the toxins to form homo-oligomers, bind to specific receptors like alkaline phosphatases and aminopeptidases before causing pores in the epithelial membrane, resulting in osmotic lysis of the cells which results in cessation of feeding and finally mortality of the insect. Cry toxins have not been reported to be toxic to higher animals such as goats, sheep and cattle in any part of the world as scientific evidences indicate that the Cry toxins do not get activated under the acidic conditions of non-target animals such as goat, sheep and cattle. In one study of acute oral toxicity study in rats, Bt cotton seed material did not induce any treatment related observable toxic effects when compared with Non-Bt cottonseeds. Histological studies in another study revealed that there were no difference in cellular architecture in liver, heart, kidney and intestine of Bt and non-Bt diet fed rats. The Cry toxins Cry1Ac, Cry1Ab, Cry2Ab, Cry1F and Cry1C are considered to be safe to human beings. The stomach of humans, being the first organ of digestion which the Bt protein encounters, is acidic and contains proteases like pepsin which degrade the Bt protein. Thus, the alkaline conditions needed for pro-toxin solubilization and protease action required for toxin activation are absent in the stomach. More importantly, the human intestine lacks the specific receptors to which the activated Bt protein binds and initiates the physiological effect. However, there are several reports that Bt genes cause some serious problems to human health. Cry1Ac toxin at higher concentration have lethal cytotoxic and genotoxic effects on the human lymphocytes. The present review is an attempt to provide details from the available literature regarding uses of Bt cotton and its implications on animal and human health.

Bioaccessibility and Gut Metabolism of Free and Melanoidin-Bound Phenolic Compounds From Coffee and Bread.

The aim of this study is to investigate the bioaccessibility and gut metabolism of free and melanoidin-bound phenolic compounds from coffee and bread. Phenolics from coffee were predominantly found in free forms (68%, mainly chlorogenic acids), whereas those from bread were mostly bound to melanoidins (61%, mainly ferulic acid). Bioacessibility of coffee total free phenolics slightly decreased during simulated digestion (87, 86, and 82% after the oral, gastric, and intestinal steps, respectively), with caffeoylquinic acids being isomerized and chlorogenic acids being partially hydrolyzed to the corresponding hydroxycinnamic acids. Bioacessibility of bread total free phenolics decreased during simulated digestion (91, 85, and 67% after the oral, gastric, and intestinal steps, respectively), probably related to complexation with the proteins in simulated gastric and intestinal fluids. Upon gut fermentation, the bioaccessibility of total free phenolics from both coffee and bread decreased, mainly after the first 4 h (56 and 50%, respectively). Caffeic and ferulic acids were the predominant metabolites found during coffee and bread gut fermentation, respectively. Melanoidin-bound phenolics from coffee and bread were progressively released after the gastric and intestinal steps, probably due to hydrolysis caused by the acidic conditions of the stomach and the action of pancreatin from the intestinal fluid. The bioaccessibilities of all phenolics from coffee and bread melanoidins after the gastric and intestinal steps were, on average, 11 and 26%, respectively. During gut fermentation, phenolics bound to both coffee and bread melanoidins were further released by the gut microbiota, whereas those from coffee were also metabolized. This difference could be related to the action of proteases on melanoproteins during gastrointestinal digestion, probably anticipating phenolics release. Nevertheless, bioaccessibilities of melanoidin-bound phenolics reached maximum values after gut fermentation for 24 h (50% for coffee and 51% for bread). In conclusion, the bioaccessibilities of coffee and bread free phenolics during simulated digestion and gut fermentation were remarkably similar, and so were the bioaccessibilities of coffee and bread melanoidin-bound phenolics.

Euphorbia trigona latex nematicidal activity on the root-knot nematode Meloidogyne incognita

The aim of this study was to test nematicidal activity of the Euphorbia trigona latex on Meloidogyne incognita juveniles and to partially purify and characterize three proteases present in this latex. Three distinct proteases were partially purificated from E. trigona latex. They were named here trigonin 1, 2 and 3. Their molecular weights were estimated at: 36, 31 and 29 kDa, for trigonin 1, 2 and 3, respectively. The pH and temperature that provided highest protease activity were pH values of 4.0, 6.0 and 9.0, and 70 °C. The crude extract containing the three proteases present in E. trigona latex reduced significantly (p &lt; 0.01) the number of live M. incognita juveniles in 96% after 24 hours treatment. The present study is the first report of E. trigona latex with nematicidal activity, therefore more studies regarding latex proteases action on nematodes are needed.

A widespread mechanism in ectomycorrhizal fungi to access nitrogen from mineral-associated proteins.

A large fraction of nitrogen (N) in forest soils is present in mineral-associated proteinaceous compounds. The strong association between proteins and minerals limits microbial accessibility to this source, which is a relatively stable reservoir of soil N. We have shown that the ectomycorrhizal (ECM) fungus Paxillus involutus can acquire N from iron oxide-associated proteins. Using tightly controlled isotopic, spectroscopic and chromatographic experiments, we demonstrated that the capacity to access N from iron oxide-associated bovine serum albumin (BSA) is shared with the ECM fungi Hebeloma cylindrosporum and Piloderma olivaceum. Despite differences in evolutionary history, growth rates, exploration types and the decomposition mechanisms of organic matter, their N acquisition mechanisms were similar to those described for P. involutus. The fungi released N from mineral-associated BSA by direct action of extracellular aspartic proteases on the mineral-associated BSA, without initial desorption of the protein. Hydrolysis was suppressed by the adsorption of proteases to minerals, but this adverse effect was counteracted by the secretion of compounds that conditioned the mineral surface. These data suggest that the enzymatic exudate-driven mechanism to access N from mineral-associated proteins is found in ECM fungi of multiple lineages and exploration types.

Punicalagin and zinc (II) ions inhibit the activity of SARS-CoV-2 3CL-protease in vitro.

Coronavirus 2019 (COVID-19) has now been declared as a worldwide pandemic. Currently, no drugs have been endorsed for its treatment; in this manner, a pressing need has been developed for any antiviral drugs that will treat COVID-19. Coronaviruses require the SARS-CoV-2 3CL-Protease (3CL-protease) for cleavage of its polyprotein to yield a single useful protein and assume a basic role in the disease progression. In this study, we demonstrated that punicalagin, the fundamental active element of pomegranate in addition to the combination of punicalagin with zinc (Zn) II, appear to show powerful inhibitory activity against SARS-CoV-2. The 3CL protease assay kit was used to quantify 3CL protease action. The tetrazolium dye, MTS, was used to evaluate cytotoxicity. Punicalagin showed inhibitory action against the 3CL-protease in a dose-dependent manner, and IC50 was found to be 6.192 μg/ml for punicalagin. Punicalagin (10 µg/mL) demonstrated a significant inhibitory activity toward 3CL-protease activity (p < 0.001), yet when punicalagin is combined with zinc sulfate monohydrate (punicalagin/Zn-II) extremely strong 3CL-protease activity (p < 0.001) was obtained. The action of 3CL-protease with punicalagin/Zn-II was decreased by approximately 4.4-fold in contrast to only punicalagin (10 µg/mL). Likewise, we did not notice any significant cytotoxicity caused by punicalagin, Zn-II, or punicalagin/Zn-II. We suggest that these compounds could be used as potential antiviral drugs against COVID-19.

A physicochemical, structural, microbiological and kinetic study of hen egg white lysozyme in complexes with alginate and chitosan

Complexes of hen egg white lysozyme (HEWL) with alginate (ALG) and chitosan (CS) (natural biodegradable polysaccharides) are important as antimicrobial agents in oral, intranasal and buccal administration. The HEWL–ALG, HEWL–ALG–Ca, [HEWL–ALG]–CS, and [HEWL–ALG–Ca]–CS complexes have been investigated using a combination of physicochemical methods (fluorescent spectroscopy, circular dichroism, microscopy, turbidimetry, microbiology, in vitro cytotoxicity assay). The complexes obtained were non-toxic negatively charged microparticles with high HEWL entrapment efficiency. The [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes were found to have the altered tertiary structure of HEWL (and/or changes in the polarity of the microenvironment of Trp residues in HEWL molecules). The HEWL entrapment in all studied complexes was carried out with retention of the HEWL activity. Under the long–term storage conditions (1.5 years at 4 °C), Ca++ cations and CS stabilized HEWL. Under physiological conditions, the [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes demonstrated sustained release of HEWL and resistance to protease action. The [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes were highly active against Gram-negative Escherichia coli cells. The data obtained are important in the development of antibacterial drugs based on lytic enzymes.