Development of iminosugar-based glycosidase inhibitors as drug candidates for SARS-CoV-2 virus via molecular modelling and in vitro studies.

We developed new iminosugar-based glycosidase inhibitors against SARS-CoV-2. Known drugs (miglustat, migalastat, miglitol, and swainsonine) were chosen as lead compounds to develop three classes of glycosidase inhibitors (α-glucosidase, α-galactosidase, and mannosidase). Molecular modelling of the lead compounds, synthesis of the compounds with the highest docking scores, enzyme inhibition tests, and in vitro antiviral assays afforded rationally designed inhibitors. Two highly active α-glucosidase inhibitors were discovered, where one of them is the most potent iminosugar-based anti-SARS-CoV-2 agent to date (EC90 = 1.94 µM in A549-ACE2 cells against Omicron BA.1 strain). However, galactosidase inhibitors did not exhibit antiviral activity, whereas mannosidase inhibitors were both active and cytotoxic. As our iminosugar-based drug candidates act by a host-directed mechanism, they should be more resilient to drug resistance. Moreover, this strategy could be extended to identify potential drug candidates for other viral infections.

Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense.

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0-11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.

In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D.

Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This study used a combined in silico virtual screening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searched first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking. Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro. After the expression and purification of PLpro, gramicidin D was screened for protease inhibition in vitro and was found to be active against PLpro. The current study's findings are significant because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable safety profile.

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A.

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl ß-d-N,N',N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2-8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy.

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis- Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators.

Enzymes are biological catalysts with many industrial applications, but natural enzymes are usually unsuitable for industrial processes because they are not optimized for the process conditions. The properties of enzymes can be improved by directed evolution, which involves multiple rounds of mutagenesis and screening. By using mathematical models to predict the structure-activity relationship of an enzyme, and by defining the optimal combination of mutations in silico, we can significantly reduce the number of bench experiments needed, and hence the time and investment required to develop an optimized product. Here, we applied our innovative sequence-activity relationship methodology (innov'SAR) to improve glucose oxidase activity in the presence of different mediators across a range of pH values. Using this machine learning approach, a predictive model was developed and the optimal combination of mutations was determined, leading to a glucose oxidase mutant (P1) with greater specificity for the mediators ferrocene-methanol (12-fold) and nitrosoaniline (8-fold), compared to the wild-type enzyme, and better performance in three pH-adjusted buffers. The kcat /KM ratio of P1 increased by up to 121 folds compared to the wild type enzyme at pH 5.5 in the presence of ferrocene methanol.

Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide.

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased kcat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.

A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries.

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.

Comparative stability of ficin and papain in acidic conditions and the presence of ethanol.

Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic-alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic-acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration.

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H.

Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0-5.0 and within the temperature range 50-60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min-1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 µmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Immobilization of Escherichia coli cells expressing 4-oxalocrotonate tautomerase for improved biotransformation of ß-nitrostyrene.

The enzyme 4-oxalocrotonate tautomerase (4-OT) encoded by the xylH gene is a part of the degradation pathway of aromatic compounds in Pseudomonas putida mt-2. 4-OT was described to catalyze Michael-type addition of acetaldehyde to ß-nitrostyrene, and the whole cell system based on recombinantly expressed 4-OT has been developed previously. In this study biocatalytic process based on Escherichia coli whole cells expressing 4-OT was significantly improved using immobilization and ex situ product recovery strategies. Whole cell immobilization in alginate beads was applied in biocatalytic production of 4-nitro-3-phenyl-butanal from ß-nitrostyrene and acetaldehyde. Immobilized biocatalyst showed wider pH activity range and could tolerate twofold higher initial concentrations of substrate in comparison to the free whole cell biocatalyst. Beads retained their initial activity over 10 consecutive biotransformations of the model reaction and remained suitable for the repetitive use with 85% of the initial activity after two months of storage. Bioprocess was further improved by utilizing Amberlite XAD-2 hydrophobic resin for the product recovery. With this modification, the amount of organic solvent was reduced 40-fold in comparison to previously reported method making this biocatalytic process greener.

Soybean hull peroxidase immobilization on macroporous glycidyl methacrylates with different surface characteristics.

Soybean hull peroxidase (SHP, E.C. 1.11.1.7) was immobilized by a glutaraldehyde and periodate method onto series of macroporous copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA), poly(GMA-co-EGDMA) with various surface characteristics and pore size diameters ranging from 44 to 200 nm. Glutaraldehyde immobilization method and poly(GMA-co-EGDMA) named SGE 20/12 with pore sizes of 120 nm gave immobilized enzyme with highest specific activity of 25 U/g. Deactivation studies showed that immobilization increased stability of SHP and that surface characteristics of the used copolymer had a major influence on a stability of immobilized enzyme at high temperatures and in an organic solvent. The highest thermostability was obtained using the copolymer SGE 20/12 with pore size of 120 nm, while the highest stability in dioxane had SHP immobilized onto copolymer SGE 10/4 with pore size of 44 nm. Immobilized SHP showed a wider pH optimum as compared to the native enzyme especially at alkaline pH values and 3.2 times increased K m value for pyrogallol. After 6 cycles of repeated use in batch reactor, immobilized SHP retained 25 % of its original activity. Macroporous copolymers with different surface characteristics can be used for fine tuning of activity and stability of immobilized SHP to obtain a biocatalyst suitable for phenol oxidation or polymer synthesis in organic solvents.

Flow cytometry-based ultra-high-throughput screening assay for cellulase activity.

We have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence-activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes, including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulase clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulase-expressing) cells in cellulase reference libraries after just one sorting round.

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

Selective Adsorption of Ionic Species Using Macroporous Monodispersed Polyethylene Glycol Diacrylate/Acrylic Acid Microgels with Tunable Negative Charge.

Monodispersed polyethylene glycol diacrylate (PEGDA)/acrylic acid (AA) microgels with a tuneable negative charge and macroporous internal structure have been produced using a Lego-inspired droplet microfluidic device. The surface charge of microgels was controlled by changing the content of AA in the monomer mixture from zero (for noncharged PEGDA beads) to 4 wt%. The macroporosity of the polymer matrix was introduced by adding 20 wt% of 600-MW polyethylene glycol (PEG) as a porogen material into the monomer mixture. The porogen was successfully leached out with acetone after UV-crosslinking, which resulted in micron-sized cylindrical pores with crater-like morphology, uniformly arranged on the microgel surface. Negatively charged PEGDA/AA beads showed improved adsorption capacity towards positively charged organic dyes (methylene blue and rhodamine B) compared to neutral PEGDA beads and high repulsion of negatively charged dye molecules (methyl orange and congo red). Macroporous microgels showed better adsorption properties than nonporous beads, with a maximum adsorption capacity towards methylene blue of 45 mg/g for macroporous PEGDA/AA microgels at pH 8.6, as compared to 23 mg/g for nonporous PEGDA/AA microgels at the same pH. More than 98% of Cu(II) ions were removed from 50 ppm solution at pH 6.7 using 2.7 mg/mL of macroporous PEGDA/AA microgel. The adsorption of cationic species was significantly improved when pH was increased from 3 to 9 due to a higher degree of ionization of AA monomeric units in the polymer network. The synthesized copolymer beads can be used in drug delivery to achieve improved loading capacity of positively charged therapeutic agents and in tissue engineering, where a negative charge of scaffolds coupled with porous structure can help to achieve improved permeability of high-molecular-weight metabolites and nutrients, and anti-fouling activity against negatively charged species.

Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination.

BACKGROUND: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. METHODS: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. RESULTS: L-arginine was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. CONCLUSIONS: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents.

Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions.

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.

Identification of SARS-CoV-2 Papain-like Protease (PLpro) Inhibitors Using Combined Computational Approach.

In the current pandemic, finding an effective drug to prevent or treat the infection is the highest priority. A rapid and safe approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 PLpro promotes viral replication and modulates the host immune system, resulting in inhibition of the host antiviral innate immune response, and therefore is an attractive drug target. In this study, we used a combined in silico virtual screening for candidates for SARS-CoV-2 PLpro protease inhibitors. We used the Informational spectrum method applied for Small Molecules for searching the Drugbank database followed by molecular docking. After in silico screening of drug space, we identified 44 drugs as potential SARS-CoV-2 PLpro inhibitors that we propose for further experimental testing.

Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer.

Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.