Peroxidase-catalyzed polymerization and depolymerization of coal in organic solvents

Peroxidases from horseradish roots (HRP) and soybean hulls (SBP) catalyze the efficient polymerization of a 4-kDa dimethylformamide (DMF)-soluble fraction of Mequininza (Spanish) lignite in 50% (v/v) DMF with an aqueous component consisting of acetate buffer, pH 5.0. Under these conditions, HRP and SBP catalyze the oxidation of free phenolic moieties in the coal matrix, thereby leading to oxidative polymerization of the low-molecular-weight coal polymers. The high fraction of nonphenolic aromatic moieties in coal inspired us to examine conditions whereby such coal components could also become oxidized. Oxidation of nonphenolic aromatic compounds was attempted using veratryl alcohol as a model substrate. SBP catalyzed the facile oxidation of veratryl alcohol at pH <3.HRP, however, was unable to elicit veratryl alcohol oxidation. The potential for SBP to catalyze interunit bond cleavage on complex polymeric substrates was examined using l-(3,4-dimethoxyphenyl)-2-(phenoxy)propan-1,3-diol (1) as a substrate. SBP catalyzed the Cα-Cβ and β-ether bond cleavage of this compound, suggesting that similar reactions on coal, itself, could lead to depolymerization. Depolymerization of a >50 Da coal fraction was achieved using SBP in 50% (v/v) DMF with an aqueous component adjusted to pH 2.2. Approximately 15% of the initial high-molecular-weight lignite fraction was depolymerized to polymers 4 Da in size. Hence, SBP is capable of catalyzing the depolymerization of coal in organic solvents, and this may have important ramifications in the generation of liquid fuels from coals.     

Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation

Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.     

CoMFA modeling of enzyme kinetics: K(m) values for sulfation of diverse phenolic substrates by human catecholamine sulfotransferase SULT1A3

Three-dimensional QSAR models were developed for predicting kinetic Michaelis constant (K(m)) values for phenolic substrates of human catecholamine sulfating sulfotransferase (SULT1A3). The K(m) values were correlated to the steric and electronic molecular fields of the substrates utilizing Comparative Molecular Field Analysis (CoMFA). The evaluated SULT1A3 substrate data set consisted of 95 different substituted phenols, catechols, catecholamines, steroids, and related structures for which the K(m) values were available. The data set was divided in three different subgroups in the initial analysis: (1). for the first CoMFA model substrates with only one reacting hydroxyl group were selected (n = 51), (2).the second model was build with structurally rigid substrates (n = 59), and (3). finally all substrates of the data set were included in the analysis (n = 95). Substrate molecules were aligned using the aromatic ring and the reacting hydroxyl group as a template. After the initial analysis different substrate alignment rules based on the existing knowledge of the SULT1A3 active site structure were evaluated. After this optimization a final CoMFA model was built including all 95 substrates of the data set. Cross-validated q(2) values (leave-one-out and leave-n-out) and coefficient contour maps were calculated for all derived CoMFA models. All four CoMFA models were statistically significant with q(2) values up to 0.624. These predictive QSAR models will provide us information about the factors that affect substrate binding at the active site of human catecholamine sulfotransferase SULT1A3.     

QSAR modeling of in vitro inhibition of cytochrome P450 3A4

We report the QSAR modeling of cytochrome P450 3A4 (CYP3A4) enzyme inhibition using four large data sets of in vitro data. These data sets consist of marketed drugs and drug-like compounds all tested in four assays measuring the inhibition of the metabolism of four different substrates by the CYP3A4 enzyme. The four probe substrates are benzyloxycoumarin, testosterone, benzyloxyresorufin, and midazolam. We first show that using state-of-the-art QSAR modeling approaches applied to only one of these four data sets does not lead to predictive models that would be useful for in silico filtering of chemical libraries. We then present the development and the testing of a multiple pharmacophore hypothesis (MPH) that is formulated as a conceptual extension of the traditional QSAR approach to modeling the promiscuous binding of a large variety of drugs to CYP3A4. In the simplest form, the MPH approach takes advantage of the multiple substrate data sets and identifies the binding of test compounds as either proximal or distal relative to that of a given substrate. Application of the approach to the in silico filtering of test compounds for potential inhibitors of CYP3A4 is also presented. In addition to an improvement in the QSAR modeling for the inhibition of CYP3A4, the results from this modeling approach provide structural insights into the drug-enzyme interactions. The existence of multiple inhibition data sets in the BioPrint database motivates the original development of the concept of a multiple pharmacophore hypothesis and provides a unique opportunity for formulating alternative strategies of QSAR modeling of the inhibition of the in vitro metabolism of CYP3A4.     

Bi‐enzyme Electrochemical Sensor for Selective Determination of Organophosphorus Pesticides with Phenolic Leaving Groups

An amperometric bi‐enzyme sensor for detection of organophosphorus pesticides (OPs) with phenolic leaving groups, which are not electroactive, is presented in this work. The biosensing platform was created by a simple, controllable, and reproducible one‐step electrodeposition onto the surface of a glassy carbon electrode of a chitosan bionanocomposite with entrapped carboxylated multi walled carbon nanotubes, organophosphorus hydrolase (OPH), and horseradish peroxidase (HRP). The OPs determination involved a sequence of OPH and HRP‐catalyzed reactions resulting in phenolic radicals production, which were quantified by registering the current of their reduction at a potential of −50 mV vs. Ag, AgCl/KCl_sat.The developed sensor was applied for the determination of prothiofos, as an example. At optimized conditions (pH 7.25 and H₂O₂ concentration 200 μmol L⁻¹), a LOD as low as 0.8 μmol L⁻¹ was attained, while the linear concentration range was extended from 2.64 μmol L⁻¹ up to 35 μmol L⁻¹. The main advantage of the proposed bi‐enzyme sensor is its selectivity toward the OPs with phenolic leaving groups, excluding the interference of the nitrophenyl‐substituted OPs.     

Combined Experimental and Theoretical Study on the Reactivity of Compounds I and II in Horseradish Peroxidase Biomimetics

For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP‐I) and Compound II (HRP‐II), we generated in situ [Fe^IV?O(TMP^+.)(2‐MeIm)]⁺ and [Fe^IV?O(TMP)(2‐MeIm)]⁰ (TMP=5,10,15,20‐tetramesitylporphyrin; 2‐MeIm=2‐methylimidazole) as biomimetics for HRP‐I and HRP‐II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at ?15 °C by utilizing rapid‐scan UV/Vis spectroscopy. Comparison of the second‐order rate constants measured for the direct reactions of the HRP‐I and HRP‐II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP‐I mimic, which is significantly higher than that of HRP‐II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP‐I mimics (^2,4Cpd I) and the closed‐shell triplet spin HRP‐II model (³Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving ^2,4Cpd I than those found for ³Cpd II are in line with the much higher oxidizing efficiency of the HRP‐I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP‐I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP‐II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during C?H bond activation processes by the HRP‐II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or oxygen rebound processes. Importantly, depending on the electronic nature of the oxidizing species, that is, ^2,4Cpd I or ³Cpd II, an interesting region‐selective conversion phenomenon between sulfoxidation and H‐atom abstraction was revealed in the course of the oxidation reaction of dimethylsulfide. The combined experimental and theoretical study on the elucidation of the intrinsic reactivity patterns of the HRP‐I and HRP‐II mimics provides a valuable tool for evaluating the particular role of the HRP active species in biological systems.     

The Oxidation of Guaiacol,Serotonin, o-Cresol,p-Cresol,m-Cresol,Homovanillic Acid,and m-Tyrosine by Horseradish Peroxidase and Hydrogen Peroxide

Abstract

A survey of 14 substrates for the fluorometric determination of the oxidative enzyme-horseradish peroxidase was carried out. The compounds which seemed to act well as substrates are serotonin, guaiacol, o-cresol, p-cresol, m-cresol, homovanillic acid and m-tyrosine. L-Epinephrine, equilenin, equilin, β-estradiol, estriol, and estrone were studied and found not to be substrates of the system.

The kinetics of the oxidation reactions were studied. They followed the Michaelis constant rate equation. The Michaelis constant, Km, was determined by a Lineweaver-Burk plot for each substrate.

It would appear that the presence of a group in the ortho, para, or meta position of the phenolic ring decreases the binding of the subtrate to the enzyme's active site, as shown by the km values obtained. It may be concluded that m-tyrosine is the best substrate (lowest km value) when compared to all the substrates which have been studied to date by the authors.     

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

In this work 3‐indoxyl phosphate (3‐IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3‐IP in presence of hydrogen peroxide (H₂O₂) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of ?0.15 V (vs. Ag pseudo‐reference electrode) when a screen‐printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V_max, K_M) for both substrates (3‐IP and H₂O₂) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86×10^?12 and 5.68×10^?12 M, respectively. Thus, 3‐IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.     

Application of molecular absorption properties of horseradish peroxidase for self-indicating enzymatic interactions and analytical methods

In this paper an in depth study is presented of the use of the horseradish peroxidase (HRP) enzyme as a self-indicating biorecognition reagent in UV-vis molecular absorption spectrometry. The HRP/H2O2 reaction mechanism in the absence of an external substrate has been clarified, and the interaction between HRP and glucose oxidase (GOx) has been studied. It has been demonstrated that GOx can act as a substrate of HRP; in both cases the kinetic constants have been obtained and mathematical models have been developed. Second, the HRP/H2O2 reaction is used to follow a H2O2-producing enzymatic reaction, the glucose reaction with GOx being used as a model. As an application of this, two methodologies have been proposed for glucose determination: with or without previous incubation of glucose with GOx. In both cases mathematical models relating HRP absorbance changes to glucose concentration have been developed and tested; both methods have been optimized, analytically characterized, and tested for glucose determination in samples. The methodology described could be applied to other heme-proteins and to other H2O2-producing enzymatic reactions. The models permit the reaction constants to be calculated. From the analytical chemistry point of view the models allow the prediction of the method sensitivity for other analytes involved in this type of reaction if the kinetic constants are known and can be used in the design of optical sensors.     

Aromatic azo compounds as spectrophotometric kinetic assay substrate for HRP

Aromatic azo compounds were efficiently cleaved by the horseradish peroxidase (HRP) into aromicdiazonium ions, during which an intense special absorption of the substrate disappeared completely. This furnished a sensitive spectrophotometric detection of 0.025-6.0 nm peroxidase. Kinetic characteristics of enzyme reaction were investigated with 14 different chemical structures of aromatic azo compounds as the substrates for HRP. Then the structure requirements for substrates were elucidated. PH dependence of enzymatic catalysis was studied with pK'(a)of 6.54 and 8.40 for Eriochrome black T, and 3.22 and 3.83 for Nitrosulfophenol S as the substrate of HRP, respectively.     

Adsorption and inactivation behavior of horseradish peroxidase on various substrates

To produce bioactive papers, i.e. papers incorporating biomolecules that are useful for analyte detection, adequate immobilization strategies should be devised. In this article, the physical immobilization behavior and activity of the enzyme horseradish peroxidase (HRP) on various papermaking substrates were studied. The papermaking substrates included amorphous and crystalline cellulose, calcium carbonate, styrene butadiene latex, polystyrene, and both negatively charged rayon and rayon with a positively charged layer. It was found that HRP adsorption improves as the hydrophobicity of the substrate increases; however, excessive hydrophobicity produces enzyme deactivation. HRP–calcium carbonate binding was weak and the enzyme loading was scant. These results provided a possible explanation for the poor analytical signals observed in pigment-coated papers when used as bioactive paper supports. Electrostatic effects played a minor role in HRP adsorption behavior.     

Non-oxidative decarboxylation of glycine derivatives by a peroxidase

Under anaerobic, peroxide-free conditions (pH 5.5, 25 degrees C), horseradish peroxidase (HRP) catalyzes the rapid, non-oxidatve decarboxylation of N-alkyl-N-phenylglycine derivatives to the corresponding N-alkyl-N-methylanilines in 100% yield. When the reaction is conducted in D2O buffer, the product contains a single deuterium in the methyl group. The reactions are very fast compared to the oxidative decarboxylation of the same substrates under standard peroxidatic conditions (i.e., hydrogen peroxide added, air present) and in fact are inhibited by peroxide and oxygen. To account for these unprecedented observations, we propose a cyclic mechanism in which ferric HRP abstracts an electron from the substrate, giving an aminium ion intermediate that decarboxylates; protonation of the resulting alpha-aminoradical on carbon gives an aminium ion that is reduced by ferrous HRP to complete the cycle.     

Self-assembled all-inclusive organic-inorganic nanoparticles enable cascade reaction for the detection of glucose

Traditional colorimetric glucose biosensor generally involves complex assay procedures. Free labile enzymes and peroxidase substrates are used separately for triggering a chromogenic reaction. These limits result in inferior enzyme stability and defective enzymatic catalytic efficiency, making it hard to routinely utilize them for the direct and fast test of glucose. In this work, we provide an all-inclusive substrates/enzymes nanoparticle employed 3,3′5,5′-tetramethylbenzidine (TMB) as chromogenic substrates and glucose oxidase (GOx)/horseradish peroxidase (HRP) as signal amplifier enzymes (TMB-GH NPs) by the molecule self-assembly technique. The “all-inclusive” nanoparticles can realize the tandem colorimetric reactions, and the oxidation product of TMB (ox-TMB) exhibits a strong NIR laser-driven photothermal effect, thus allowing quantitative photothermal detection of glucose. Owing to the restriction of the molecular motion of GOx, HRP, and TMB, the distance of mass transfer between substrates was shortened largely, leading to improved catalytic activity for glucose. Overall, our strategy will simplify the analysis procedure, furthermore, these integrated nanoparticles not only display higher stability and activity than that of the free GOx/HRP system and possesses an excellent performance for colorimetric and photothermal bioassay of glucose simultaneously. We believe that this unique technique will give good inspirations to develop simple and precise methods for bioassay.     

Inactivation of horseradish peroxidase by phenoxyl radical attack

To test the hypothesis that horseradish peroxidase (HRP) can be inactivated by phenoxyl radicals upon reaction with H(2)O(2)/phenol, we probed HRP-catalyzed phenol oxidation at various phenol/H(2)O(2) concentrations. To this end the total protein, phenolic product, active protein, and iron concentrations in the aqueous phase were determined by protein assay, phenol-(14)C isotopic labeling, resonance Raman and atomic absorption spectroscopy, respectively. Additionally, resonance Raman and FTIR measurements were carried out to probe possible structural changes of the enzyme during the reaction. The data obtained provide the first experimental support for the hypothesis that HRP can be inactivated by a phenoxyl radical attack. The heme macrocycle destruction involving deprivation of the heme iron occurs as a result of the reaction. An intermediate type of the active protein was observed by Raman difference spectra at low concentrations which features a stabilization of the quantum mixed state of the heme iron and a significant amount of phenoxylphenol-type oligomers in solution and probably also in the heme pocket. This work provides a basis for evaluating the relative contributions of different HRP inactivation mechanisms and is thus critical for optimizing engineering applications involving HRP reactions.     

Mechanistic insight into the activity of tyrosinase from variable-temperature studies in an aqueous/organic solvent

The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4% methanol-glycerol (7:1, v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61+/-9 kJ mol(-1) irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10% to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein-substrate interactions. The substrate 4-hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH(double dagger) approximately = 150 kJ mol(-1) and deltaS(double dagger) approximately = 280 J K(-1) mol(-1), due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.     

New combined model for the prediction of regioselectivity in cytochrome P450/3A4 mediated metabolism

Cytochrome P450 3A4 metabolizes nearly 50% of the drugs currently in clinical use with a broad range of substrate specificity. Early prediction of metabolites of xenobiotic compounds is crucial for cost efficient drug discovery and development. We developed a new combined model, MLite, for the prediction of regioselectivity in the cytochrome P450 3A4 mediated metabolism. In the model, the ensemble catalyticphore- based docking method was implemented for the accessibility prediction, and the activation energy estimation method of Korzekwa et al. was used for the reactivity prediction. Four major metabolic reactions, aliphatic hydroxylation, N-dealkylation, O-dealkylation, and aromatic hydroxylation reaction, were included and the reaction data, metabolite information, were collected for 72 well-known substrates. The 47 drug molecules were used as the training set, and the 25 well-known substrates were used as the test set for the ensemble catalyticphore-based docking method. MLite predicted correctly about 76% of the first two sites in the ranking list of the test set. This predictability is comparable with that of another combined model, MetaSite, and the recently published QSAR model proposed by Sheridan et al. MLite also offers information about binding configurations of the substrate-enzyme complex. This may be useful in drug modification by the structure-based drug design.     

Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO<Subscript>2</Subscript> nanoparticles

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO₂ particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H₂O₂ forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO₂ particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10⁻⁶–7.3×10⁻⁴ g/L and with a detection limit of 1.5×10⁻⁶ g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results. Supported by the Projects of Scientific Research Fund of Hunan Provincial Education Department of China (Grant Nos. 05B020 and 06C098)     

In-silico investigation of phenolic compounds from leaves of Phillyrea angustifolia L. as a potential inhibitor against the SARS-CoV-2 main protease (Mpro PDB ID:5R83) using a virtual screening method

There is currently a global COVID-19 pandemic caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and its variants. This highly contagious viral disease continues to pose a major health threat global. The discovery of vaccinations is not enough to prevent their spread and dire consequences. To take advantage of the current drugs and isolated compounds, and immediately qualifying approach is required. The aim of our research is evaluation the potency for natural antiviral compounds against the SARS CoV-2 M^pro. Molecular docking of four phenolic compounds from Phillyrea angustifolia leaves with SARS-CoV-2 M^pro has been conducted. Similarly, the stability of selected ligand–protein interactions has been determined using MD simulations. Moreover, the quantitative structure–activity relationship (QSAR), MMGBSA binding energies, pharmacokinetics, and drug-likeness predictions for selected phenolic have been reported. The selected phenolic compounds (Luteolin-7-O-glucoside, Apigenin-7-O-glucoside, Demethyl-oleuropein, and Oleuropein aglycone) revealed strong binding contacts in the two active pockets of a target protein of SARS-CoV-2 M^pro with the docking scores and highest binding energies with a binding energy of ?8.2 kcal/mol; ?7.8 kcal/mol; ?7.2 kcal/mol and ?7.0 kcal/mol respectively. Both Demethyloleoeuropein and Oleuropein aglycone can interact with residues His41 and Cys145 (catalytic dyad) and other amino acids of the binding pocket of M^pro. According to QSAR, studies on pharmacokinetics and drug-like properties suggested that oleuropein aglycone could be the best inhibitor of SARS-CoV-2 for new drug design and development. Further in vivo, in vitro, and clinical studies are highly needed to examine the potential of these phenolic compounds in the fight against COVID-19.