Evaluation of Antiviral Effect against SARS-CoV-2 Propagation by Crude Polysaccharides from Seaweed and Abalone Viscera In Vitro.

Crude polysaccharides, extracted from two seaweed species (Hizikia fusiforme and Sargassum horneri) and Haliotis discus hannai (abalone) viscera, were evaluated for their inhibitory effect against SARS-CoV-2 propagation. Plaque titration revealed that these crude polysaccharides efficiently inhibited SARS-CoV-2 propagation with IC50 values ranging from 0.35 to 4.37 µg/mL. The crude polysaccharide of H. fusiforme showed the strongest antiviral effect, with IC50 of 0.35 µg/mL, followed by S. horneri and abalone viscera with IC50 of 0.56 and 4.37 µg/mL, respectively. In addition, immunofluorescence assay, western blot, and quantitative RT-PCR analysis verified that these polysaccharides could inhibit SARS-CoV-2 replication. In Vero E6 cells, treatment with these crude polysaccharides before or after viral infection strongly inhibited the expression level of SARS-CoV-2 spikes, nucleocapsid proteins, and RNA copies of RNA-dependent RNA-polymerase and nucleocapsid. These results show that these crude marine polysaccharides effectively inhibit SARS-CoV-2 propagation by interference with viral entry.

Antiviral Activity of Two Marine Carotenoids against SARS-CoV-2 Virus Entry In Silico and In Vitro.

The marine carotenoids fucoxanthin and siphonaxanthin are powerful antioxidants that are attracting focused attention to identify a variety of health benefits and industry applications. In this study, the binding energy of these carotenoids with the SARS-CoV-2 Spike-glycoprotein was predicted by molecular docking simulation, and their inhibitory activity was confirmed with SARS-CoV-2 pseudovirus on HEK293 cells overexpressing angiotensin-converting enzyme 2 (ACE2). Siphonaxanthin from Codium fragile showed significant antiviral activity with an IC50 of 87.4 µM against SARS-CoV-2 pseudovirus entry, while fucoxanthin from Undaria pinnatifida sporophyll did not. The acute toxicities were predicted to be relatively low, and pharmacokinetic predictions indicate GI absorption. Although further studies are needed to elucidate the inhibition of viral infection by siphonaxanthin, these results provide useful information in the application of these marine carotenoids for the treatment and prevention of COVID-19.

Inhibition of SARS-CoV-2 Virus Entry by the Crude Polysaccharides of Seaweeds and Abalone Viscera In Vitro.

Much attention is being devoted to the potential of marine sulfated polysaccharides as antiviral agents in preventing COVID-19. In this study, sulfated fucoidan and crude polysaccharides, extracted from six seaweed species (Undaria pinnatifida sporophyll, Laminaria japonica, Hizikia fusiforme, Sargassum horneri, Codium fragile, Porphyra tenera) and Haliotis discus hannai (abalone viscera), were screened for their inhibitory activity against SARS-CoV-2 virus entry. Most of them showed significant antiviral activities at an IC50 of 12~289 µg/mL against SARS-CoV-2 pseudovirus in HEK293/ACE2, except for P. tenera (IC50 > 1000 µg/mL). The crude polysaccharide of S. horneri showed the strongest antiviral activity, with an IC50 of 12 µg/mL, to prevent COVID-19 entry, and abalone viscera and H. fusiforme could also inhibit SARS-CoV-2 infection with an IC50 of 33 µg/mL and 47 µg/mL, respectively. The common properties of these crude polysaccharides, which have strong antiviral activity, are high molecular weight (>800 kDa), high total carbohydrate (62.7~99.1%), high fucose content (37.3~66.2%), and highly branched polysaccharides. These results indicated that the crude polysaccharides from seaweeds and abalone viscera can effectively inhibit SARS-CoV-2 entry.

Screening of Human CYP1A2 and CYP3A4 Inhibitors from Seaweed In Silico and In Vitro.

Phenolic compounds and carotenoids are potential inhibitors of cytochrome P450s. Sixteen known compounds, phenolic compounds and carotenoids from seaweed were examined for potential inhibitory capacity against CYP1A2 and CYP3A4 in silico and in vitro. Morin, quercetin, and fucoxanthin inhibited the enzyme activity of CYP1A2 and CYP3A4 in a dose-dependent manner. The IC50 values of morin, quercetin, and fucoxanthin were 41.8, 22.5, and 30.3 µM for CYP1A2 and 86.6, 16.1, and 24.4 µM for CYP3A4, respectively. Siphonaxanthin and hesperidin did not show any significant effect on CYP1A2, but they slightly inhibited CYP3A4 activity at high concentrations. In silico modeling of CYP's binding site revealed that the potential inhibitors bound in the cavity located above the distal surface of the heme prosthetic group through the 2a or 2f channel of CYPs. This study presents an approach for quickly predicting CYP inhibitory activity and shows the potential interactions of compounds and CYPs through in silico modeling.

Surface display of heme- and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation.

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a heme- and diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. Surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins, into practically useful whole-cell biocatalysts for extensive biotechnological applications including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and bio-chip development.

Functional expression in Bacillus subtilis of mammalian NADPH-cytochrome P450 oxidoreductase and its spore-display.

The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P(groE) and P(tac). The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications.

Continuous spectrofluorometric and spectrophotometric assays for NADPH-cytochrome P450 reductase activity using 5-cyano-2,3-ditolyl tetrazolium chloride.

Mammalian NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 enzymes and other several microsomal enzymes. It also catalyzes the one-electron reduction of many chemicals and drugs. Reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by CPR was assessed as a method for monitoring CPR activity. The electrons released from NADPH by CPR were transferred to CTC in the reaction medium, and CTC reduction activity could be assessed spectrophotometrically and spectrofluorometrically. The reduction kinetics of CTC follows classical Michaelis-Menten kinetics (K(m) = 50 microM, k(cat) = 2,520 min(-1)). This method offers a continuous assay of the enzymatic activity of CPR.

Functional expression of mammalian NADPH-cytochrome P450 oxidoreductase on the cell surface of Escherichia coli.

To develop a whole-cell oxidoreductase system without the practical limitation of substrate/product transport, easy preparation, stability of enzymes, and low expression levels, we here report the development of a whole cell biocatalyst displaying rat NADPH-cytochrome P450 oxidoreductase (CPR, 77-kDa) on the surface of Escherichia coli by using ice-nucleation protein from Pseudomonas syringae. Surface localization and functionality of the CPR were verified by flow cytometry, electron microscopy, and measurements of enzyme activities. The results of this study comprise the first report of microbial cell-surface display of diflavin-containing mammalian enzymes. This system will allow us to select and develop oxidoreductases, containing bulky and complex prosthetic groups of FAD and FMN, into practically useful whole-cell biocatalysts for broad biological and biotechnological applications including the selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, and bio-chip development.

Functional expression of human cytochrome P450 enzymes in Escherichia coli.

Knowledge regarding cytochrome P450 (P450) is crucial to the fields of drug therapy and drug development, as well as in our understanding of the mechanisms underlying the metabolic activation of potentially toxic and carcinogenic compounds. Escherichia coli is the most extensively utilized host in the production of recombinant human P450 enzymes. However, the recovery of substantial yields of functionally active P450 proteins remains problematic. Mammalian P450 protein was first expressed in 1991, via the modification of the N-terminal amino acid sequences in E. coli cells. Since that time, a variety of strategies have been established for the functional expression of recombinant P450s in E. coli, including N-terminal modification, the use of molecular chaperones, and culturing at lower temperatures. In all cases, human P450 expressed in E. coli cells has been shown to efficiently catalyze the oxidation of representative substrates at efficient rates. These recombinant P450s are applicable to studies which estimate the kinetic parameters of drug oxidation, and have also been used to determine the metabolic pathways of drugs and carcinogens exploited by human P450s. Despite the potential of P450s in various pharmaceutical and biotechnological fields, however, a host of substantial challenges must be overcome before these enzymes can be routinely utilized. Intrinsically, these enzymes are not very active, and exhibit poor stability. In this review, we have described current developments in the heterologous expression of human P450 enzymes.

A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome b5, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of A610. MTT reduction followed classical Michaelis-Menten kinetics (K(m)= 20 microM, k(cat)= 1,910 min(-1)). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

Temperature effect on the functional expression of human cytochromes P450 2A6 and 2E1 in Escherichia coli.

Human cytochromes P450 (CYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various CYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under 30 degrees C). Expression levels of CYPs 2A6 and 2E1 at 37 degrees C were compared to those at 280 degrees C, which is a usual temperature used in most bacterial expression systems for human CYP expression. Within 18 h the expression levels of CYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at 37 degrees C, respectively, which are compatible with those of 36 h culture at 280 degrees C. The activities of CYPs expressed at 37 degrees C were also comparable to those expressed at 28 degrees C. The present over-expression system can be useful for rapid production of large amounts of active human CYPs 2A6 and 2E1 in E. coli.

A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 1,1-diphenyl-2-picrylhydrazyl.

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring A(520) reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was 4.09mM(-1) cm(-1). DPPH reduction followed classical Michaelis-Menten kinetics (K(m) = 28 microM, k(cat) = 1690 min(-1)). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive.

Roles of human liver cytochrome P450 3A4 and 1A2 enzymes in the oxidation of myristicin.

The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated (r(2)=0.87) with nifedipine oxidation (a marker of CYP3A4). With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect. The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2. These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene.

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion.

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.