A Chinese Herb Prescription "Fang-gan Decoction" Protects Against Damage to Lung and Colon Epithelial Cells Caused by the SARS-CoV-2 Spike Protein by Regulating the TGF-ß/Smad2/3 and NF-κB Pathways.

Objective To explore the effects and mechanisms of a traditional Chinese medicine (TCM) prescription, "Fang-gan Decoction" (FGD), in protecting against SARS-CoV-2 spike protein-induced lung and intestinal injuries in vitro and in vivo.Methods Female BALB/c mice and three cell lines pretreated with FGD were stimulated with recombinant SARS-CoV-2 spike protein (spike protein). Hematoxylin-eosin (HE) staining and pathologic scoring of tissues, cell permeability and viability, and angiotensin-converting enzyme 2 (ACE2) expression in the lung and colon were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory factors in serum and cell supernatant. The expression of NF-κB p65, p-NF-κB p65, p-IκBα, p-Smad2/3, TGF-ß1, Caspase3, and Bcl-2 was evaluated by Western blotting.Results FGD protected against the damage to the lung and colon caused by the spike protein in vivo and in vitro according to the pathologic score and cell permeability and viability (P<0.05). FGD up-regulated ACE2 expression, which was reduced by the spike protein in the lung and colon, significantly improved the deregulation of inflammatory markers caused by the spike protein, and regulated the activity of TGF-ß/Smads and NF-κB signaling.Conclusion Traditional Chinese medicine has a protective effect on lung and intestinal tissue injury stimulated by the spike protein through possible regulatory functions of the NF-κB and TGF-ß1/Smad pathways with tissue type specificity.

Marine algal antagonists targeting 3CL protease and spike glycoprotein of SARS-CoV-2: a computational approach for anti-COVID-19 drug discovery.

The COVID-19 pandemic has severely destructed human life worldwide, with no suitable treatment until now. SARS-CoV-2 virus is unprecedented, resistance against number of therapeutics and spreading rapidly with high mortality, which warrants the need to discover new effective drugs to combat this situation. This current study is undertaken to explore the antiviral potential of marine algal compounds to inhibit the viral entry and its multiplication using computational analysis. Among the proven drug discovery targets of SARS-CoV-2, spike glycoprotein and 3-chymotrypsin-like protease are responsible for the virus attachment and viral genome replication in the host cell. In this study, the above-mentioned drug targets were docked with marine algal compounds (sulfated polysaccharides, polysaccharide derivatives and polyphenols) using molecular docking tools (AutoDockTools). The obtained results indicate that κ-carrageenan, laminarin, eckol, trifucol and ß-D-galactose are the top-ranking compounds showing better docking scores with SARS-CoV-2 targets, than the current experimental COVID-19 antiviral drugs like dexamethasone, remdesivir, favipiravir and MIV-150. Further, molecular dynamic simulation, ADMET and density functional theory calculations were evaluated to substantiate the findings. To the best of our knowledge, this is the first report on in silico analysis of aforesaid algal metabolites against SARS-CoV-2 targets. This study concludes that these metabolites can be curative for COVID-19 in the hour of need after further validations in in vitro and in vivo testings.Communicated by Ramaswamy H. Sarma.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Computational study, synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells.

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1ß Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2.

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

Age-dependent effects of the recombinant spike protein/SARS-CoV-2 on the M-CSF- and IL-34-differentiated macrophages in vitro.

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M-CSF- and IL-34-differentiated macrophages. The Spike-protein upregulated SASP expression in young and aged male M-CSF-macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to the Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M-CSF-macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M-CSF-macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M-CSF-macrophages, indicating that CatK may be also involved in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Identification of the peptide derived from S1 domain that inhibits type I and type II feline infectious peritonitis virus infection.

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). A therapeutic drug that is effective against FIP has not yet been developed. Peptides based on viral protein amino acid sequences have recently been attracting attention as new antiviral drugs. In the present study, we synthesized 30 overlapping peptides based on the amino acid sequence of the S1 domain of the type I FIPV strain KU-2 S protein, and investigated their inhibitory effects on FIPV infection. To evaluate the inhibitory effects on type I FIPV infection of these peptides, we investigated a method to increase the infection efficiency of poorly replicative type I FIPV. The efficiency of type I FIPV infection was increased by diluting the virus with medium containing a polycation. Of the 30 peptides, I-S1-8 (S461-S480), I-S1-9 (S471-S490), I-S1-10 (S481-S500), I-S1-16 (S541-S560), and I-S1-22 (S601-S620) significantly decreased the infectivity of FIPV strain KU-2 while I-S1-9 and I-S1-16 exhibited marked inhibitory effects on FIPV infection. The inhibitory effects on FIPV infection of these 2 peptides on other type I and type II FIPV strains, feline herpesvirus (FHV), and feline calicivirus (FCV) were also examined. These 2 peptides specifically inhibited type I and type II FIPV, but did FHV or FCV infection. In conclusion, the possibility of peptides derived from the S protein of type I FIPV strain KU-2 as anti-FIPV agents effective not only for type I, but also type II FIPV was demonstrated in vitro.