Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α

Inhaled nebulized interferon (IFN)-α and IFN-β have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5′-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3′-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 μM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.     

Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.     

Geraniin Inhibits the Entry of SARS-CoV-2 by Blocking the Interaction between Spike Protein RBD and Human ACE2 Receptor

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite the development of vaccines, the emergence of SARS-CoV-2 variants and the absence of effective therapeutics demand the continual investigation of COVID-19. Natural products containing active ingredients may be good therapeutic candidates. Here, we investigated the effectiveness of geraniin, the main ingredient in medical plants Elaeocarpus sylvestris var. ellipticus and Nephelium lappaceum, for treating COVID-19. The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (hACE2) receptor to initiate virus entry into cells; viral entry may be an important target of COVID-19 therapeutics. Geraniin was found to effectively block the binding between the SARS-CoV-2 spike protein and hACE2 receptor in competitive enzyme-linked immunosorbent assay, suggesting that geraniin might inhibit the entry of SARS-CoV-2 into human epithelial cells. Geraniin also demonstrated a high affinity to both proteins despite a relatively lower equilibrium dissociation constant (K_D) for the spike protein (0.63 μM) than hACE2 receptor (1.12 μM), according to biolayer interferometry-based analysis. In silico analysis indicated geraniin’s interaction with the residues functionally important in the binding between the two proteins. Thus, geraniin is a promising therapeutic agent for COVID-19 by blocking SARS-CoV-2’s entry into human cells.     

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.     

miR-218 Inhibits Erythroid Differentiation and Alters Iron Metabolism by Targeting ALAS2 in K562 Cells

microRNAs (miRNAs) are involved in a variety of biological processes. The regulatory function and potential role of miRNAs targeting the mRNA of the 5′-aminolevulinate synthase 2 (ALAS2) in erythropoiesis were investigated in order to identify miRNAs which play a role in erythroid iron metabolism and differentiation. Firstly, the role of ALAS2 in erythroid differentiation and iron metabolism in human erythroid leukemia cells (K562) was confirmed by ALAS2 knockdown. Through a series of screening strategies and experimental validations, it was identified that hsa-miR-218 (miR-218) targets and represses the expression of ALAS2 by binding to the 3′-untranslated region (UTR). Overexpression of miR-218 repressed erythroid differentiation and altered iron metabolism in K562 cells similar to that seen in the ALAS2 knockdown in K562 cells. In addition to iron metabolism and erythroid differentiation, miR-218 was found to be responsible for a reduction in K562 cell growth. Taken together, our results show that miR-218 inhibits erythroid differentiation and alters iron metabolism by targeting ALAS2 in K562 cells.     

MicroRNA-Mediated Regulation of the Virus Cycle and Pathogenesis in the SARS-CoV-2 Disease

In the last few years, microRNA-mediated regulation has been shown to be important in viral infections. In fact, viral microRNAs can alter cell physiology and act on the immune system; moreover, cellular microRNAs can regulate the virus cycle, influencing positively or negatively viral replication. Accordingly, microRNAs can represent diagnostic and prognostic biomarkers of infectious processes and a promising approach for designing targeted therapies. In the past 18 months, the COVID-19 infection from SARS-CoV-2 has engaged many researchers in the search for diagnostic and prognostic markers and the development of therapies. Although some research suggests that the SARS-CoV-2 genome can produce microRNAs and that host microRNAs may be involved in the cellular response to the virus, to date, not enough evidence has been provided. In this paper, using a focused bioinformatic approach exploring the SARS-CoV-2 genome, we propose that SARS-CoV-2 is able to produce microRNAs sharing a strong sequence homology with the human ones and also that human microRNAs may target viral RNA regulating the virus life cycle inside human cells. Interestingly, all viral miRNA sequences and some human miRNA target sites are conserved in more recent SARS-CoV-2 variants of concern (VOCs). Even if experimental evidence will be needed, in silico analysis represents a valuable source of information useful to understand the sophisticated molecular mechanisms of disease and to sustain biomedical applications.     

Metal Complexes as Antiviral Agents for SARS-CoV-2

The severe acute respiratory syndrome – coronavirus 2 (SARS-CoV-2), the infectious agent responsible for COVID-19 – has caused more than 2.5 million deaths worldwide and triggered a global pandemic. Even with successful vaccines being delivered, there is an urgent need for novel treatments to combat SARS-CoV-2, and other emerging viral diseases. While several organic small molecule drug candidates are in development, some effort has also been devoted towards the application of metal complexes as potential antiviral agents against SARS-CoV-2. Herein, the metal complexes that have been reported to show antiviral activity against SARS-CoV-2 or one of its target proteins are described and their proposed mechanisms of action are discussed.     

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.     

Could Histamine H1 Receptor Antagonists Be Used for Treating COVID-19?

COVID-19 has rapidly become a pandemic worldwide, causing extensive and long-term health issues. There is an urgent need to identify therapies that limit SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Unbalanced lung inflammation is a common feature in severe COVID-19 patients; therefore, reducing lung inflammation can undoubtedly benefit the clinical manifestations. Histamine H1 receptor (H1 receptor) antagonists are widely prescribed medications to treat allergic diseases, while recently it has emerged that they show significant promise as anti-SARS-CoV-2 agents. Here, we briefly summarize the novel use of H1 receptor antagonists in combating SARS-CoV-2 infection. We also describe the potential antiviral mechanisms of H1 receptor antagonists on SARS-CoV-2. Finally, the opportunities and challenges of the use of H1 receptor antagonists in managing COVID-19 are discussed.     

Iterated Virtual Screening-Assisted Antiviral and Enzyme Inhibition Assays Reveal the Discovery of Novel Promising Anti-SARS-CoV-2 with Dual Activity

Unfortunately, COVID-19 is still a threat to humankind and has a dramatic impact on human health, social life, the world economy, and food security. With the limited number of suggested therapies under clinical trials, the discovery of novel therapeutic agents is essential. Here, a previously identified anti-SARS-CoV-2 compound named Compound 13 (1,2,5-Oxadiazole-3-carboximidic acid, 4,4′-(methylenediimino) bis,bis[[(2-hydroxyphenyl)methylene]hydrazide) was subjected to an iterated virtual screening against SARS-CoV-2 M^pro using a combination of Ligand Designer and PathFinder. PathFinder, a computational reaction enumeration tool, was used for the rapid generation of enumerated structures via default reaction library. Ligand designer was employed for the computerized lead optimization and selection of the best structural modification that resulted in a favorable ligand–protein complex. The obtained compounds that showed the best binding to M^pro were re-screened against TMPRSS2, leading to the identification of 20 shared compounds. The compounds were further visually inspected, which resulted in the identification of five shared compounds M1–5 with dual binding affinity. In vitro evaluation and enzyme inhibition assay indicated that M3, an analogue of Compound 13 afforded by replacing the phenolic moiety with pyridinyl, possesses an improved antiviral activity and safety. M3 displayed in vitro antiviral activity with IC₅₀ 0.016 µM and M^pro inhibition activity with IC₅₀ 0.013 µM, 7-fold more potent than the parent Compound 13 and potent than the antivirals drugs that are currently under clinical trials. Moreover, M3 showed potent activity against human TMPRSS2 and furin enzymes with IC₅₀ 0.05, and 0.08 µM, respectively. Molecular docking, WaterMap analysis, molecular dynamics simulation, and R-group analysis confirmed the superiority of the binding fit to M3 with the target enzymes. WaterMap analysis calculated the thermodynamic properties of the hydration site in the binding pocket that significantly affects the biological activity. Loading M3 on zinc oxide nanoparticles (ZnO NPs) increased the antiviral activity of the compound 1.5-fold, while maintaining a higher safety profile. In conclusion, lead optimized discovery following an iterated virtual screening in association with molecular docking and biological evaluation revealed a novel compound named M3 with promising dual activity against SARS-CoV-2. The compound deserves further investigation for potential clinical-based studies.     

Reciprocal Changes in miRNA Expression with Pigmentation and Decreased Proliferation Induced in Mouse B16F1 Melanoma Cells by l-Tyrosine and 5-Bromo-2′-Deoxyuridine

Background: Many microRNAs have been identified as critical mediators in the progression of melanoma through its regulation of genes involved in different cellular processes such as melanogenesis, cell cycle control, and senescence. However, microRNAs’ concurrent participation in syngeneic mouse B16F1 melanoma cells simultaneously induced decreased proliferation and differential pigmentation by exposure to 5-Brd-2′-dU (5’Bromo-2-deoxyuridine) and L-Tyr (L-Tyrosine) respectively, is poorly understood. Aim: To evaluate changes in the expression of microRNAs and identify which miRNAs in-network may contribute to the functional bases of phenotypes of differential pigmentation and reduction of proliferation in B16F1 melanoma cells exposed to 5-Brd-2′-dU and L-Tyr. Methods: Small RNAseq evaluation of the expression profiles of miRNAs in B16F1 melanoma cells exposed to 5-Brd-2′-dU (2.5 μg/mL) and L-Tyr (5 mM), as well as the expression by qRT-PCR of some molecular targets related to melanogenesis, cell cycle, and senescence. By bioinformatic analysis, we constructed network models of regulation and co-expression of microRNAs. Results: We confirmed that stimulation or repression of melanogenesis with L-Tyr or 5-Brd-2′-dU, respectively, generated changes in melanin concentration, reduction in proliferation, and changes in expression of microRNAs 470-3p, 470-5p, 30d-5p, 129-5p, 148b-3p, 27b-3p, and 211-5p, which presented patterns of coordinated and reciprocal co-expression, related to changes in melanogenesis through their putative targets Mitf, Tyr and Tyrp1, and control of cell cycle and senescence: Cyclin D1, Cdk2, Cdk4, p21, and p27. Conclusions: These findings provide insights into the molecular biology of melanoma of the way miRNAs are coordinated and reciprocal expression that may operate in a network as molecular bases for understanding changes in pigmentation and decreased proliferation induced in B16F1 melanoma cells exposed to L-Tyr and 5-Brd-2′-dU.     

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.     

Innate Immune Response to SARS-CoV-2 Infection: From Cells to Soluble Mediators

The vulnerability of humankind to SARS-CoV-2 in the absence of a pre-existing immunity, the unpredictability of the infection outcome, and the high transmissibility, broad tissue tropism, and ability to exploit and subvert the immune response pose a major challenge and are likely perpetuating the COVID-19 pandemic. Nevertheless, this peculiar infectious scenario provides researchers with a unique opportunity for studying, with the latest immunological techniques and understandings, the immune response in SARS-CoV-2 naïve versus recovered subjects as well as in SARS-CoV-2 vaccinees. Interestingly, the current understanding of COVID-19 indicates that the combined action of innate immune cells, cytokines, and chemokines fine-tunes the outcome of SARS-CoV-2 infection and the related immunopathogenesis. Indeed, the emerging picture clearly shows that the excessive inflammatory response against this virus is among the main causes of disease severity in COVID-19 patients. In this review, the innate immune response to SARS-CoV-2 infection is described not only in light of its capacity to influence the adaptive immune response towards a protective phenotype but also with the intent to point out the multiple strategies exploited by SARS-CoV-2 to antagonize host antiviral response and, finally, to outline inborn errors predisposing individuals to COVID-19 disease severity.     

Peptide Derivatives of the Zonulin Inhibitor Larazotide (AT1001) as Potential Anti SARS-CoV-2: Molecular Modelling,Synthesis and Bioactivity Evaluation

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (M^pro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds M^pro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.     

Autophagy,Unfolded Protein Response,and Neuropilin-1 Cross-Talk in SARS-CoV-2 Infection: What Can Be Learned from Other Coronaviruses

The COVID-19 pandemic is caused by the 2019–nCoV/SARS-CoV-2 virus. This severe acute respiratory syndrome is currently a global health emergency and needs much effort to generate an urgent practical treatment to reduce COVID-19 complications and mortality in humans. Viral infection activates various cellular responses in infected cells, including cellular stress responses such as unfolded protein response (UPR) and autophagy, following the inhibition of mTOR. Both UPR and autophagy mechanisms are involved in cellular and tissue homeostasis, apoptosis, innate immunity modulation, and clearance of pathogens such as viral particles. However, during an evolutionary arms race, viruses gain the ability to subvert autophagy and UPR for their benefit. SARS-CoV-2 can enter host cells through binding to cell surface receptors, including angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1). ACE2 blockage increases autophagy through mTOR inhibition, leading to gastrointestinal complications during SARS-CoV-2 virus infection. NRP1 is also regulated by the mTOR pathway. An increased NRP1 can enhance the susceptibility of immune system dendritic cells (DCs) to SARS-CoV-2 and induce cytokine storm, which is related to high COVID-19 mortality. Therefore, signaling pathways such as mTOR, UPR, and autophagy may be potential therapeutic targets for COVID-19. Hence, extensive investigations are required to confirm these potentials. Since there is currently no specific treatment for COVID-19 infection, we sought to review and discuss the important roles of autophagy, UPR, and mTOR mechanisms in the regulation of cellular responses to coronavirus infection to help identify new antiviral modalities against SARS-CoV-2 virus.     

MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.     

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-β in Triggering Inflammatory Signals in Cells of Distant Organ

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-β (FGB) are highly abundant in exosomes from COVID-19 patients’ plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C–C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.