SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl- concentration.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupts the epithelial barrier and triggers airway inflammation. The envelope (E) protein, a core virulence structural component of coronaviruses, may play a role in this process. Pathogens could interfere with transepithelial Cl- transport via impairment of the cystic fibrosis transmembrane conductance regulator (CFTR), which modulates nuclear factor κB (NF-κB) signaling. However, the pathological effects of SARS-CoV-2 E protein on airway epithelial barrier function, Cl- transport and the robust inflammatory response remain to be elucidated. Here, we have demonstrated that E protein down-regulated the expression of tight junctional proteins, leading to the disruption of the airway epithelial barrier. In addition, E protein triggered the activation of Toll-like receptor (TLR) 2/4 and downstream c-Jun N-terminal kinase (JNK) signaling, resulting in an increased intracellular Cl- concentration ([Cl-]i) via up-regulating phosphodiesterase 4D (PDE4D) expression in airway epithelial cells. This elevated [Cl-]i contributed to the heightened airway inflammation through promoting the phosphorylation of serum/glucocorticoid regulated kinase 1 (SGK1). Moreover, blockade of SGK1 or PDE4 alleviated the robust inflammatory response induced by E protein. Overall, these findings provide novel insights into the pathogenic role of SARS-CoV-2 E protein in airway epithelial damage and the ongoing airway inflammation during SARS-CoV-2 infection.

The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.

Discovery of natural catechol derivatives as covalent SARS-CoV-2 3CLpro inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a threat to public health, and extensive research by scientists worldwide has also prompted the development of antiviral therapies. The 3C-like protease (3CLpro) is critical for SARS-CoV-2 replication and acts as an effective target for drug development. To date, numerous of natural products have been reported to exhibit inhibitory effects on 3CLpro, which encourages us to identify other novel inhibitors and elucidate their mechanism of action. In this study, we first screened an in-house compound library of 101 natural products using FRET assay, and found that oleuropein showed good inhibitory activity against SARS CoV-2 3CLpro with an IC50 value of 4.18 µM. Further studies revealed that the catechol core is essential for activity and can covalently bind to SARS-CoV-2 3CLpro. Among other 45 catechol derivatives, wedelolactone, capsazepine and brazilin showed better SARS-CoV-2 3CLpro inhibitory activities with IC50 values of 1.35 µM, 1.95 µM and 1.18 µM, respectively. These catechol derivatives were verified to be irreversible covalent inhibitors by time-dependent experiments, enzymatic kinetic studies, dilution and dialysis assays. It also exhibited good selectivity towards different cysteine proteases (SARS-CoV-2 PLpro, cathepsin B and cathepsin L). Subsequently, the binding affinity between brazilin and SARS-CoV-2 3CLpro was determined by SPR assay with KD value of 0.80 µM. Molecular dynamic (MD) simulations study showed the binding mode of brazilin in the target protein. In particular, brazilin displayed good anti-SARS-CoV-2 activity in A549-hACE2-TMPRSS2 cells with EC50 values of 7.85 ± 0.20 µM and 5.24 ± 0.21 µM for full time and post-infection treatments, respectively. This study provides a promising lead compound for the development of novel anti-SARS-CoV-2 drugs.

Discovery of highly potent covalent SARS-CoV-2 3CLpro inhibitors bearing 2-sulfoxyl-1,3,4-oxadiazole scaffold for combating COVID-19.

The coronavirus disease (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a major public health crisis, posing a significant threat to human well-being. Despite the availability of vaccines, COVID-19 continues to spread owing to the emergence of SARS-CoV-2 mutants. This highlights the urgent need for the discovery of more effective drugs to combat COVID-19. As an important target for COVID-19 treatment, 3C-like protease (3CLpro) plays a crucial role in the replication of SARS-CoV-2. In our previous research, we demonstrated the potent inhibitory activities of compound A1, which contains a 2-sulfonyl-1,3,4-oxadiazole scaffold, against SARS-CoV-2 3CLpro. Herein, we present a detailed investigation of structural optimization of A1 and conduct a study on the structure-activity relationship. Among the various compounds tested, sulfoxide D6 demonstrates a potent irreversible inhibitory activity (IC50 = 0.030 µM) against SARS-CoV-2 3CLpro, as well as a favorable selectivity towards host cysteine proteases such as cathepsin B and cathepsin L. Utilizing mass spectrometry-based peptide profiling, we found that D6 covalently binds to Cys145 of SARS-CoV-2 3CLpro. Some representative compounds, namely C11, D9 and D10 also demonstrates antiviral activity against SARS-CoV-2 in Vero E6 cells. Overall, the investigation of the 2-sulfoxyl-1,3,4-oxadiazole scaffold as a novel cysteine reactive warhead would provide valuable insights into the design of potent covalent 3CLpro inhibitors for COVID-19 treatment.

Intranasal booster using an Omicron vaccine confers broad mucosal and systemic immunity against SARS-CoV-2 variants.

The highly contagious SARS-CoV-2 Omicron subvariants severely attenuated the effectiveness of currently licensed SARS-CoV-2 vaccines based on ancestral strains administered via intramuscular injection. In this study, we generated a recombinant, replication-incompetent human adenovirus type 5, Ad5-S-Omicron, that expresses Omicron BA.1 spike. Intranasal, but not intramuscular vaccination, elicited spike-specific respiratory mucosal IgA and residential T cell immune responses, in addition to systemic neutralizing antibodies and T cell immune responses against most Omicron subvariants. We tested intranasal Ad5-S-Omicron as a heterologous booster in mice that previously received intramuscular injection of inactivated ancestral vaccine. In addition to inducing serum broadly neutralizing antibodies, there was a significant induction of respiratory mucosal IgA and neutralizing activities against Omicron subvariants BA.1, BA.2, BA.5, BA.2.75, BF.7 as well as pre-Omicron strains Wildtype, Beta, and Delta. Serum and mucosal neutralizing activities against recently emerged XBB, BQ.1, and BQ.1.1 could also be detected but were much lower. Nasal lavage fluids from intranasal vaccination contained multimeric IgA that can bind to at least 10 spike proteins, including Omicron subvariants and pre-Omicron strains, and possessed broadly neutralizing activities. Intranasal vaccination using Ad5-S-Omicron or instillation of intranasal vaccinee's nasal lavage fluids in mouse nostrils protected mice against Omicron challenge. Taken together, intranasal Ad5-S-Omicron booster on the basis of ancestral vaccines can establish effective mucosal and systemic immunity against Omicron subvariants and multiple SARS-CoV-2 variants. This candidate vaccine warrants further development as a safe, effective, and user-friendly infection and transmission-blocking vaccine.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19.

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Heterologous immunization with adenovirus vectored and inactivated vaccines effectively protects against SARS-CoV-2 variants in mice and macaques.

To cope with the decline in COVID-19 vaccine-induced immunity caused by emerging SARS-CoV-2 variants, a heterologous immunization regimen using chimpanzee adenovirus vectored vaccine expressing SARS-CoV-2 spike (ChAd-S) and an inactivated vaccine (IV) was tested in mice and non-human primates (NHPs). Heterologous regimen successfully enhanced or at least maintained antibody and T cell responses and effectively protected against SARS-CoV-2 variants in mice and NHPs. An additional heterologous booster in mice further improved and prolonged the spike-specific antibody response and conferred effective neutralizing activity against the Omicron variant. Interestingly, priming with ChAd-S and boosting with IV reduced the lung injury risk caused by T cell over activation in NHPs compared to homologous ChAd-S regimen, meanwhile maintained the flexibility of antibody regulation system to react to virus invasion by upregulating or preserving antibody levels. This study demonstrated the satisfactory compatibility of ChAd-S and IV in prime-boost vaccination in animal models.

Suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy.

In addition to investigating the virology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), discovering the host-virus dependencies are essential to identify and design effective antiviral therapy strategy. Here, we report that the SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) and provide evidence indicating that prevention of ACE2 SUMOylation can block SARS-CoV-2 infection. E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. TOLLIP deficiency results in the stabilization of ACE2 and elevated SARS-CoV-2 infection. In conclusion, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination as a potential way to combat SARS-CoV-2 infection.

SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl- accumulation in respiratory epithelium.

SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl- is a crucial regulator of host defense, whereas the role of Cl- signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl- concentration ([Cl-]i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl-]i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl-]i. Our findings suggested that Cl- acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl- signaling pathway might be a novel therapeutic strategy for COVID-19.

A Mouse-Adapted Model of HCoV-OC43 and Its Usage to the Evaluation of Antiviral Drugs.

The human coronavirus OC43 (HCoV-OC43) is one of the most common causes of common cold but can lead to fatal pneumonia in children and elderly. However, the available animal models of HCoV-OC43 did not show respiratory symptoms that are insufficient to assist in screening antiviral agents for respiratory diseases. In this study, we adapted the HCoV-OC43 VR-1558 strain by serial passage in suckling C57BL/6 mice and the resulting mouse-adapted virus at passage 9 (P9) contained 8 coding mutations in polyprotein 1ab, spike (S) protein, and nucleocapsid (N) protein. Pups infected with the P9 virus significantly lost body weight and died within 5 dpi. In cerebral and pulmonary tissues, the P9 virus replication induced the production of G-CSF, IFN-γ, IL-6, CXCL1, MCP-1, MIP-1α, RANTES, IP-10, MIP-1ß, and TNF-α, as well as pathological alterations including reduction of neuronal cells and typical symptoms of viral pneumonia. We found that the treatment of arbidol hydrochloride (ARB) or Qingwenjiere Mixture (QJM) efficiently improved the symptoms and decreased n gene expression, inflammatory response, and pathological changes. Furthermore, treating with QJM or ARB raised the P9-infected mice's survival rate within a 15 day observation period. These findings suggested that the new mouse-adapted HCoV-OC43 model is applicable and reproducible for antiviral studies of HCoV-OC43.

Potential drug discovery for COVID-19 treatment targeting Cathepsin L using a deep learning-based strategy.

Cathepsin L (CTSL), a cysteine protease that can cleave and activate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, could be a promising therapeutic target for coronavirus disease 2019 (COVID-19). However, there is still no clinically available CTSL inhibitor that can be used. Here, we applied Chemprop, a newly trained directed-message passing deep neural network approach, to identify small molecules and FDA-approved drugs that can block CTSL activity to expand the discovery of CTSL inhibitors for drug development and repurposing for COVID-19. We found 5 molecules (Mg-132, Z-FA-FMK, leupeptin hemisulfate, Mg-101 and calpeptin) that were able to significantly inhibit the activity of CTSL in the nanomolar range and inhibit the infection of both pseudotype and live SARS-CoV-2. Notably, we discovered that daptomycin, an FDA-approved antibiotic, has a prominent CTSL inhibitory effect and can inhibit SARS-CoV-2 pseudovirus infection. Further, molecular docking calculation showed stable and robust binding of these compounds with CTSL. In conclusion, this study suggested for the first time that Chemprop is ideally suited to predict additional inhibitors of enzymes and revealed the noteworthy strategy for screening novel molecules and drugs for the treatment of COVID-19 and other diseases with unmet needs.

Combinational benefit of antihistamines and remdesivir for reducing SARS-CoV-2 replication and alleviating inflammation-induced lung injury in mice.

COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Angiotensin-converting enzyme 2 in peripheral lung club cells modulates the susceptibility to SARS-CoV-2 in chronic obstructive pulmonary disease.

Accumulating evidence has confirmed that chronic obstructive pulmonary disease (COPD) is a risk factor for development of severe pathological changes in the peripheral lungs of patients with COVID-19. However, the underlying molecular mechanisms remain unclear. Because bronchiolar club cells are crucial for maintaining small airway homeostasis, we sought to explore whether the altered susceptibility to SARS-CoV-2 infection of the club cells might have contributed to the severe COVID-19 pneumonia in COPD patients. Our investigation on the quantity and distribution patterns of angiotensin-converting enzyme 2 (ACE2) in airway epithelium via immunofluorescence staining revealed that the mean fluorescence intensity of the ACE2-positive epithelial cells was significantly higher in club cells than those in other epithelial cells (including ciliated cells, basal cells, goblet cells, neuroendocrine cells, and alveolar type 2 cells). Compared with nonsmokers, the median percentage of club cells in bronchiolar epithelium and ACE2-positive club cells was significantly higher in COPD patients. In vitro, SARS-CoV-2 infection (at a multiplicity of infection of 1.0) of primary small airway epithelial cells, cultured on air-liquid interface, confirmed a higher percentage of infected ACE2-positive club cells in COPD patients than in nonsmokers. Our findings have indicated the role of club cells in modulating the pathogenesis of SARS-CoV-2-related severe pneumonia and the poor clinical outcomes, which may help physicians to formulate a novel therapeutic strategy for COVID-19 patients with coexisting COPD.

Neuropilin-1-Mediated SARS-CoV-2 Infection in Bone Marrow-Derived Macrophages Inhibits Osteoclast Differentiation.

In humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause medical complications across various tissues and organs. Despite the advances to understanding the pathogenesis of SARS-CoV-2, its tissue tropism and interactions with host cells have not been fully understood. Existing clinical data have revealed disordered calcium and phosphorus metabolism in Coronavirus Disease 2019 (COVID-19) patients, suggesting possible infection or damage in the human skeleton system by SARS-CoV-2. Herein, SARS-CoV-2 infection in mouse models with wild-type and beta strain (B.1.351) viruses is investigated, and it is found that bone marrow-derived macrophages (BMMs) can be efficiently infected in vivo. Single-cell RNA sequencing (scRNA-Seq) analyses of infected BMMs identify distinct clusters of susceptible macrophages, including those related to osteoblast differentiation. Interestingly, SARS-CoV-2 entry on BMMs is dependent on the expression of neuropilin-1 (NRP1) rather than the widely recognized receptor angiotensin-converting enzyme 2 (ACE2). The loss of NRP1 expression during BMM-to-osteoclast differentiation or NRP1 neutralization and knockdown can significantly inhibit SARS-CoV-2 infection in BMMs. Importantly, it is found that authentic SARS-CoV-2 infection impedes BMM-to-osteoclast differentiation. Collectively, this study provides evidence for NRP1-mediated SARS-CoV-2 infection in BMMs and establishes a potential link between disturbed osteoclast differentiation and disordered skeleton metabolism in COVID-19 patients.

Inactivated Rabies Virus Vectored MERS-Coronavirus Vaccine Induces Protective Immunity in Mice, Camels, and Alpacas.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent coronavirus that has caused frequent zoonotic events through camel-to-human spillover. An effective camelid vaccination strategy is probably the best way to reduce human exposure risk. Here, we constructed and evaluated an inactivated rabies virus-vectored MERS-CoV vaccine in mice, camels, and alpacas. Potent antigen-specific antibody and CD8+ T-cell responses were generated in mice; moreover, the vaccination reduced viral replication and accelerated virus clearance in MERS-CoV-infected mice. Besides, protective antibody responses against both MERS-CoV and rabies virus were induced in camels and alpacas. Satisfyingly, the immune sera showed broad cross-neutralizing activity against the three main MERS-CoV clades. For further characterization of the antibody response induced in camelids, MERS-CoV-specific variable domains of heavy-chain-only antibody (VHHs) were isolated from immunized alpacas and showed potent prophylactic and therapeutic efficacies in the Ad5-hDPP4-transduced mouse model. These results highlight the inactivated rabies virus-vectored MERS-CoV vaccine as a promising camelid candidate vaccine.

Investigating Derivatives of Tanshinone IIA Sulfonate Sodium and Chloroxine for Their Inhibition Activities against the SARS-CoV-2 Papain-like Protease.

SARS-CoV-2 has caused a global pandemic of COVID-19, posing a huge threat to public health. The SARS-CoV-2 papain-like cysteine protease (PLpro) plays a significant role in virus replication and host immune regulation, which is a promising antiviral drug target. Several potential inhibitors have been identified in vitro. However, the detailed mechanism of action and structure-activity relationship require further studies. Here, we investigated the structure-activity relationships of the series of derivatives of tanshinone IIA sulfonate sodium (TSS) and chloroxine based on biochemical analysis and molecular dynamics simulation. We found that compound 7, a derivative of chloroxine, can disrupt PLpro-ISG15 interaction and exhibits an antiviral effect for SARS-CoV-2 variants (wild type, delta, and omicron) at the low micromolar level. These studies confirmed that inhibiting PLpro-ISG15 interaction and, thus, restoring the host's innate immunity are effective methods for fighting against viral infection.

Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models.

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.