ENDO-LYSOSOME-TARGETED NANOPARTICLE DELIVERY OF ANTIVIRAL THERAPY FOR CORONAVIRUS INFECTIONS (preprint)

SARS-CoV-2 can infect cells through endocytic uptake, a process which can be targeted by inhibition of lysosomal proteases. However, clinically this approach fared poorly with an oral regimen of hydroxychloroquine that was accompanied by significant toxicity due to off-target effects. We rationalized that an organelle-targeted approach will avoid toxicity while increasing the concentration of the drug at the target. Here we describe a lysosome-targeted, mefloquine-loaded poly(glycerol monostearate-co-{varepsilon}-caprolactone) nanoparticle (MFQ-NP) for pulmonary delivery via inhalation. Mefloquine is a more effective inhibitor of viral endocytosis than hydroxychloroquine in cellular models of COVID-19. MFQ-NPs are less toxic than molecular mefloquine, 100-150 nm in diameter, and possess a negative surface charge which facilitates uptake via endocytosis allowing inhibition of lysosomal proteases. MFQ-NPs inhibit coronavirus infection in mouse MHV-A59 and human OC43 coronavirus model systems and inhibit SARS-CoV-2-WA1 and its Omicron variant in a human lung epithelium model. This study demonstrates that organelle-targeted delivery is an effective means to inhibit viral infection.

Identification of a Papain-Like Protease Inhibitor with Potential for Repurposing in Combination with an Mpro Protease Inhibitor for Treatment of SARS-CoV-2 (preprint)

SARS-CoV-2 requires two cysteine proteases for viral polypeptide processing to allow maturation and replication: the 3Clike protease also known as the Main protease (Mpro) and the papain-like protease (PLpro). In addition to its critical role in viral replication, PLpro removes post-translational modifications like ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins through its deubiquitinase domain, leading to host immunosuppression and increased ability of the virus to evade the host antiviral immune response. Through screening of a custom clinical compound library, we identified eltrombopag (DDL701), a thrombopoietin receptor antagonist, as having PLpro inhibitory activity that is sustained in the presence of the Mpro inhibitor nirmatrelvir. DDL701 also suppressed both the deubiquitinase and ISG15 cleavage activities of PLpro. In addition, DDL701 partially restored interferon-{beta} induction, an element of the host immune response, in an in vitro model system. Further, modeling and docking studies suggest DDL701 interacts with the active site region of the PLpro enzyme and pilot pharmacokinetic studies indicate it is brain permeable. DDL701 is already approved for treatment of thrombocytopenia and has previously been shown to achieve human plasma levels after oral dosing that is above the IC50 needed for it to exert its PLpro inhibitory activity in vivo. In addition, it has also been reported to have antiviral efficacy against SARS-CoV-2. DDL-701 thus represents a drug that can immediately be repurposed and undergo clinical evaluation as a PLpro inhibitor that may be most effectively used in a protease inhibitor cocktail with an Mpro inhibitor such as nirmatrelvir (Paxlovid) for the treatment of COVID19.

Hippo Signaling Pathway Activation during SARS-CoV-2 Infection Contributes to Host Antiviral Response (preprint)

SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

Mitoquinone mesylate targets SARS-CoV-2 and associated lung inflammation through host pathways (preprint)

To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo. Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy. One-Sentence SummaryMitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

Structure-based design of antisense oligonucleotides that inhibit SARS-CoV-2 replication (preprint)

Antisense oligonucleotides (ASOs) are an emerging class of drugs that target RNAs. Current ASO designs strictly follow the rule of Watson-Crick base pairing along target sequences. However, RNAs often fold into structures that interfere with ASO hybridization. Here we developed a structure-based ASO design method and applied it to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our method makes sure that ASO binding is compatible with target structures in three-dimensional (3D) space by employing structural design templates. These 3D-ASOs recognize the shapes and hydrogen bonding patterns of targets via tertiary interactions, achieving enhanced affinity and specificity. We designed 3D-ASOs that bind to the frameshift stimulation element and transcription regulatory sequence of SARS-CoV-2 and identified lead ASOs that strongly inhibit viral replication in human cells. We further optimized the lead sequences and characterized structure-activity relationship. The 3D-ASO technology helps fight coronavirus disease-2019 and is broadly applicable to ASO drug development.

ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling (preprint)

ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.

Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition (preprint)

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, despite limited overall sequence conservation, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.34 - 0.76], p = 0.001). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner. Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks.

Deleterious effects of SARS-CoV-2 infection on human pancreatic cells (preprint)

COVID-19 pandemic has infected more than 46 million people worldwide and caused more than 1.2 million deaths. It is transmitted by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and affects the respiratory tract as well as extra-pulmonary systems, including the pancreas, that express the virus entry receptor, Angiotensin-Converting Enzyme 2 (ACE2) receptor. Importantly, the endocrine and exocrine pancreas, the latter composed of ductal and acinar cells, express high levels of ACE2, which correlates to impaired functionality characterized as acute pancreatitis observed in some cases presenting with COVID-19. Since acute pancreatitis is already one of the most frequent gastrointestinal causes of hospitalization in the U.S. and the majority of studies investigating the effects of SARS-CoV-2 on the pancreas are clinical and observational, we utilized human iPSC technology to investigate the potential deleterious effects of SARS-CoV-2 infection on iPSC-derived pancreatic cultures containing endocrine and exocrine cells. Interestingly, SARS-CoV-2 is capable of infecting iPSC-derived pancreatic cells, thus perturbing their normal molecular and cellular phenotypes. The infection increased a key inflammatory cytokine, CXCL12, known to be involved in pancreas dysfunction. Transcriptome analysis of infected pancreatic cultures confirmed that SARS-CoV-2 hijacks the ribosomal machinery in these cells. Notably, the SARS-CoV-2 infectivity of the pancreas is confirmed in post-mortem tissues from COVID-19 patients, which showed co-localization of SARS-CoV-2 in pancreatic endocrine and exocrine cells and increased the expression of some pancreatic ductal stress response genes. Thus, we demonstrate for the first time that SARS-CoV-2 can directly infect human iPSC-derived pancreatic cells with supporting evidence of presence of the virus in post-mortem pancreatic tissue of confirmed COVID-19 human cases. This novel model of iPSC-derived pancreatic cultures will open new avenues for the comprehension of the SARS-CoV-2 infection and potentially establish a platform for endocrine and exocrine pancreas-specific antiviral drug screening.

A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro (preprint)

We recently discovered a superantigen-like motif, similar to Staphylococcal enterotoxin B (SEB), near the S1/S2 cleavage site of SARS-CoV-2 Spike protein, which might explain the multisystem-inflammatory syndrome (MIS-C) observed in children and cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif, and in particular its PRRA insert, to inhibit infection by blocking the access of host cell proteases, TMPRSS2 or furin, to the cleavage site. The high affinity of 6D3 for the furin-cleavage site originates from a poly-acidic segment at its heavy chain CDR2, a feature shared with SARS-CoV-2-neutralizing mAb 4A8. The affinity of 6D3 and 4A8 for this site points to their potential utility as therapeutics for treating COVID-19, MIS-C, or common cold caused by human coronaviruses (HCoVs) that possess a furin-like cleavage site.

Cold plasma for SARS-CoV-2 Inactivation (preprint)

SARS-CoV-2 infectious virions are viable on various surfaces (e.g., plastic, metals, cardboard) for several hours. This presents a transmission cycle for the human infection that can be broken by developing new inactivation approaches. We employed an efficient cold atmospheric plasma (CAP) with argon feed gas to inactivate SARS-CoV-2 on various surfaces including plastic, metal, cardboard, basketball composite leather, football leather, and baseball leather. These results demonstrate the great potential of CAP as a safe and effective means to prevent virus transmission and infections.

Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stemcell-derived airway repair response (preprint)

Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon {beta}-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery (preprint)

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. One Sentence SummaryA novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication (preprint)

Emergence of a highly contagious novel coronavirus, SARS-CoV-2 that causes COVID-19, has precipitated the current global health crisis with over 479,000 deaths and more than 9.3 million confirmed cases. Currently, our knowledge of the mechanisms of COVID-19 disease pathogenesis is very limited which has hampered attempts to develop targeted antiviral strategies. Therefore, we urgently need an effective therapy for this unmet medical need. Viruses hijack and dysregulate cellular machineries in order for them to replicate and infect more cells. Thus, identifying and targeting dysregulated signaling pathways that have been taken over by viruses is one strategy for developing an effective antiviral therapy. We have developed a high-throughput drug screening system to identify potential antiviral drugs targeting SARS-CoV-2. We utilized a small molecule library of 430 protein kinase inhibitors, which are in various stages of clinical trials. Most of the tested kinase antagonists are ATP competitive inhibitors, a class of nucleoside analogs, which have been shown to have potent antiviral activity. From the primary screen, we have identified 34 compounds capable of inhibiting viral cytopathic effect in epithelial cells. Network of drug and protein relations showed that these compounds specifically targeted a limited number of cellular kinases. More importantly, we have identified mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response (DDR) pathways as key cellular signaling pathways critical for SARS-CoV-2 infection. Subsequently, a secondary screen confirmed compounds such as Berzosertib (VE-822), Vistusertib (AZD2014), and Nilotinib with anti SARS-CoV-2 activity. Finally, we found that Berzosertib, an ATR kinase inhibitor in the DDR pathway, demonstrated potent antiviral activity in a human epithelial cell line and human induced pluripotent stem cell (hIPSC)-derived cardiomyocytes. These inhibitors are already in clinical trials of phase 2 or 3 for cancer treatment, and can be repurposed as promising drug candidates for a host-directed therapy of SARS-CoV-2 infection. In conclusion, we have identified small molecule inhibitors exhibiting anti SARS-CoV-2 activity by blocking key cellular kinases, which gives insight on important mechanism of host-pathogen interaction. These compounds can be further evaluated for the treatment of COVID-19 patients following additional in vivo safety and efficacy studies. DisclosuresNone declared.

Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection (preprint)

Coronavirus disease 2019 (COVID-19) is a viral pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is predominantly defined by respiratory symptoms, but cardiac complications including arrhythmias, heart failure, and viral myocarditis are also prevalent. Although the systemic ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well-understood. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and immunofluorescence demonstrated that SARS-CoV-2 can enter and replicate within hiPSC-CMs, localizing at perinuclear locations within the cytoplasm. Viral cytopathic effect induced hiPSC-CM apoptosis and cessation of beating after 72 hours of infection. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro, establishing a model for elucidating the mechanisms of infection and potentially a cardiac-specific antiviral drug screening platform.

Insights into Cross-species Evolution of Novel Human Coronavirus 2019-nCoV and Defining Immune Determinants for Vaccine Development (preprint)

Novel Coronavirus (nCoV) outbreak in the city of Wuhan, China during December 2019, has now spread to various countries across the globe triggering a heightened containment effort. This human pathogen is a member of betacoronavirus genus carrying 30 kilobase of single positive-sense RNA genome. Understanding the evolution, zoonotic transmission, and source of this novel virus would help accelerating containment and prevention efforts. The present study reported detailed analysis of 2019-nCoV genome evolution and potential candidate peptides for vaccine development. This nCoV genotype might have been evolved from a bat-CoV by accumulating non-synonymous mutations, indels, and recombination events. Structural proteins Spike (S), and Membrane (M) had extensive mutational changes, whereas Envelope (E) and Nucleocapsid (N) proteins were very conserved suggesting differential selection pressures exerted on 2019-nCoV during evolution. Interestingly, 2019-nCoV Spike protein contains a 39 nucleotide sequence insertion relative to SARS-like bat-SL-CoVZC45/2017. Furthermore, we identified eight high binding affinity (HBA) CD4 T-cell epitopes in the S, E, M and N proteins, which can be commonly recognized by HLA-DR alleles of Asia and Asia-Pacific Region population. These immunodominant epitopes can be incorporated in universal subunit CoV vaccine. Diverse HLA types and variations in the epitope binding affinity may contribute to the wide range of immunopathological outcomes of circulating virus in humans. Our findings emphasize the requirement for continuous surveillance of CoV strains in live animal markets to better understand the viral adaptation to human host and to develop practical solutions to prevent the emergence of novel pathogenic CoV strains.