SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design (preprint)

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 Angstroms and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 Angstroms, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Goblet Cell Hyperplasia Increases SARS-CoV-2 Infection in COPD (preprint)

SARS-CoV-2 has become a major problem across the globe, with approximately 50 million cases and more than 1 million deaths and currently no approved treatment or vaccine. Chronic obstructive pulmonary disease (COPD) is one of the underlying conditions in adults of any age that place them at risk for developing severe illness associated with COVID-19. We established an airway epithelium model to study SARS-CoV-2 infection in healthy and COPD lung cells. We found that both the entry receptor ACE2 and the co-factor transmembrane protease TMPRSS2 are expressed at higher levels on nonciliated goblet cell, a novel target for SARS-CoV-2 infection. We observed that SARS-CoV-2 infected goblet cells and induced syncytium formation and cell sloughing. We also found that SARS-CoV-2 replication was increased in the COPD airway epithelium likely due to COPD associated goblet cell hyperplasia. Our results reveal goblet cells play a critical role in SARS-CoV-2 infection in the lung.

Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerase (PARP), blocks replication of the SARS-CoV-2 human coronavirus in vitro. (preprint)

By late 2020, the coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 has caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new PARP inhibitor, stenoparib, which was recently advanced to Stage II clinical trials for treatment of ovarian cancer. This is an initial report on the activity of stenoparib against human respiratory coronaviruses, including SARS-CoV-2, in vitro. Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to multiplication of the HCoV-NL63 human seasonal respiratory coronavirus. Compared to remdesivir, which inhibits the viral replicon subsequent to cell entry, stenoparib is inhibitory to virus entry and post-entry processes as determined by time-of-addition (TOA) experiments. Moreover, a 10 M dosage of stenoparib, which is far below its 25.5 M half-maximally effective concentration (EC50), when combined with 0.5 M remdesivir suppressed coronavirus growth by 90.7%, indicating a potentially synergistic effect for this drug combination. Thus, stenoparib as a standalone or as a component of combinatorial therapy with remdesivir may be a valuable addition to the arsenal against COVID-19.

A practical PPE decontamination method using warm air and ambient humidity (preprint)

Personal protective equipment (PPE) remains in short supply. Current decontamination methods are complex, slow, expensive and particularly ill-suited for low to middle income nations where the need is greatest. We propose a low temperature, ambient humidity decontamination method (WASP-D) based on the thirty minute or less half-life of Sars-CoV-2 (and other common pathogens) at temperatures above 45C, combined with the observation that most PPE are designed to be safely transported and stored at temperatures below 50C. Decontamination at 12 hours, 46C (115F) and ambient humidity should consistently reduce SARS-CoV-2 viral load by a factor of 10-6, without negatively affecting PPE materials or performance.

ORF3a mediated-incomplete autophagy facilitates SARS-CoV-2 replication (preprint)

SARS-CoV-2 is the causative agent for the COVID-19 pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin-1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 28 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate Beclin-1-Vps34-Atg14 complex but selectively inhibit Beclin-1-Vps34-UVRAG complex. Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raises the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease (preprint)

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Ozone exposure upregulates the expression of host susceptibility protein TMPRSS2 to SARS-CoV-2 (preprint)

Background: SARS-CoV-2, a novel coronavirus, and the etiologic agent for the current global health emergency, causes acute infection of the respiratory tract leading to severe disease and significant mortality. Ever since the start of SARS-CoV-2, also known as COVID-19 pandemic, countless uncertainties have been revolving around the pathogenesis and epidemiology of the SARS-CoV-2 infection. While air pollution has been shown to be strongly correlated to increased SARS-CoV-2 morbidity and mortality, whether environmental pollutants such as ground level ozone affects the susceptibility of individuals to SARS-CoV-2 is not yet established. Objective: To investigate the impact of ozone inhalation on the expression levels of signatures associated with host susceptibility to SARS-CoV-2. Methods: We analyzed lung tissues collected from mice that were sub-chronically exposed to air or 0.8ppm ozone for three weeks (4h/night, 5 nights/week), and analyzed the expression of signatures associated with host susceptibility to SARS-CoV-2. Results: SARS-CoV-2 entry into the host cells requires proteolytic priming by the host-derived protease, transmembrane protease serine 2 (TMPRSS2). The TMPRSS2 protein and Tmprss2 transcripts were significantly elevated in the extrapulmonary airways, parenchyma, and alveolar macrophages from ozone-exposed mice. A significant proportion of additional known SARS-CoV-2 host susceptibility genes were upregulated in alveolar macrophages and parenchyma from ozone-exposed mice. Conclusions: Our data indicate that the unhealthy levels of ozone in the environment may predispose individuals to severe SARS-CoV-2 infection. Given the severity of this pandemic, and the challenges associated with direct testing of host-environment interactions in clinical settings, we believe that this mice-ozone-exposure based study informs the scientific community of the potentially detrimental effects of the ambient ozone levels determining the host susceptibility to SARS-CoV-2.

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease (preprint)

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.