COVID Moonshot: Open Science Discovery of SARS-CoV-2 Main Protease Inhibitors by Combining Crowdsourcing, High-Throughput Experiments, Computational Simulations, and Machine Learning (preprint)

Herein we provide a living summary of the data generated during the COVID Moonshot project focused on the development of SARS-CoV-2 main protease (Mpro) inhibitors. Our approach uniquely combines crowdsourced medicinal chemistry insights with high throughput crystallography, exascale computational chemistry infrastructure for simulations, and machine learning in triaging designs and predicting synthetic routes. This manuscript describes our methodologies leading to both covalent and non-covalent inhibitors displaying protease IC50 values under 150 nM and viral inhibition under 5 uM in multiple different viral replication assays. Furthermore, we provide over 200 crystal structures of fragment-like and lead-like molecules in complex with the main protease. Over 1000 synthesized and ordered compounds are also reported with the corresponding activity in Mpro enzymatic assays using two different experimental setups. The data referenced in this document will be continually updated to reflect the current experimental progress of the COVID Moonshot project, and serves as a citable reference for ensuing publications. All of the generated data is open to other researchers who may find it of use.

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies (preprint)

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2 (preprint)

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the world's first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-Crypt platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

Evidence for adaptive evolution in the receptor-binding domain of seasonal coronaviruses (preprint)

Seasonal coronaviruses (OC43, 229E, NL63 and HKU1) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. It is not known to what extent reinfection by these viruses is due to waning immune memory or antigenic drift of the viruses. Here, we address the influence of antigenic drift on immune evasion of seasonal coronaviruses. We provide evidence that at least two of these viruses, OC43 and 229E, are undergoing adaptive evolution in regions of the viral spike protein that are exposed to human humoral immunity. This suggests that reinfection may be due, in part, to positively-selected genetic changes in these viruses that enable them to escape recognition by the immune system. It is possible that, as with seasonal influenza, these adaptive changes in antigenic regions of the virus would necessitate continual reformulation of a vaccine made against them.

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells (preprint)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19.

Evaluation of nafamostat mesylate safety and inhibition of SARS-CoV-2 replication using a 3-dimensional human airway epithelia model (preprint)

In the current COVID-19 pandemic context, Ensysce and its subsidiary Covistat have been working to repurpose nafamostat mesylate as an effective oral and inhalation treatment against SARS-CoV-2 infection. Prior reports used cell lines to demonstrate the antiviral potential of nafamostat against coronaviral infections and determined its mechanism of action through inhibition of transmembrane protease serine 2 (TMPRSS2). We selected a biologically relevant pre-clinical experimental model of SARS-CoV-2 lung infection using a 3D human reconstituted airway epithelial model of nasal origin to characterize the effects of nafamostat on tissue-level cellular ultrastructure and viral infection kinetics. Our results confirm the not only the relevance of this model for the preclinical evaluation of safety and efficacy of antiviral candidates, but also the highly potent nature of nafamostat SARS-CoV-2 antiviral activity. The studies described herein provided evidence demonstrating the therapeutic potential of nafamostat against COVID-19, as well as its safety upon exposure to lung airway cellular.

Lopinavir-ritonavir is not an effective inhibitor of the main protease activity of SARS-CoV-2 in vitro (preprint)

COVID-19 has caused over 900,000 deaths worldwide as of September 2020, and effective medicines are urgently needed. Lopinavir was identified as an inhibitor of the HIV protease, and a lopinavir-ritonavir combination therapy was reported to be beneficial for the treatment of SARS and MERS. However, recent clinical tests could not prove that lopinavir-ritonavir therapy was an effective treatment for COVID-19. In this report, we examined the effect of lopinavir and ritonavir to the activity of the purified main protease (Mpro) protein of SARS-CoV-2, the causative virus of COVID-19. Unexpectedly, lopinavir and ritonavir did not inhibit Mpro activity. These results will aid the drug candidate selection for ongoing and future COVID-19 clinical trials.

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors (preprint)

There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (Mpro) is one of the most extensively studied drug targets. Mpro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, Mpro has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that Mpro inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as Mpro inhibitors, with several of which also showed antiviral activity in cell culture. In this study, we investigated the mechanism of action of six previously reported Mpro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of Mpro by these six compounds is non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit Mpro, but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 2Apro and 3Cpro from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC50 values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 Mpro inhibitors, and urge the scientific community to be stringent with hit validation.

Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function (preprint)

Background: The emergence of a novel coronavirus (SARS-CoV-2) associated with severe acute respiratory disease (COVID-19) has prompted efforts to understand the genetic basis for its unique characteristics and its jump from non-primate hosts to humans. Tests for positive selection can identify apparently nonrandom patterns of mutation accumulation within genomes, highlighting regions where molecular function may have changed during the origin of a species. Several recent studies of the SARS-CoV-2 genome have identified signals of conservation and positive selection within the gene encoding Spike protein based on the ratio of synonymous to nonsynonymous substitution. Such tests cannot, however, detect changes in the function of RNA molecules. Methods: Here we apply a test for branch-specific oversubstitution of mutations within narrow windows of the genome without reference to the genetic code. Results: We recapitulate the finding that the gene encoding Spike protein has been a target of both purifying and positive selection. In addition, we find other likely targets of positive selection within the genome of SARS-CoV-2, specifically within the genes encoding Nsp4 and Nsp16. Homology-directed modeling indicates no change in either Nsp4 or Nsp16 protein structure relative to the most recent common ancestor. Thermodynamic modeling of RNA stability and structure, however, indicates that RNA secondary structure within both genes in the SARS-CoV-2 genome differs from those of RaTG13, the reconstructed common ancestor, and Pan-CoV-GD (Guangdong). These SARS-CoV-2-specific mutations may affect molecular processes mediated by the positive or negative RNA molecules, including transcription, translation, RNA stability, and evasion of the host innate immune system. Our results highlight the importance of considering mutations in viral genomes not only from the perspective of their impact on protein structure, but also how they may impact other molecular processes critical to the viral life cycle.

Copper-oxide impregnated respiratory masks may significantly reduce the risk of SARS-CoV-2 cross-contamination (preprint)

The use of protective respiratory face masks has been adopted universally as an important measure in the fight against COVID-19. Masks become contaminated by symptomatic and asymptomatic SARS-CoV-2 infected individuals and the virus can remain viable on the surface of the masks for several days. Although the regular respiratory face masks are single use disposable masks, these masks are being largely reused and not often discarded after use by the general population. Mask touching during use, reuse and disposal occurs frequently, and this can lead to increased risk of infection and further transmission. N95s and regular surgical masks were produced in which the external layers were made with nonwoven fabric impregnated with copper-oxide microparticles. The masks reduced the infectious titers of SARS-CoV-2 by more than 99.9% within 1 minute of contact, as determined by TCID50 assay and serial PCR assays. The use of masks capable of rendering the SARS-CoV-2 non-infectious within minutes, may significantly reduce the risk of viral transmission and infection.

The Infectious Nature of Patient-Generated SARS-CoV-2 Aerosol (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission causing coronavirus disease 2019 (COVID-19) may occur through multiple routes. We collected aerosol samples around six patients admitted into mixed acuity wards in April of 2020 to identify the risk of airborne SARS-CoV-2. Measurements were made to characterize the size distribution of aerosol particles, and size-fractionated, aerosol samples were collected to assess the presence of infectious virus in particles sizes of >4.1 m, 1-4 m, and <1 m in the patient environment. Samples were analyzed by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR), cell culture, western blot, and transmission electron microscopy (TEM). SARS-CoV-2 RNA was detected in all six rooms in all particle size fractions (>4.1 m, 1-4 m, and <1 m). Increases in viral RNA during cell culture of the virus from recovered aerosol samples demonstrated the presence of infectious, replicating virions in three <1 m aerosol samples (P<0.05). Viral replication of aerosol was also observed in the 1-4 m stage but did not reach statistical significance (0.05

Transmission Potential of SARS-CoV-2 in Viral Shedding Observed at the University of Nebraska Medical Center (preprint)

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in late 2019, and its resulting coronavirus disease, COVID-19, was declared a pandemic by the World Health Organization on March 11, 2020. The rapid global spread of COVID-19 represents perhaps the most significant public health emergency in a century. As the pandemic progressed, a continued paucity of evidence on routes of SARS-CoV-2 transmission has resulted in shifting infection prevention and control guidelines between clasically-defined airborne and droplet precautions. During the initial isolation of 13 individuals with COVID-19 at the University of Nebraska Medical Center, we collected air and surface samples to examine viral shedding from isolated individuals. We detected viral contamination among all samples, indicating that SARS-CoV-2 may spread through both direct (droplet and person-to-person) as well as indirect mechanisms (contaminated objects and airborne transmission). Taken together, these finding support the use of airborne isolation precautions when caring for COVID-19 patients.