The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir (preprint)

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6- to 72-fold). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting.

A SCID mouse model to evaluate the efficacy of antivirals against SARS-CoV-2 infection (preprint)

Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2.

Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model (preprint)

Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.

Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern. (preprint)

The in vitro effect of GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir against the various SARS-CoV-2 VOCs, including Omicron, was determined. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The number of fluorescent pixels of GFP signal, determined by high-content imaging on day 4 post-infection, was used as read-out, and the EC50 of each compound on a viral isolate of each VOC was calculated. These experiments were performed in the presence of the Pgp-inhibitor CP-100356 in order to limit compound efflux. A SARS-CoV-2 strain grown from the first Belgian patient sample was used as ancestral strain. All the other isolates were obtained from patients in Belgium as well. Our results indicate that GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir retain their activity against the VOCs Alpha, Beta, Gamma, Delta and Omicron. This is in accordance with the observation that the target proteins of these antivirals are highly conserved.

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern (preprint)

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the main viral protease (Mpro, 3CLpro) that can be dosed orally; the compound is in clinical development. We demonstrate that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels. The trough drug concentration at this efficacious dose were above the in vitro efficacious concentrations.

A Highly Potent Antibody Effective Against SARS-CoV-2 Variants of Concern (preprint)

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies and reduced sensitivity to vaccine-induced immunity. Here, we screened B cells from COVID-19 donors and identified P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOC) identified to date. Structural characterization of P5C3 Fab in complex with the Spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 showed complete prophylactic protection in the SARS-CoV-2 infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals.Funding: This CARE project has received funding from the Innovative MedicinesInitiative 2 Joint Undertaking (JU) under grant agreement No 101005077. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and BILL & MELINDA GATES FOUNDATION, GLOBAL HEALTH DRUG DISCOVERYINSTITUTE, UNIVERSITY OF DUNDEE. Furthermore, funding was also provided through the Lausanne University Hospital, through the Swiss Vaccine Research Institute to G.P., and through the EPFL COVID fund to D.T.Conflict of Interest: None to declare. Ethical Approval: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD).

Identification and Evaluation of Potential SARS-CoV-2 Antiviral Agents Targeting mRNA Cap Guanine N7-Methyltransferase (preprint)

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 μM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay

A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents (preprint)

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35{degrees}C than 37{degrees}C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN ({beta}1 and {lambda}1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model (preprint)

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 M). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Nelfinavir markedly improves lung pathology in SARS-CoV-2-infected Syrian hamsters despite a lack of an antiviral effect (preprint)

In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The FDA-approved HIV protease inhibitor Nelfinavir is one of the drugs that has been reported to inhibit in vitro SARS-CoV2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 mg/kg BID or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] did not reduce viral RNA loads nor infectious virus titres in the lungs (as compared to the vehicle control at the end of treatment) the drug markedly improved virus-induced lung pathology at doses that were well tolerated. Yet, a massive interstitial infiltration of neutrophils was observed in the lungs of treated (infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology that is unrelated to an antiviral effect warrants further exploration in the context of the treatment of COVID-19.

Environmentally-induced mdig is a major contributor to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism (preprint)

The novel {beta}-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 101 million people and resulted in 2.2 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provide evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is a master regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. These data, accordingly, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen (preprint)

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

Molnupiravir (EIDD-2801) inhibits SARS-CoV2 replication in Syrian hamsters model (preprint)

Since its emergence in Wuhan, China in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide resulting in a global pandemic with >1.5 million deaths until now. In the search for small molecule inhibitors of SARS-CoV-2, drug repurposing is being extensively explored. Molnupiravir (EIDD-2801) is an orally bioavailable nucleoside analog that possesses a relatively broad-spectrum antiviral activity including against coronaviruses. We here studied the effect of EIDD-2801 in a well-established Syrian hamster SARS-CoV2 infection model. Treatment of SARS-CoV-2-infected hamsters with 200 mg/kg BID of EIDD-2801 for four consecutive days, starting from the day of infection, significantly reduced infectious virus titers and viral RNA loads in the lungs and markedly improved lung histopathology. When onset of treatment was delayed until 1 or 2 days after infection, a very modest antiviral effect was observed. The potential of EIDD-2801 for the treatment and or prevention of SARS-CoV2 deserves further attention.