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.

Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses.

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.

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 °C than 37 °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 (β1 and λ1) 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.

Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve ∼18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range. Enterovirus A71 (EV71) is a small virus (ϳ30 nm) with a singlestranded positive-sense RNA genome of ϳ7.4 kb that belongs to the genus Enterovirus of the family Picornaviridae (1, 2). EV71 is the etiologic agent of hand, foot, and mouth disease (HFMD), a mild syndrome that most frequently affects children younger than 6 years and that is characterized by the development of fever with skin vesicles on the palms and feet, as well as ulcers on the oral mucosa (3). Unlike other HFMD-associated enteroviruses, EV71 can also cause severe neurological problems, such as aseptic meningitis and brain stem encephalitis, which can lead to cardiopulmonary failure and death (4-6). After having suffered from such neurological complications, survivors often have permanent neurological sequelae, such as delayed neurodevelopment, reduced cognitive function, and polio-like paralysis (7). Similar to other human enteroviruses, such as poliovirus, transmission of EV71 occurs through the fecal-oral route (8).In recent years, large outbreaks of EV71 have been reported throughout the world, and they have been particularly severe in the Pacific region of Asia, with a high number of fatal cases among children (9-11). So far, there is no drug on the market to treat or prevent this infection. An inactivated EV71 vaccine was recently approved in China (12), but it may induce only limited crossneutralization between EV71 genogroups, which does not make it suitable for widespread use.Recently, we reported on the anti-HIV activity of a dendrimer family containing different central scaffolds and multiple (9 to 18) peripheral tryptophan (Trp) groups (Fig. 1, compounds 1 to 11) that are linked to the dendrimer branches through an amino group. These compounds were shown to inhibit an early step in the replication cycle of HIV by interacting with glycoproteins gp120 and gp41 of the HIV envelope (13). Further exploration in virus-cell-based assays for broad-spectrum antiviral activity against other viruses (herpes simplex viruses 1 and 2, vaccinia virus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus, reovirus 1, Sindbis virus, Punta Toro virus, cytomegalovirus, influenza virus A [subtypes H1N1 and H3N2], influenza virus B, feline coronavirus, and feline herpes virus) did not reveal any inhibitory activity, except when evaluated against EV71, a virus whose structure and mechanism of replication are completely different than those of HIV. This unexpected and intriguing observation prompted us to inv...

There are, besides Remdesivir (RDV), no approved antivirals for the treatment and/or prophylaxis 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 (HAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI) and infected at the apical side with either one of two different SARS-CoV-2 isolates. The 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°C than at 37°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. GS-441524 (the parent nucleoside of Remdesivir), EIDD-1931 (the active metabolite of Molnupiravir) and IFN (β1 and λ1) all resulted in a dose-dependent inhibition of viral RNA and infectious virus titers at the apical side. However, AT-511 (a guanosine nucleotide previously reported to inhibit SARS-CoV-2) failed to inhibit viral replication. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic targets for the treatment of COVID-19. Nirmatrelvir is the first 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization. We recently reported on the in vitro selection of SARS-CoV-2 3CLpro resistant virus (L50F-E166A-L167F; 3CLprores) that is cross-resistant with nirmatrelvir and other 3CLpro inhibitors. Here, we demonstrate that the 3CLprores virus replicates efficiently in the lungs of intranasally infected female Syrian hamsters and causes lung pathology comparable to that caused by the WT virus. Moreover, hamsters infected with 3CLprores virus transmit the virus efficiently to co-housed non-infected contact hamsters. Importantly, at a dose of 200 mg/kg (BID) of nirmatrelvir, the compound was still able to reduce the lung infectious virus titers of 3CLprores-infected hamsters by 1.4 log10 with a modest improvement in the lung histopathology as compared to the vehicle control. Fortunately, resistance to Nirmatrelvir does not readily develop in clinical setting. Yet, as we demonstrate, in case drug-resistant viruses emerge, they may spread easily which may thus impact therapeutic options. Therefore, the use of 3CLpro inhibitors in combination with other drugs may be considered, especially in immunodeficient patients, to avoid the development of drug-resistant viruses.