Nirmatrelvir-resistant SARS-CoV-2 is efficiently transmitted in female Syrian hamsters and retains partial susceptibility to treatment.

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

Rational modifications, synthesis and biological evaluation of new potential antivirals for RSV designed to target the M2-1 protein

Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract diseases in infants and young children, with potentially serious and fatal consequences associated with severe infections. Despite extensive research efforts invested in the identification of therapeutic measures, no vaccine is currently available, while treatment options are limited to ribavirin and palivizumab, which both present significant limitations. While clinical and pre-clinical candidates mainly target the viral fusion protein, the nucleocapsid protein or the viral polymerase, our focus has been the identification of new antiviral compounds targeting the viral M2-1 protein, thanks to the presence of a zinc-ejecting group in their chemical structure. Starting from an anti-RSV hit we had previously identified with an in silico structure-based approach, we have designed, synthesised and evaluated a new series of dithiocarbamate analogues, with which we have explored the antiviral activity of this scaffold. The findings presented in this work may provide the basis for the identification of a new antiviral lead to treat RSV infections

Progress in human picornavirus research : New findings from the AIROPico consortium

Several research groups in Europe are active on different aspects of human picornavirus research. The AIROPico (Academia-Industry R&D Opportunities for Picornaviruses) consortium combined the disciplines of pathogenesis, diagnostics and therapy development in order to fill the gaps in our understanding of how picornaviruses cause human disease and how to combat them. AIROPico was the first EU consortium dedicated to human picornavirus research and development, and has largely accelerated and improved R&D on picornavirus biology, diagnostics and therapy. In this article, we present the progress on pathogenesis, diagnostics and treatment strategy developments for human picornaviruses resulting from the structured, translational research approach of the AIROPico consortium. We here summarize new insights in protection against infection by maternal or cross-protective antibodies, the visualisation of interactions between virus and neutralizing antibodies by cryoEM structural imaging, and the outcomes from a picornavirus-infected human 3D organoid. Progress in molecular detection and a fast typing assay for rhinovirus species are presented, as well as the identification of new compounds potentially interesting as therapeutic compounds.Peer reviewe

Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C−C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development

Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery.

Massive efforts on both vaccine development and antiviral research were launched to combat the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We contributed, amongst others, by the development of a high-throughput screening (HTS) antiviral assay against SARS-CoV-2 using a fully automated, high-containment robot system. Here, we describe the development of this novel, convenient and phenotypic dual-reporter virus-cell-based high-content imaging assay using the A549+hACE2+TMPRSS2_mCherry reporter lung carcinoma cell line and an ancestral SARS-CoV-2_Wuhan_mNeonGreen reporter virus. Briefly, by means of clonal selection, a host cell subclone was selected that (i) efficiently supports replication of the reporter virus with high expression, upon infection, of the NeonGreen fluorescent reporter protein, (ii) that is not affected by virus-induced cytopathogenic effects and, (iii) that expresses a strong fluorescent mCherry signal in the nucleus. The selected clone matched these criteria with an infection rate on average of 75% with limited cell death. The average (R)Z'-factors of the assay plates were all >0.8, which indicates a robust assay suitable for HTS purposes. A selection of reference compounds that inhibits SARS-CoV-2 replication in vitro were used to validate this novel dual-reporter assay and confirms the data reported in the literature. This assay is a convenient and powerful tool for HTS of large compound libraries against SARS-CoV-2

Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells

The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of Mpro. This design led to the most potent ABP for Mpro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize Mpro activity and target engagement by drugs in infected cells

Rational design of novel nucleoside analogues reveals potent antiviral agents for EV71

Different viruses belonging to distinct viral families, such as enterovirus 71, rely on the host methyltransferase METTL3 for the completion of fundamental cytoplasmic stages of their life cycle. Modulation of the activity of this enzyme could therefore provide a broad-spectrum approach to interfere with viral infections caused by viruses that depend on its activity for the completion of their viral cycle. With the aim to identify antiviral therapeutics with this effect, a series of new nucleoside analogues was rationally designed to act as inhibitors of human METTL3, as a novel approach to interfere with a range of viral infections. Guided by molecular docking studies on the SAM binding pocket of the enzyme, 24 compounds were prepared following multiple-step synthetic protocols, and evaluated for their ability to interfere with the replication of different viruses in cell-based systems, and to directly inhibit the activity of METTL3. While different molecules displayed moderate inhibition of the human methyltransferase in vitro, multiple novel, potent and selective inhibitors of enterovirus 71 were identified