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The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.Significance statementThis study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1Bdomains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structurefunction analysis using SPR and cryo-EM.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity., The MANCO project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 101003651). This work made use of the Dutch national e infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) - 398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has so far infected more than 160 million people globally, and continues to undergo genomic evolution. Emerging SARS-CoV-2 variants show increased infectivity and may lead to resistance against immune responses of previously immunized individuals or existing therapeutics, especially antibody-based therapies. Several monoclonal antibody therapeutics authorized for emergency use or in development have been shown to lose potency against some SARS-CoV-2 variants. Cocktails of two different monoclonal antibodies constitute a promising approach to protect against novel emerging variants as long as both antibodies are potent, but come with increased development complexity and therefore cost. As an alternative, we developed two multispecific DARPin® therapeutics, each combining three independent DARPin® domains binding the SARS-CoV-2 spike protein in one molecule, to potently neutralize the virus and overcome virus escape. Here, we show in a panel of in vitro studies that the multispecific DARPin® therapeutic design incorporated in our clinical candidate ensovibep (MP0420), achieved high neutralization potencies against the circulating SARS-CoV-2 variants B.1.1.7 (UK variant), B.1.351 (South African variant), P.1 (Brazilian variant), B.1.429 (South Californian variant), B.1.526 (New York variant), R.1 (Japanese variant), A.23.1 (Ugandan variant), and B.1.617 (Indian variant), and there is strong evidence that ensovibep also potently neutralizes the Indian variant B.1.618 based on the analysis of the key point mutations in the spike protein of this variant. Additionally, viral passaging experiments show potent protection by ensovibep and MP0423 against development of escape mutations. Finally, we demonstrate that the cooperative binding of the individual modules in a multispecific DARPin® antiviral is key for potent virus inhibition and protection from escape variants. These results, combined with the relatively small size and high production yields of DARPin® molecules, suggest that ensovibep is a highly valuable alternative to monoclonal antibody cocktails for global supply and demonstrate the strength of the DARPin® platform for achieving potent and lasting virus inhibition for SARS-CoV-2 and possibly other viruses.

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.