The COVID-19 (Coronavirus disease-2019) pandemic, caused by the SARS-CoV-2 coronavirus, is a significant threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and MERS-CoV. Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analysis for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 Orf9b, an interaction we structurally characterized using cryo-EM. Combining genetically-validated host factors with both COVID-19 patient genetic data and medical billing records identified important molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

The SARS-CoV-2 virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.