Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. 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 diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 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 crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.