"Molecular Masks" for ACE2 to Effectively and Safely Block SARS-CoV-2 Virus Entry.

Coronavirus Disease 2019 (COVID-19) remains a global health crisis, despite the development and success of vaccines in certain countries. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, uses its spike protein to bind to the human cell surface receptor angiotensin-converting enzyme 2 (ACE2), which allows the virus to enter the human body. Using our unique cell screening technology, we identified two ACE2-binding peptoid compounds and developed dimeric derivatives (ACE2P1D1 and ACE2P2D1) that effectively blocked spike protein-ACE2 interaction, resulting in the inhibition of SARS-CoV-2 pseudovirus entry into human cells. ACE2P1D1 and ACE2P2D1 also blocked infection by a D614G mutant pseudovirus. More importantly, these compounds do not decrease ACE2 expression nor its enzyme activity (which is important in normal blood pressure regulation), suggesting safe applicability in humans.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

Comprehensive and systemic optimization for improving the yield of SARS-CoV-2 spike pseudotyped virus.

Virus neutralization assay is principally conducted by measuring the ability of the antibodies in patient sera to prevent the infection of susceptible cells by the virus. As SARS-CoV-2 is classified as a risk group 3 pathogen, neutralization assay using a live virus needs to be handled in a biosafety level 3 laboratory. To overcome this limitation, pseudotyped viruses have been developed as an alternative for the live SARS-CoV-2. However, one of the issues that we and others have encountered during the production of pseudotyped virus with SARS-CoV-2 spike protein was the low virus yield. In our own experience, we were only able initially to produce a stock with a virus titer that is more than two orders of magnitude lower than what we usually get with a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector. We have conducted a series of improvements, including using a C-terminal truncated form of spike protein and a D614G mutated spike. Together, these have led to a significant improvement in the yield of the pseudotyped virus. Finally, our data show that using a high-affinity ACE2-expressing cell line resulted in a reduction in detection sensitivity of the neutralization assay.