Stapled peptides based on Human Angiotensin-Converting Enzyme 2 (ACE2) potently inhibit SARS-CoV-2 infection in vitro

Abstract

Human angiotensin-converting enzyme 2 (ACE2) is the primary receptor of SARS-CoV-2 to enter the host cells and start the infection process. Therefore, it is prudent to design therapeutics based on the critical binding region of ACE2, which is a ~30 aa long helix with a kink in the middle. However, the small peptide in solution may lose its helical conformation and subsequently lose its binding potential to the SARS-CoV-2 RBD, which it utilizes to bind to that helical region. Here we report the design of four stapled peptides based on that helix, which is expected to bind to SARS-CoV-2 with high affinity and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 - 94% helicity). On the contrary, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus based single-cycle assay in HT1080 and human lung cells, A549. Three of the four stapled peptides showed potent antiviral activity in HT1080 (IC50: 1.9 – 4.1 μ) and A549 cells (IC50: 2.2 – 2.8 μ). It is noteworthy that the stapled peptide, NYBSP-3, which showed the least helical content, also had the lowest antiviral activity in both cell lines. The linear peptides NYBSP-C and SBP1, reported recently to bind SARS-CoV-2 with KD of ~47nM affinity, showed no antiviral activity. Most significantly, none of the stapled peptides show any appreciable cytotoxicity at the highest dose tested. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T1/2) of >289 min.

Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK (Alpha), South Africa (Beta), and India (Delta)

“…Similarly, we used MERScoronavirus spike protein S2 polyclonal antibody (Invitrogen), to immunodetect the spike protein S2 of the MERS-CoV pseudovirus (Figure 2b). For SARS-CoV-2, we found a specific band at 80 kDa, which identifies the subdomain S2, and a second band at about 190 kDa, which corresponds to the full-length S protein (S1 + S2), as previously reported 24,45,46 . In SARS-CoV pseudovirus lysate, the same antibody detected a lighter band at 80 kDa representing the S2 subunit and a 190 kDa band, which corresponds to the full-length of the spike S protein (Figure 2a).…”

“…Due to the surface exposure of the spike protein, it is the primary target for neutralizing antibodies and vaccines. Both S1, primarily the RBD, and S2 proteins, especially the HR1 domain, have been targeted for identifying and designing novel drugs [17][18][19][20][21][22][23][24][25] . However, the RBD of the S1 subdomain of the spike proteins among various coronaviruses is less conserved 26 .…”

“…The antiviral activity of the NBCoV small molecules was evaluated in a single-cycle infection assay by infecting different cell types with the SARS-CoV-2, SARS-CoV, or MERS-CoV pseudoviruses as previously described with minor modifications 24,45 .…”

Chloroquine and Hydroxychloroquine Interact Differently with ACE2 Domains Reported to Bind with the Coronavirus Spike Protein: Mediation by ACE2 Polymorphism

“…Our ADME and pharmacokinetic results confirmed previous data about pharmacokinetics and pharmacogenomics of CQ and HCQ [ 46 ]. The importance of the pharmacokinetic, ADME, and QSAR (for quantitative structure–activity relationship) assays was previously largely reported in the design and the assessment of several drugs [ 41 , 47 , 48 ].…”

SARS-CoV-2 cell entry and targeted antiviral development

“…To overcome these limitations, further engineering was conducted by fusing ACE2 to the Fc region of the human immunoglobulin IgG1, in order to increase its plasma stability 93 , 94 . Alternatively, the N-terminal helix of ACE2 which contains most of the contacting residues at the binding site was mimicked, and the designed peptides exhibit high anti-SARS-CoV-2 activity in vitro 95 , 96 , 97 . Vice versa , a SARS-CoV-2 RBD-derived hexapeptide YKYRYL has also been proposed to possess inhibitory effect against virus entry by interfering with RBD–ACE2 interaction 98 .…”