Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro

Abstract

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 μM) and A549/ACE2 (IC50: 2.2 to 2.8 μM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 μM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 μM. 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. IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.

Stapled ACE2 peptidomimetics designed to target the SARS‐CoV‐2 spike protein do not prevent virus internalization

“…There have been several reports based on the linear peptide fragment of the ACE2 PD α1 helix [13,23] Both biophysical and pseudoviral techniques have been used to assess the binding and activity, revealing that the peptides showed micromolar affinity and anti-viral activity.…”

“…While preparing this manuscript, Curreli et al reported the design and synthesis of double stapled peptide analogues also based on native ACE2 PD α1. [23] We also synthesized double stapled analogues of the α1 helix, however these were isolated as crude mixtures of cis/trans alkene isomers and when tested in the pseudovirus inhibition assay and they showed no anti-viral activity (data not shown). In their studies, like us, they confirmed that the peptide designed by G. Zhang et al did not show any binding.…”

Targeting SARS-CoV-2 Receptor Binding Domain with Stapled Peptides: An In Silico Study

“…The antiviral activity and superior α-helicity observed in the experimental study of Curreli et al 40 (which possess a double-stapled) might be partly understood by the results of our MD simulation study. According to Figure 4 , in which the initial and final structures from the MD studies of the control peptide and modification 15 are superimposed, the second stapled at the right side of the designed peptide helps to sustain this part of the structure in place in the complex with the S protein.…”

“…This sequence was altered according to the proposed modifications (see Figure S1 (SI) for more details regarding the structures of all of the proposed peptides) followed by structure energy minimization in all positions where modifications were assigned (as described before for the receptor). Additionally, for comparison purposes, the best hydrocarbon stapled peptide based on the hACE2 helix published by Curreli and co-workers, 40 peptide NYBSP-4, was also modeled using the same protocol employed with the designed peptides. The NYBSP-4 peptide was also docked and underwent MD simulations using the same approaches described below.…”

Keep out! SARS-CoV-2 entry inhibitors: their role and utility as COVID-19 therapeutics

“…Their data show that peptide fragments (P8, P9, P10) from the α-helix of the ACE2 peptidase domain (PD) that are rationally modified with residues that have a propensity for helical folding show high binding affinity and antiviral activity against authentic SARS-CoV-2 in the nanomolar range [118]. A study by Curreli et al also showed that peptides from a similar region of ACE2 that are structurally stabilized with double stapling show inhibitory activity against pseudotyped and live SARS-CoV-2 in the low micromolar range [119]. For peptides that are based on the binding motif of S1 RBD, particularly the RBM as shown in Fig.…”