A peptide array pipeline for the development of Spike-ACE2 interaction inhibitors

Abstract

In humans, coronaviruses are the cause of endemic illness and have been the causative agents of more severe epidemics. Most recently, SARS-CoV-2 was the causative agent of the COVID19 pandemic. Thus, there is a high interest in developing therapeutic agents targeting various stages of the coronavirus viral life cycle to disrupt viral propagation. Besides the development of small-molecule therapeutics that target viral proteases, there is also interest molecular tools to inhibit the initial event of viral attachment of the SARS-CoV-2 Spike protein to host ACE2 surface receptor. Here, we leveraged known structural information and peptide arrays to develop an in vitro peptide inhibitor of the Spike-ACE2 interaction. First, from previous co-crystal structures of the Spike-ACE2 complex, we identified an initial 24-residue long region (sequence: STIEEQAKTFLDKFNHEAEDLFYQ) on the ACE2 sequence that encompasses most of the known contact residues. Next, we scanned this 24-mer window along the ACE2 N-terminal helix and found that maximal binding to the SARS-CoV-2 receptor binding domain (CoV2-RBD) was increased when this window was shifted nine residues in the N-terminal direction. Further, by systematic permutation of this shifted ACE2-derived peptide we identified mutations to the wildtype sequence that confer increased binding of the CoV2-RBD. Among these peptides, we identified binding peptide 19 (referred to as BP19; sequence: SLVAVTAAQSTIEEQAKTFLDKFI) as an in vitro inhibitor of the Spike-ACE2 interaction with an IC₅₀ of 2.08 ± 0.38 μM. Overall, BP19 adds to the arsenal of Spike-ACE2 inhibitors, and this study highlights the utility of systematic peptide arrays as a platform for the development of coronavirus protein inhibitors.

Keywords: ACE2; Peptide array; Peptide inhibitor; Spike.

Fig. 1

Structure-guided development of ACE2-derived Spike-binding peptides. (A) Crystal structure (PDB: 6M0J) of human ACE2 N-terminal peptidase domain (S19 – D615; grey) bound to the SARS-CoV-2 RBD (R319 – F541; blue). ACE2 contact residues residing in the N-terminal helix have side chains displayed as pink and more distal contact residues are shown as green. The part of the N-terminal helix used as an initial window (IW) peptide is shown as black with the sequence underneath. (B) Monitoring binding of rCoV2-RBD towards 24-mer peptides representing a sliding window of the IW peptide + /- 9 residues along the hACE2 sequence.

Fig. 2

Binding profile of rCoV2-RBD to a permutation array of the 9 ^N ACE2-derived peptide. Binding of 0.3 μM rCoV2-RBD was assessed towards the 24-mer permutation array (n = 480 peptides) overnight at 4 °C and detected by chemiluminescence with anti-HisProbe. Binding signal was quantified by densitometry with ImageLab. Individual binding signals were normalized to the average wildtype peptide signal to visualize Fold Changes. Deviation of the binding signal between all individual wildtype peptides (n = 24 peptides) is shown. The wildtype 9 ^N sequence and individual amino acid substitutions are shown on the horizontal and vertical axes, respectively.

Fig. 3

Dose-responsive inhibition of the in vitro ACE2-Spike interaction by BP19. (A) The binding reactions of Spike and ACE2 occurred in the presence of either no peptide and or a concentration range of BP19 (1–128 μM). Binding activity between Spike and ACE2 was monitored by ELISA and shown as a percentage compared to that without BP19. Half-maximal inhibitory concentration (IC50) is shown within figure. (B) Docking of BP19 (magenta) with the CoV2-RBD (blue) using HPEPDOCK Server. The ACE2 protein (grey) from the original crystal structure is shown, and the initial window peptide within this protein is colored black.