Engineering defensin α‐helix to produce high‐affinitySARS‐CoV‐2 spike protein binding ligands

Abstract

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein.Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alphahelix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7 C), high-affinity binding to the spike protein with apparent K d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands.

A Rationally Designed Synthetic Antiviral Peptide Binder Targeting the Receptor-Binding Domain of SARS-CoV-2

“…In this context, mimetics of the N-terminal α-helix of ACE2 have been actively pursued as they have the potential to disrupt the interaction of the S-protein of SARS-CoV-2 with the host cell ACE2 receptor with minimal immunogenicity. 63 So far, several ACE2-derived peptides ≤30 aa have been described as inhibitors of PPIs between SARS-CoV-2 and the ACE2 receptor. Multiple sequence alignments of a few such peptides indicate that, from a design perspective, SR16 has a unique sequence that is not a potential allergen 36 and is predicted 64 to be free from aggregation (Figure S10).…”

“…Therefore, the search for other potential therapeutic leads for a better understanding of the pathology of the virus appears to be interesting, at least from the perspective of academics. In this context, mimetics of the N-terminal α-helix of ACE2 have been actively pursued as they have the potential to disrupt the interaction of the S-protein of SARS-CoV-2 with the host cell ACE2 receptor with minimal immunogenicity . So far, several ACE2-derived peptides ≤30 aa have been described as inhibitors of PPIs between SARS-CoV-2 and the ACE2 receptor.…”

Antibody mimetics: The next generation antibody engineering, a retrospective and prospective analysis

“…Antibody mimetics, typically larger and more structurally intricate, recognize and bind to specific targets with high affinity and specificity. They exhibit various modes of action, such as blocking protein-protein interactions, [53] inhibiting enzymatic activity, [54] or inducing cell signaling, [55] while peptidomimetics typically act as ligands or modulators of specific receptors or signaling pathways. [56,57] Here, we define protein/peptide-based antibody mimetics as the core concept in this field (Figure 5…”