Engineering Defensin α-helix to produce high-affinity SARS-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 peptidomimetic 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 and highly stable proteins, and engineered the amino acid residues on its conformationally constrained alpha-helices to mimic the critical residues on the ACE2 helix 1 that interacts with the Spike-protein. The engineered proteins were soluble and purified to homogeneity with high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability, high-affinity binding to the Spike protein with dissociation constants in the low nanomolar range, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical peptidomimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing high-affinity ligands.

Virtual screening of knottin and defensin peptides perceives hits against the SARS CoV-2 RBD domain and hACE2 interaction

“…There are numerous reports of SARS CoV-2 spike's RBD domain peptide inhibitors, including human native ACE2 α-helix and its decoys [5][6] , de-novo designed high affinity binders [7][8] , D-peptide mimicking ACE2 helix 9 , helical bundle 10,11 , computationally modeled peptides 12,13, , in silico screening of peptides possessing antimicrobial properties such as antiviral 14,15 , cyclic 16,17, & stapled peptides 18,19 as well as covalently engineered minibinders 20 . Also, there are multiple earlier reports of in silico investigations and in vitro studies of human defensins [21][22][23] , and an engineered defensin α-helix 24 provided a straightforward way to identify potential peptide-based therapeutics.…”