Engineering Defensin α-helix to produce high-affinity SARS-CoV-2 Spike protein binding ligands

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.