Structural analysis of a peptide-based simplified model reproducing SARS-CoV-2 S RBD/ACE2 binding site

SARS-CoV-2, the pathogen causing the COVID-19 outbreak, is an RNA virus which infects lung cells through the binding of its surface glycoprotein spike (S) with the angiotensin-converting enzyme 2 (ACE2).The moiety of S involved in the interaction -namely receptor binding motif (RBM) -has been subjected to a remarkable number of point mutations throughout the rapid evolution of the virus.Therefore, the description of a model that individuates the interacting residues between S and ACE2 needs to be updated as soon as new variants of interest emerge.To this aim, the synthesis of peptide molecules reproducing short moieties involved in the interaction, rather than the production of the full protein construct, allowed the development of a simplified system that would afford to study the change in the SARS-CoV-2 S RBD/ACE2 binding related to the frequent mutations. 1-3 In this work, we synthesized and studied the structure of short amino acid sequences, mimicking the two proteins' critical portions.Variations in the residues were easily managed through the one-point alteration of the sequences.Nuclear magnetic resonance (NMR) spectroscopy provides insights into ACE2 and SARS-CoV-2 S RBD deriving peptides' structures with their related variants (Alpha, Beta, and Gamma).Moreover, these sequences proved to be highly immunogenic, as many monoclonal antibodies target this moiety and give an individual susceptibility to the different variants.To study this effect, we used these peptides to functionalize the monomers of H-chain of human ferritin (hHFt), a protein able to assemble in an ordered 24-mer nanocage with high thermal and chemical stability in physiological conditions.The decoration of the outer surface of the nanocage with the S-derived epitopes can provide a tool able to elicit an immune answer in view of the development of novel vaccine strategies that can also be applied to other critical pathologies, for which there is no therapeutical strategy. Conclusions.In this poster, we report a methodology for studying large binding complexes using short peptide sequences.The starting model was the SARS-CoV-2 S RBD/ACE2 complex.We extracted two sets of peptide sequences mimicking the binding domains of the two proteins (including the mutations of the variants of concern in SARS-CoV-2 S) and studied the peptides' conformation in solutions.Moreover, we functionalized chitosan beads with SMIMICS OR and tested their binding potential with ACE 21- 42 using STD-NMR, reporting a sizeable binding.With the aim of using these S-deriving sequences in a pharmacological active tool, we functionalized the human H chain of the ferritin (hHFt) with the spike peptides, which is a very interesting molecule for the drug delivery and for the development of immunoreactive devices.We successfully produced six modified hHFt sequences and tested their binding with ACE 21-42 using 1D CPMG.These preliminary results will be used as the basis for a thorough study of the potential of these macromolecules in novel vaccine development strategies.