Abstract
Coronavirus disease 19 (COVID-19) is an emerging global health crisis. With over 7 million confirmed cases to date, this pandemic continues to expand, spurring research to discover vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact interface. Using molecular dynamics simulations based on the recently solved cryo-EM structure of ACE2 in complex with SARS-CoV-2-RBD, we observed that the ACE2 peptidase domain (PD) α1 helix is important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we chemically synthesized a 23-mer peptide fragment of the ACE2 PD α1 helix (SBP1) composed entirely of proteinogenic amino acids. Chemical synthesis of SBP1 was complete in 1.5 hours, and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer interferometry (BLI) revealed that SBP1 associates with micromolar affinity to insect-derived SARS-CoV-2-RBD protein obtained from Sino Biological. Association of SBP1 was not observed to an appreciable extent to HEK cell-expressed SARS-CoV-2-RBD proteins and insect-derived variants acquired from other vendors. Moreover, competitive BLI assays showed SBP1 does not outcompete ACE2 binding to Sino Biological insect-derived SARS-CoV-2-RBD. Further investigations are ongoing to gain insight into the molecular and structural determinants of the variable binding behavior to different SARS-CoV-2-RBD protein variants.
Competing Interest Statement
Bradley L. Pentelute is a co-founder of Resolute Bio and Amide Technologies.
Footnotes
We revised and expanded our binding affinity studies of our lead SBP1 peptide with various SARS-CoV-2-RBD isoforms obtained from different commercial sources and added the following experiments. Using bio-layer interferometry (BLI), we determined that N-terminal biotinylated SBP1 binds Sino Biological insect-derived SARS-CoV-2-RBD with micromolar affinity. We also found, however, that N-terminal biotinylated SBP1 does not associate with HEK-expressed SARS-CoV-2-RBD or insect-derived variants purchased from other commercial sources. Although biotinylated SBP1 binds Sino Biological insect-derived SARS-CoV-2-RBD when immobilized on BLI streptavidin tips, no specific disruption of the SARS-CoV-2-RBD/ACE2 interaction was observed in solution in a BLI competition assay. Title, abstract and author list was also updated to reflect the new studies and findings.