Discovery of All‑d‑Peptide Inhibitors of SARS-CoV‑2 3C-like Protease

During the replication process of SARS-CoV-2, the main protease of the virus [3-chymotrypsin-like protease (3CL^pro)] plays a pivotal role and is essential for the life cycle of the pathogen. Numerous studies have been conducted so far, which have confirmed 3CL^pro as an attractive drug target to combat COVID-19. We describe a novel and efficient next-generation sequencing (NGS) supported phage display selection strategy for the identification of a set of SARS-CoV-2 3CL^pro targeting peptide ligands that inhibit the 3CL protease, in a competitive or noncompetitive mode, in the low μM range. From the most efficient l-peptides obtained from the phage display, we designed all-d-peptides based on the retro-inverso (ri) principle. They had IC₅₀ values also in the low μM range and in combination, even in the sub-micromolar range. Additionally, the combination with Rutinprivir decreases 10-fold the IC₅₀ value of the competitive inhibitor. The inhibition modes of these d-ri peptides were the same as their respective l-peptide versions. Our results demonstrate that retro-inverso obtained all-d-peptides interact with high affinity and inhibit the SARS-CoV-2 3CL protease, thus reinforcing their potential for further development toward therapeutic agents. The here described d-ri peptides address limitations associated with current l-peptide inhibitors and are promising lead compounds. Further optimization regarding pharmacokinetic properties will allow the development of even more potent d-peptides to be used for the prevention and treatment of COVID-19.