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

Abstract

During the replication process of SARS-CoV-2, the main protease of the virus [3-chymotrypsin-like protease (3CLpro)] plays a pivotal role and is essential for the life cycle of the pathogen. Numerous studies have been conducted so far, which have confirmed 3CLpro 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 3CLpro 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.

SARS‐CoV‐2 main protease inhibitors: What is moving in the field of peptides and peptidomimetics?

“…This led to the identification of stable peptides with IC 50 values in the micromolar range (1.5-9 μM). 25 More importantly, the combination of the corresponding all-L and all-D derivatives cooperatively inhibits the Mpro, displaying submicromolar potency. The remarkable stability of some of these D-retro-inverso peptides in simulating gastric and intestinal fluids, as well as in human plasma and liver microsomes, makes them a promising starting point for further optimization.…”

“…A phage display selection strategy was used for the recognition of 16‐mer L‐amino acid containing peptides able to inhibit SARS‐CoV‐2 Mpro, and then, the best compounds were transformed into all‐D‐amino acid containing peptide retro‐inverse analogs. This led to the identification of stable peptides with IC 50 values in the micromolar range (1.5–9 μM) 25 . More importantly, the combination of the corresponding all‐L and all‐D derivatives cooperatively inhibits the Mpro, displaying submicromolar potency.…”

On the origins of SARS-CoV-2 main protease inhibitors

“…Aside from all the inhibitors described above, which are arguably continuations of the research made on the inhibition of various viral proteases, including SARS-CoV-1 M pro , one original approach involved the generation of a large number of random peptidic sequences which were then assayed. If a similar but more virtual-based approach failed in an early instance, 383 this also led to all D-peptides 384,385 or to macrocyclic 11-14-mer peptides featuring modified amino acids 386 endowed with affinities for the SARS-CoV-2 M pro catalytic site. In the latter case, the corresponding X-ray based structure (PDB 7Z4S) may be of future use to deconstruct such rather large peptides into more drug-like compounds.…”

Mechanism of Protease Resistance of D-Amino Acid Residue Containing Cationic Antimicrobial Heptapeptides

“…Although the knowledge of imparting protease resistance to peptides by using the D -amino acid residues is common and widely used, the mechanism of acquiring stability against proteases is vaguely understood. There are very few studies, − to the best of our knowledge, which actually demonstrate the mechanism of enhanced stability development in atomic details and with thermodynamic understanding. In the present study, we have developed a library of short cationic peptides (P4A-C and P5A-C) containing few or all- D -amino acid residues from two moderately active AMPs P4 and P5 earlier developed by our research group, with an intention of developing protease-resistant AMPs.…”