Discovery of dual S-RBD/NRP1-targeting peptides: structure-based virtual screening, synthesis, biological evaluation, and molecular dynamics simulation studies.

Both receptor-binding domain in spike protein (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human neuropilin-1 (NRP1) are important in the virus entry, and their concomitant inhibition may become a potential strategy against the SARS-CoV-2 infection. Herein, five novel dual S-RBD/NRP1-targeting peptides with nanomolar binding affinities were identified by structure-based virtual screening. Particularly, RN-4 was found to be the most promising peptide targeting S-RBD (Kd = 7.4 ± 0.5 nM) and NRP1-BD (the b1 domain of NRP1) (Kd = 16.1 ± 1.1 nM) proteins. Further evidence in the pseudovirus infection assay showed that RN-4 can significantly inhibit the SARS-CoV-2 pseudovirus entry into 293 T cells (EC50 = 0.39 ± 0.09 µM) without detectable side effects. These results suggest that RN-4, a novel dual S-RBD/NRP1-targeting agent, holds potential as an effective therapeutic to combat the SARS-CoV-2 infection.

Non-covalent cyclic peptides simultaneously targeting Mpro and NRP1 are highly effective against Omicron BA.2.75.

Available vaccine-based immunity may at high risk of being evaded due to substantial mutations in the variant Omicron. The main protease (Mpro) of SARS-CoV-2 and human neuropilin-1 (NRP1), two less mutable proteins, have been reported to be crucial for SARS-CoV-2 replication and entry into host cells, respectively. Their dual blockade may avoid vaccine failure caused by continuous mutations of the SARS-CoV-2 genome and exert synergistic antiviral efficacy. Herein, four cyclic peptides non-covalently targeting both Mpro and NRP1 were identified using virtual screening. Among them, MN-2 showed highly potent affinity to Mpro (K d = 18.2 ± 1.9 nM) and NRP1 (K d = 12.3 ± 1.2 nM), which was about 3,478-fold and 74-fold stronger than that of the positive inhibitors Peptide-21 and EG3287. Furthermore, MN-2 exhibited significant inhibitory activity against Mpro and remarkable anti-infective activity against the pseudotyped variant Omicron BA.2.75 without obvious cytotoxicity. These data demonstrated that MN-2, a novel non-covalent cyclic peptide, is a promising agent against Omicron BA.2.75.

Dual-targeting cyclic peptides of receptor-binding domain (RBD) and main protease (Mpro) as potential drug leads for the treatment of SARS-CoV-2 infection.

The receptor-binding domain (RBD) and the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) play a crucial role in the entry and replication of viral particles, and co-targeting both of them could be an attractive approach for the treatment of SARS-CoV-2 infection by setting up a "double lock" in the viral lifecycle. However, few dual RBD/Mpro-targeting agents have been reported. Here, four novel RBD/Mpro dual-targeting peptides, termed as MRs 1-4, were discovered by an integrated virtual screening scheme combining molecular docking-based screening and molecular dynamics simulation. All of them possessed nanomolar binding affinities to both RBD and Mpro ranging from 14.4 to 39.2 nM and 22.5-40.4 nM, respectively. Further pseudovirus infection assay revealed that the four selected peptides showed >50% inhibition against SARS-CoV-2 pseudovirus at a concentration of 5 µM without significant cytotoxicity to host cells. This study leads to the identification of a class of dual RBD/Mpro-targeting agents, which may be developed as potential and effective SARS-CoV-2 therapeutics.

A mechanism for matrikine regulation in acute inflammatory lung injury.

Proline-glycine-proline (PGP) and its acetylated form (Ac-PGP) are neutrophil chemoattractants generated by collagen degradation, and they have been shown to play a role in chronic inflammatory disease. However, the mechanism for matrikine regulation in acute inflammation has not been well established. Here, we show that these peptides are actively transported from the lung by the oligopeptide transporter, PEPT2. Following intratracheal instillation of Ac-PGP in a mouse model, there was a rapid decline in concentration of the labeled peptide in the bronchoalveolar lavage (BAL) over time and redistribution to extrapulmonary sites. In vitro knockdown of the PEPT2 transporter in airway epithelia or use of a competitive inhibitor of PEPT2, cefadroxil, significantly reduced uptake of Ac-PGP. Animals that received intratracheal Ac-PGP plus cefadroxil had higher levels of Ac-PGP in BAL and lung tissue. Utilizing an acute LPS-induced lung injury model, we demonstrate that PEPT2 blockade enhanced pulmonary Ac-PGP levels and lung inflammation. We further validated this effect using clinical samples from patients with acute lung injury in coculture with airway epithelia. This is the first study to our knowledge to determine the in vitro and in vivo significance of active matrikine transport as a mechanism of modulating acute inflammation and to demonstrate that it may serve as a potential therapeutic target.