Developing Staple Peptide Therapeutics for Targeting the Main Protease SARS-CoV-2

In December of 2019, the recently discovered SARS-CoV-2 virus started a worldwide pandemic that has since killed around 7 million people. This single-stranded RNA (ssRNA) virus translates 29 proteins in the host cell. Researchers have quickly isolated important proteins from this group, including 3-chymotrypsin-like protease (3CLpro), which is essential for viral replication. At the cleavage sites, 3CLpro cleaves polyproteins. This results in the release of specific proteins that control the viral life cycle. Staple peptides are synthetic peptides that have had an amino acid modification creating a covalent cross link which preserves helical structure and additional serum stability. The inclusion of alpha-methyl phenylalanine in the staple peptide, a TLP-3 analogue, increases stability, protease resistance, and raises the possibility of a 3CLpro targeting inhibitor. To screen inhibitors targeting the 3CLpro, several efficient techniques were established. Due to its cost and accessibility, fluorescence resonance energy transfer (FRET) based assay is used most of the time to measure inhibitor affinity for the 3CLpro. However, in this study selected ion monitoring (SIM) combined with liquid chromatography-mass spectroscopy (LC-MS) was also utilized. The 50% inhibitory concentrations (IC50) for the linear peptide (TLP3L1) inhibitor of 3CLPro were first determined using the SIM based LC-MS method to evaluate substrate degradation and product generation, and the results were compared using FRET assay. TLP3L1\u27s estimated 50% inhibitory concentration was 6.26 µM, which agreed with the IC50 value determined by the FRET assay (6.53 µM). The staple analogue TLP3S1\u27s estimated IC50 value, as determined by the LCMS assay, was 0.3046 µM