Publication date: Sep 05, 2025
The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6cD7His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6cD7His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (y = 725. 29x – 52. 356; R = 0. 998) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K of 22. 01 +/- 3. 5 μM, k of 0. 021 s, and catalytic efficiency (k/K) of 946 M. s. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC of 0. 88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.
| Concepts | Keywords |
|---|---|
| Accurate | 3CLpro |
| Antiviral | fluorescence assay |
| Recombinant | magnetic bead-based substrate |
| Viral | protease activity assay |
Semantics
| Type | Source | Name |
|---|---|---|
| disease | IDO | assay |
| disease | IDO | role |
| drug | DRUGBANK | Sodium lauryl sulfate |