Cost-effective synthesis of a chromogenic substrate for SARS-CoV-2 Mpro and development of a miniaturized high-throughput inhibition assay.

The SARS-CoV-2 main protease (Mpro) is a critical therapeutic target for antiviral drug development. Here, we report the synthesis and application of a chromogenic substrate handmade for Mpro activity assays. The peptide-pNA conjugate was synthesized via Fmoc-based solid-phase peptide synthesis, followed by solution-phase coupling with pNA. Due to the low nucleophilicity of pNA's aromatic amino group, Oxyma was used to enhance coupling efficiency and reduce side reactions. The crude product showed a yield of 60 % and purity of 75 %. The coupling reaction was optimized and monitored by thin-layer chromatography, purified by silica gel column chromatography, and confirmed by RP-HPLC. Also, a miniaturized enzymatic assay was then developed using this substrate, optimized for 96-well microplates with minimal reagent consumption (50 µL total volume). Optimal assay performance was achieved with 50 µg/mL of Mpro and 400 µg/mL of substrate, providing a robust signal at 405 nm. Importantly, the cost of synthesizing this substrate in-house represents an approximately 18-fold reduction compared to the commercial alternative. This substantial improvement in accessibility and affordability transforms the assay into a democratized scientific tool, enabling resource-limited laboratories to conduct high-throughput screening with robust and reproducible results. Moreover, the methodology is scalable, compatible with automation, and broadly applicable to drug discovery efforts targeting SARS-CoV-2 and other viral proteases.