The DIFF Biotech patented fluorescence reporting system offers a direct positive screening method at the cellular level for drug activity, effectively balancing convenience and accuracy in large-scale screening. Unlike traditional FRET methods, it eliminates substrate dependency, reducing screening costs by over 60%compared to FRET methods (internal data).

The DIFF Biotech patented protease inhibitor detection system is based on fluorescence reporting technology using GFP-derived proteins. The core feature of this system is that fluorescence signals are only emitted when inhibitors effectively target the protein. As inhibitor efficacy increases, fluorescence intensity also increases, providing a reliable foundation for quantitative analysis.

This fluorescence reporting system is suitable for Biosafety Level 1 (BSL-1) laboratories, ensuring safe and convenient operations.

Additionally, the system is compatible with high-throughput screening platforms. It can be used in conjunction with existing drug libraries, significantly improving the efficiency and accuracy of novel antiviral drug screening. This system is based on a co-expression mechanism using custom plasmids. Taking the novel coronavirus as an example, it combines recombinant green fluorescent protein (rGFP) with the coronavirus 3CL protease. In this system, the rGFP sequence incorporates specific cleavage sites for the 3CL protease. When the 3CL protease is functional, it specifically cleaves rGFP, causing the loss of fluorescence functionality and a decrease in fluorescence intensity.

Conversely, when the inhibitor effectively suppresses the activity of the 3CL protease, the 3CL protease fails to cleave rGFP, thus maintaining fluorescence, resulting in increased fluorescence intensity. Drug efficacy is positively correlated with fluorescence intensity, providing a more intuitive positive screening method.

System Principle

Accurate Test Results:

When using live virus for testing, the IC50 value for GC376 is 4.69 μM; using DIFF Biotech’s screening system, the IC50 value is 6.44 μM. The IC50 values obtained by the two methods are highly similar.

Intuitive Test Results Visible to the Naked Eye:

After adding different concentrations of novel coronavirus 3CL protease inhibitors, the GFP expression in cells at 24h, 48h, and 72h is shown in the following figure:

This plasmid system does not require virus involvement during drug screening, freeing experiments from the need to isolate test strains. It eliminates the necessity for high biosafety level laboratories (P2/P3/P4), enabling cellular-level drug screening：

Good Signal-to-Noise Ratio and Reliable Results for High-Throughput Screening:

A typical laboratory can screen tens of thousands of candidate compounds in one week, significantly shortening the early R&D cycle.

Suitable for discovering new drugs targeting the proteases of various viruses