Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates.

Area of Science:

• Biochemistry
• Assay Development
• Chemical Biology

Background:

• Fluorescence-based assays are crucial for drug discovery but are often hindered by compound interference.
• Traditional coumarin-based substrates are susceptible to interference from compounds absorbing UV light.
• Red-shifted fluorescent dyes offer a potential solution to minimize spectral overlap and interference.

Purpose of the Study:

• To develop and validate novel rhodamine-based fluorogenic substrates for protease assays.
• To enable multiplexed protease activity assessment using tandem rhodamine substrates.
• To create dual-substrate assays combining coumarin and rhodamine for high-throughput screening (HTS).

Main Methods:

• Synthesis of rhodamine-based fluorogenic substrates, including bis-(Leu)(2)-Rhod110 and bis-(Ala-Pro)-Rhod110.
• Design of novel tandem rhodamine substrates (e.g., Ala-Pro-Rhod110-Leu) for multiplexing.
• Development of endpoint HTS assays incorporating both coumarin and rhodamine substrates for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin.

Main Results:

• Successfully developed and characterized rhodamine-based substrates for leucine aminopeptidase and dipeptidyl aminopeptidase.
• Demonstrated multiplexed protease assays using novel tandem rhodamine substrates.
• Established dual-substrate HTS assays capable of screening compound libraries (LOPAC) and natural product extracts.

Conclusions:

• Rhodamine-based substrates effectively mitigate fluorescence interference in protease assays.
• Novel multiplexed and dual-substrate assay formats enhance screening capabilities for complex biological samples.
• These advancements facilitate robust high-throughput screening of diverse chemical libraries against protease targets.