Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Legumain (Asp-rich endosomal proteinase, AEP) is a lysosomal cysteine protease implicated in diseases like inflammation, arteriosclerosis, and cancer.
• Its upregulation makes AEP a significant molecular target for developing novel chemical markers and therapeutics.

Purpose of the Study:

• To develop highly selective chemical probes for targeting legumain.
• To establish a robust methodology for designing selective protease probes, addressing challenges in conventional profiling.

Main Methods:

• Utilized a hybrid combinatorial substrate library (HyCoSuL) to identify P1-Asp fluorogenic substrates and biotin-labeled inhibitors for legumain.
• Introduced a Counter Selection Substrate Library (CoSeSuL) approach to enhance selectivity against related proteases, particularly caspases.
• Validated probe selectivity using M38L and HEK293 cell lines.

Main Results:

• Successfully generated P1-Asp fluorogenic substrates and biotin-labeled inhibitors targeting legumain.
• The CoSeSuL approach significantly improved probe selectivity by minimizing cross-reactivity with caspases.
• Validated the efficacy and selectivity of the developed legumain probes in cellular models.

Conclusions:

• The CoSeSuL methodology provides a powerful strategy for designing highly selective legumain probes.
• This approach offers a generalizable principle for developing selective probes for other challenging protease families.
• The developed probes serve as valuable tools for studying legumain's role in disease and for potential therapeutic development.