Novel broad-spectrum activity-based probes to profile malarial cysteine proteases

Area of Science:

• Biochemistry
• Parasitology
• Drug Discovery

Background:

• Clan CA cysteine proteases (papain-like proteases) are crucial in the malaria parasite's life cycle and represent potential drug targets.
• Developing selective inhibitors for Plasmodium falciparum clan CA proteases is challenging due to subfamily diversity and potential off-target effects on human cathepsins.

Purpose of the Study:

• To develop novel, broad-spectrum activity-based probes for clan CA cysteine proteases.
• To validate these probes for studying protease function and inhibitor specificity in both parasite and mammalian cells.

Main Methods:

• Synthesis of novel dipeptydic vinyl sulfone probes with N-terminal tryptophan and P1-fluorophore.
• Application of probes for activity-based protein profiling in Plasmodium falciparum and mammalian cell lysates.
• Assessment of probe cell permeability and specificity in living cells.

Main Results:

• The new probes efficiently label both endopeptidase and dipeptidyl aminopeptidase subfamilies of clan CA proteases.
• Probes demonstrate effectiveness in both Plasmodium falciparum and mammalian cells.
• Probe cell permeability and specificity are significantly influenced by the choice of fluorophore.

Conclusions:

• These novel probes serve as improved broad-spectrum tools for clan CA protease research.
• The probes facilitate inhibitor specificity determination in living cells, aiding antimalarial drug development.
• Fluorophore selection is critical for optimizing probe performance in biological systems.