Competitive profiling for enzyme inhibitors using chemical probes

Area of Science:

• Chemical Biology
• Enzyme Inhibitor Discovery
• Microbial Pathogenesis

Background:

• Pseudomonas aeruginosa produces quorum sensing molecules and virulence factors derived from quinolone biosynthesis.
• The PqsD enzyme is crucial for the biosynthesis of these essential quinolone compounds, making it a key target for drug development.
• Traditional screening methods for enzyme inhibitors can be inefficient and costly.

Purpose of the Study:

• To describe the development and application of activity-based probes (ABPs) for PqsD inhibitor screening.
• To present a competitive profiling strategy for identifying potent PqsD inhibitors.
• To provide detailed protocols for ABP synthesis and enzyme labeling.

Main Methods:

• Synthesis of an α-chloroacetamide probe with a terminal alkyne tag for selective PqsD labeling.
• Covalent labeling of the active site cysteine of PqsD using the developed ABP.
• Competitive inhibition assays where inhibitors prevent probe labeling, quantified via fluorescence and click chemistry.

Main Results:

• The ABP selectively labeled the active site cysteine of PqsD.
• The competitive profiling strategy successfully identified potent PqsD inhibitors.
• The method demonstrated applicability in vitro and in live cells.

Conclusions:

• Activity-based probes are effective tools for competitive profiling of PqsD inhibitors.
• This strategy offers advantages over traditional screening methods for enzyme inhibitor discovery.
• The developed protocols facilitate the identification of novel inhibitors targeting P. aeruginosa virulence.