An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Area of Science:

• Biochemistry
• Microbiology
• Chemical Biology

Background:

• Staphylococcus aureus (S. aureus) is a major human pathogen causing significant mortality.
• Fluorophosphonate binding hydrolases (Fphs) are key S. aureus enzymes involved in lipid metabolism and host colonization.
• Fphs represent valuable targets for developing novel diagnostics and therapeutics due to their specific expression in Staphylococcus.

Purpose of the Study:

• To develop and screen cyclic peptide libraries for potent and selective inhibitors of Fph enzymes.
• To create fluorescent probes for visualizing and targeting FphB activity in live S. aureus.
• To explore the utility of genetically encoded electrophiles in mRNA display for covalent ligand discovery.

Main Methods:

• Utilized mRNA display with a genetically encoded oxadiazolone (Ox) electrophile to screen diverse cyclic peptide libraries.
• Employed counter-selection strategies with wild-type (WT) and catalytic-dead FphB to enhance target selectivity.
• Synthesized and validated fluorescent probes based on selected peptide inhibitors for active site labeling.

Main Results:

• Identified potent cyclic peptides containing the Ox residue that covalently inhibit Fph enzymes.
• Achieved high selectivity for FphB through iterative selection and counter-selection processes.
• Developed fluorescent probes that label the FphB active site in live S. aureus at low nanomolar concentrations.

Conclusions:

• Demonstrated the efficacy of genetically encoded electrophiles in mRNA display for identifying covalent binding ligands.
• Introduced potent and selective fluorescent probes for FphB, a critical enzyme in S. aureus.
• Highlighted the potential of these probes for future diagnostic and therapeutic applications against S. aureus infections.