Assay platform for clinically relevant metallo-β-lactamases

Area of Science:

• Microbiology and Infectious Diseases
• Drug Discovery and Development
• Biochemistry

Background:

• Metallo-β-lactamases (MBLs) are critical enzymes conferring resistance to β-lactam antibiotics.
• The rise of MBLs necessitates the development of novel inhibitors and effective screening platforms.
• Existing screening methods often lack the sensitivity and scope required for broad-spectrum MBL inhibitor discovery.

Purpose of the Study:

• To establish a robust screening platform for identifying metallo-β-lactamase inhibitors.
• To develop and validate novel fluorogenic substrates for MBL activity detection.
• To identify potential pan-MBL inhibitors effective against clinically relevant MBL variants.

Main Methods:

• Preparation of clinically relevant MBLs: NDM-1, IMP-1, SPM-1, and VIM-2.
• Synthesis and characterization of umbelliferone-derived fluorogenic cephalosporin substrates.
• Comparative analysis of fluorogenic substrates against chromogenic substrates (CENTA, nitrocefin, imipenem).
• Inhibitor screening using the developed fluorogenic substrate platform.

Main Results:

• Successfully prepared a panel of clinically significant MBLs.
• Developed sensitive fluorogenic substrates with improved kinetic parameters compared to chromogenic alternatives.
• Identified 4-chloroisoquinolinols as potent inhibitors with potential pan-MBL activity.
• Demonstrated the utility of the fluorogenic assay for efficient inhibitor screening.

Conclusions:

• The developed fluorogenic substrate assay provides a sensitive and efficient platform for MBL inhibitor discovery.
• Umbelliferone-derived cephalosporins are effective substrates for screening MBLs.
• 4-chloroisoquinolinols represent promising candidates for broad-spectrum MBL inhibitors, addressing a critical unmet medical need.