Optimization of CoaD Inhibitors against Gram-Negative Organisms through Targeted Metabolomics

Area of Science:

• Microbiology and Molecular Biology
• Medicinal Chemistry
• Biochemistry

Background:

• Rising global concern over drug-resistant Gram-negative bacteria necessitates novel antibiotic development.
• Optimizing antibiotic candidates requires balancing target affinity and cellular potency, often presenting divergent structure-activity relationships (SARs).

Purpose of the Study:

• To develop novel mass spectrometry assays for monitoring bacterial enzyme CoaD activity during inhibitor lead optimization.
• To investigate cellular potency and efflux liabilities of CoaD inhibitors to guide optimization against Gram-negative pathogens.

Main Methods:

• Developed mass spectrometry assays to quantify CoA metabolites, correlating with cellular CoaD enzymatic activity.
• Utilized assays to assess compound effects at concentrations significantly below minimum inhibitory concentration (MIC).
• Generated efflux pump mutants to evaluate inhibitor susceptibility to bacterial efflux mechanisms.

Main Results:

• Mass spectrometry assays enabled monitoring of cellular enzyme inhibition, revealing effects at sub-MIC levels.
• Efflux pump mutant analysis provided insights into permeability and efflux liabilities of CoaD inhibitors.
• Optimization guided by these methods resulted in measurable MICs against wild-type Escherichia coli.

Conclusions:

• Mass spectrometry-based metabolite assays are valuable tools for early-stage antibiotic lead optimization.
• Understanding cellular potency and efflux is crucial for developing effective antibiotics against Gram-negative bacteria.
• This integrated approach facilitates the discovery of novel antibacterial agents targeting essential bacterial enzymes.