Multidrug resistant gram-negative infections are on the rise, and new classes of drugs for treatment are lacking. Developing new antibiotics for gram-negative bacteria remains challenging as these organisms are impermeable to most small molecules. An inner phospholipid membrane (IM) and an extra outer membrane (OM) composed of lipopolysaccharide are believed to form an ‘orthogonal permeability barrier’. Compounds that cross the OM are additionally subject to highly promiscuous efflux pumps that further restrict accumulation. To be effective, compounds must possess physicochemical properties that balance OM/IM permeability and efflux pump recognition to achieve therapeutic concentrations in the target compartment.
Traditional small-molecule antibiotic screens infer compound accessibility based on antibacterial activity. They lack spatial information regarding subcellular compound accumulation in periplasmic versus cytoplasmic compartments. Methods to screen novel molecules for improved permeability, or to improve the properties of inactive/ weakly-active compounds that may be good candidates for drug development would aid in the discovery of novel gram-negative antibiotics.
A recent paper in ACS Infect. Dis. describes an approach using bioorthogonal click chemistry-coupled mass spectrometry (MS) to measure compound accumulation with subcellular resolution. Azide-reactive biotin-BCN, selectively localized to the periplasmic or cytoplasmic compartment of gram-negative strains heterologously-expressing streptavidin, was used as a probe to screen a panel of azide-bearing molecules of known permeability. The production of ‘click’ products was monitored by MS. Appropriate compartment-specific positive (AZT for cytoplasmic; azidocillin for periplasmic) and negative (PMA for cell impermeable) azide-bearing controls showed expected profiles demonstrating the accuracy and sensitivity of the approach for quantifying subcellular distribution profiles. This approach can be easily adapted for high-throughput screening of hundreds to thousands of azide-bearing compounds, or similar bioorthogonal pairs in other bacteria or within organelles of eukaryotic cells.