Screening new anti-cell wall antibiotics
D-amino acid-based probes enabled quantification of peptidoglycan (the main content of bacterial cell wall) synthesis activity and thus can be used to investigate the anti-peptidoglycan synthesis ability of small molecules. Nowadays new antibiotic discovery heavily relies on screening the isolated natural products from the environment, such as bacterial secretions or plant secretions. Efficient drug screening requires target-specific but low-cost approaches. D-amino acid-based probes have outstanding specificity toward bacterial peptidoglycan synthesis and are also easy to be made, fulfilling the requirements of being a new high-throughput screening approach for antibiotic discovery.
Fluorogenic D-amino acids (RfDAAs) are thought to be the best probe candidates for the screening applications because washing and fixation are not required for visualizing their signal. Although such a novel, high-throughput approach has not been fully demonstrated, we have proposed a protocol to pursue the experiments. [1][2] This includes 1) distributing growing bacterial cells into transparent 48/96-well plates, 2) adding antibiotic-RfDAA mixture to each well, 3) incubating for 10-50% generation time, 4) air-drying the culture medium to mount the cell onto the surface of the plate, and 5) imaging the cells and quantifying the fluorescence intensity. In this approach, compounds without anti-cell wall ability do not inhibit RfDAA labeling and thus show strong fluorescence intensity; while compounds inhibit cell wall synthesis lead to failed labeling and less (or no) fluorescence signal can be detected.
References
[1] Radkov et al. Imaging Bacterial Cell Wall Biosynthesis. Annu. Rev. Biochem., 2018, 87:22.1-22.4.
[2] Hsu et al. D-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis. Acc Chem Res. 2019. doi: 10.1021/acs.accounts.9b00311.