Several strategies are currently available for investigating mechanisms of action during antibiotic drug discovery. The macromolecular synthesis assay—which uses radioactive labeling to determine whether a compound blocks RNA, DNA, lipid, or cell wall synthesis—is a common starting point, but it falls short in terms of accuracy and throughput. Other ways to determine a drug’s mechanism are to isolate either resistant or sensitized mutants or to use transcriptional profiling. Each of these strategies has its own strengths and drawbacks, not the least of which is that they require users to generate large amounts of the compound of interest.
Now, a new, one-step imaging technique that analyzes bacterial cell shape could enable researchers to more rapidly identify the mechanisms by which compounds kill bacteria. The approach, described online September 17 in Proceedings of the National Academy of Sciences, will allow industry and academic scientists to pick out new drug candidates based on the pathways they target or uncover new mechanisms of action to pursue in antibiotic development as bacteria develop resistance to existing treatments .
Showing that a particular drug kills bacteria is not difficult, but learning how exactly an antibiotic works is a slow and laborious process, noted senior author Joseph Pogliano, a molecular biologist at the University of California, San Diego.
Pogliano and his long-time collaborator Kit Pogliano (who is also his wife) were applying antibiotics to bacteria to better understand how they grow and divide. They wound up learning more about the antibiotics themselves and, as a consequence, decided to refocus, applying antibiotics to E. coli systematically and looking for differences in the cells’ appearance.
In their study, the researchers applied 41 different compounds from 26 different classes to E. coli. After staining the cultures with fluorescent proteins and using ImageJ analysis software, they measured 14 different parameters of morphology, including the area, perimeter, and circularity of both the membranes and the DNA.
To their surprise, each antibiotic—even those with activity that seems unrelated to morphology, such as inhibiting protein synthesis—affected the cell shape in a unique way after two hours of exposure. In a double-blinded follow-up experiment, the assay correctly assigned each of 18 antibiotics to their targets.
The method, called “bacterial cytological profiling,” was also able to identify the mechanism of action of spirohexenolide A, a natural product compound that kills methicillin-resistant Staphylococcus aureus and other species through a previously unknown mechanism. The drug’s effect on E. coli shape was similar to that of a known drug, nisin, which kills by poking holes in the bacterial membrane. The group confirmed the mechanism in a follow-up study.
“Professor Pogliano’s recent paper in PNAS represents a remarkable advance in determining the mode of action of antibacterial compounds,” noted Thomas Keating, a principal scientist at AstraZeneca Infection Innovative Medicines, who was not involved with the new study.
Keating, who is now collaborating with Pogliano to study the modes of action of new compounds in E. coli and additional species, added, “His method should prove a valuable tool in the hunt for new medicines.”
The new assay, like others already available, does not identify precise targets—just pathways. Follow-up studies will be needed to zero in on the molecular target, although narrowing down the list in pathways containing only a few enzymes should be easier.
The Poglianos have founded a start-up, Linnaeus Bioscience in San Diego, to sell bacterial cytological profiling as a service, which Pogliano says could be used at any point in the pipeline, from a primary screen of 500,000 compounds to the lead optimization of just a handful.
“Our goal was to see that it be used as widely as possible,” Pogliano said. “We wanted to have an avenue where it could be commercialized so that companies could use it to find better drugs for treating antibiotic-resistant bacteria, especially Gram negatives.”
He added that the technology will continue to improve as the group adds to its library of cell morphology profiles. In the meantime, they are also using the technology for their own drug discovery projects, screening natural product libraries to find a replacement for penicillin.
1. Nonejuie, P., M. Burkart, K. Pogliano, and J. Pogliano. 2013. Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules. PNAS. Published online before print September 17, 2013, doi: 10.1073/pnas.1311066110