Chemical Biology: Uncaging an antibiotic

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A 'caged' version of a protein synthesis inhibitor, when released at a high concentration in a restricted region, can be employed to precisely inhibit biological processes.

Regulation of protein synthesis is important in many biological processes, but it is difficult to study because of a lack of methodologies to interfere selectively with protein synthesis with temporal and spatial precision. To address this problem, a team of researchers led by Timothy Dore at the University of Georgia and Erin Schuman at the California Institute of Technology used a caged version of the antibiotic anisomycin, an inhibitor of protein synthesis that blocks the peptide bond–forming reaction in eukaryotic ribosomes.

This caged compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), comprises a photolabile protecting group covalently bound to the antibiotic anisomycin. Exposure to light cleaves away the protecting group, thus 'uncaging' the protein synthesis inhibitor.

For the work described in Chemistry & Biology, the team synthesized three different caged anisomycins and found that Bhc-Aniso had the fastest uncaging kinetics and was sufficiently sensitive to two-photon excitation for biological use. They tested Bhc-Aniso in an in vitro protein translation system, CHO cells, neurons and HEK293 cells, and found that photoreleased anisomycin can inhibit protein synthesis with spatial specificity. In cells expressing a green fluorescent protein reporter, the maximum decrease in fluorescence was within 100 µm of the center of the uncaging spot, whereas cells outside of the uncaging spot had constant fluorescence or a slight increase as more reporter was synthesized over the course of the experiment.

The successful use of Bhc-Aniso in neurons will allow researchers to study the role of local protein synthesis in this system. "When and where in the neuron is protein synthesis used to bring about changes? How does protein synthesis regulate synaptic strength and axonal outgrowth? These are questions we'd like to answer," says Schuman.

This protecting group potentially has broad-ranging applications in the study of other physiological processes. According to Dore, "Anything that has an amine, an alcohol, a phosphate, a ketone, an aldehyde or a carboxylate can be attached to Bhc... It's a general protecting group." This strategy could, for example, be used in drug development by administering the caged drug and then studying how the system responds when the drug is selectively activated in various cellular locations.

So far, Dore has developed several caging groups, and he plans to apply them to biological systems: "We are working on strategies and using different caging groups to enable proteins, RNA, drugs—any biological effector you can imagine—to be activated in a spatially and temporally controlled manner, hopefully using two-photon excitation, which would potentially enable subcellular localization of the release."

Irene Kaganman

References

Goard, M. et al. Light-mediated inhibition of protein synthesis. Chem. Biol. 12, 685–693 (2005). | Article | PubMed |