High-throughput screening of natural products discovers inhibitors of ribosome biogenesis

Cells must double their ribosome abundance every cell cycle. Thus, cancer cells have a high demand for ribosomes to match their high proliferation rate. For this reason, ribosome biogenesis has become an alluring target for cancer interventions. As the result of an evolutionary arms race, nature has produced a rich source of bioactive molecules and thus screening natural product fractions presents an opportunity to discover novel compounds that target ribosome biogenesis. In collaboration with the NIH's National Center for Advancing Translational Science (NCATS) we have screened the NCI's natural products library for inhibitors of ribosome biogenesis using a ribosome biogenesis reporter system called RiboSNAP. The RiboSNAP reporter incorporates a SNAP tag on an endogenous ribosomal protein that can be covalently labeled with fluorescent substrates in live cells. Substrates can then be washed out leaving newly synthesized SNAP tagged protein free for subsequent labeling with different substrates to distinguish between old and new ribosomes. This allows for the tracking of newly produced ribosomes from the nucleolus to the cytoplasm as a readout for ribosome biogenesis. Inhibitor discovery is predicated on the finding that inhibition of ribosome biogenesis throughout the pathway results in retention of newly translated SNAP tagged RPL28 protein in the nucleolus and/or nucleus. Previous work by our group and others has determined that nucleolar morphology serves as an indicator of ribosome biogenesis inhibitor mechanism of action and therefore different puncta morphologies indicate the inhibition of different targets. This image-based analysis suggests our screening efforts have identified not only inhibitors of rRNA transcription, but also rRNA processing and ribosome maturation. Once screening is complete, our efforts will shift towards purification of the active compounds and target identification to further our understanding of how ribosome biogenesis responds to perturbations and to develop cancer interventions. (This work was supported by 5UG3CA290312-01)