Inhibiting Drg1

The cytoplasmic AAA-ATPase Drg1 consists of an N-terminal domain and two AAA-domains and forms hexamers in solution. We demonstrated previously that Drg1 plays a key role in eukaryotic ribosome biogenesis. Drg1 binds to large ribosomal subunit precursor particle shortly after their export from the nucleus into the cytoplasm where it is required for the release of shuttling proteins and export factors. Very recently we could show that Drg1 is recruited to pre-60S particles by direct interaction with the shuttling pre-ribosome maturation factors Rlp24. This interaction results in strong stimulation of the ATPase activity of Drg1. The enhanced ATPase activity is used for specific extraction of Rlp24 from pre-60S particles. Our unpublished results demonstrate that this enhanced ATPase activity of Drg1 can specifically be inhibited by a low molecular weight inhibitor. Since only the stimulated ATPase activity, but not the basal activity of Drg1 is blocked, the inhibitor seems to exhibit a novel mode of action. This project aims to unravel the exact mechanism of inhibition of Drg1. This aim will be achieved by up to date biochemical and structural biological methods. The detailed description of the mode of inhibition of Drg1 by the compound will support the later development of specific high affinity inhibitors targeting the activated form of other AAA-ATPases. Such inhibitors are thought to possess considerable clinical potential for therapy of human diseases, including cancer. In addition, ribosome biogenesis provides a promising future target for antimicrobial and anti-tumor chemotherapy. Due to its key role in the process, Drg1 represents an excellent target for blocking ribosome biogenesis with low molecular weight inhibitors. By investigating the effects of its inhibition on upstream and downstream processes the results of this project will also contribute to evaluate the potential of inhibitors acting within the ribosome biogenesis pathway.

The protein Drg1 of the bakers yeast Saccharomyces cerevisiae belongs to the AAA-family of ATPases. This type of proteins exhibits ATP dependent chaperon activity which often is used for remodelling of macromolecular complexes. Our previous work has shown that Drg1 contains two ATPase domains (D1 and D2) and forms hexamers in solution. In the cytoplasm Drg1 binds to ribosomal pre-60S particles shortly after their export from the nucleus and catalyses the ATP dependent release of the shuttling pre-ribosome maturation factor Rlp24 for subsequent recycling into the nucleus. Since this release reaction is a prerequisite for all downstream maturation steps and for the recycling of shuttling proteins, a blockage of the activity of Drg1 is lethal for the cells. Indeed we could previously show that Drg1 is the target of the low molecular weight inhibitor diazaborine, which turned out to be the first inhibitor of eukaryotic ribosome biogenesis. Since ribosome biogenesis is crucial for any living cell, targeting this pathway has significant potential for the development of inhibitors for the treatment of tumours and infectious diseases. This project was therefore designed to gain deeper insights into the mechanism of action of the inhibitor diazaborine and to analyse the detailed consequences of inhibition of Drg1 for the ribosome biogenesis pathway. Using purified Drg1, we could measure the binding affinity for the inhibitor and show that it specifically binds into the D2 AAA domain of Drg1 to inhibit ATP hydrolysis in this site. Since ATP hydrolysis in the D2 domain is crucial for the release of Rlp24 from the pre-ribosome, the inhibitor blocks this release reaction, as we could demonstrate in an in vitro system using purified pre-ribosomal particles and Drg1. Moreover we investigated the consequences of inhibition of Drg1 by diazaborine for the ribosome biogenesis pathway. The extremely fast onset of inhibition of Drg1 together with the entrapment of shuttling proteins in the cytoplasm allowed us to follow the extremely dynamic ribosome biogenesis pathway with up to now unanticipated temporal resolution. This enabled us to shed more light on temporal and functional linkages during the ribosome biogenesis processes. Our results were published in high-ranking journals and presented at international conferences where they gained significant interest in the research community.