Deeds Pilot Project Summary

The proteasome is a large macromolecular machine that serves as the proteolytic component of the primary protein degradation pathway in eukaryotes, archaea, and actinomycete bacteria. It has emerged as a major drug target in a number of diseases, particularly in the treatment of cancer and tuberculosis infections. Currently-available active-site inhibitors of the tuberculosis proteasome tend to significantly inhibit the human proteasome or other human proteases, and to our knowledge there are no proteasome inhibitors that are being actively investigated to target tuberculosis infections. The goal of this Pilot Project is to pursue two independent approaches for developing more specific tuberculosis proteasome inhibitors. Cells do not synthesize the proteasome as a single, active unit, but rather as a set of protein subunits that must be assembled into a specific structure in order to function. In particular, the proteasome Core Particle (CP), which is the catalytic component of the complex, is not active until fully assembled. While the overall architecture of the human and tuberculosis proteasomes are similar, the tuberculosis CP has protein interaction interfaces that are very different from those present in the human proteasome, and assembles via a completely different pathway. It has thus been suggested that an assembly inhibitor might provide greater specificity for the tuberculosis proteasome. Working with the Computational Chemical Biology (CCB) core of the CBID COBRE, we have developed the first known assembly inhibitor of the bacterial CP. While this molecule shows clear inhibition of assembly, it lacks both solubility and potency, which has hindered further characterization and testing. A major goal of this proposal is to work with the CCB and Synthetic Chemical Biology (SCB) cores to improve the solubility and efficacy of this inhibitor to support future validation and development. The second approach that we are pursuing focuses on a natural product called fellutamide B, which shows very high activity against both the tuberculosis and human CPs. Crystal structures indicate that fellutamide B binds these two CP’s in very different ways, and the second goal of this proposal is to work with the CCB and SCB cores to leverage this difference and generate derivatives that are specific for tuberculosis. If successful, these studies will provide two separate classes of tool compounds for probing proteasome function and assembly in bacterial cells. Compounds discovered through this work should also serve as the starting point for the eventual development of novel classes of therapeutics targeting CP assembly.