Screening for selective small molecule inhibitors of the proteasome using activity-based probes

The proteasome's role in fundamental biological processes ranging from control of the cell cycle to production of peptides for display to immune cells has been uncovered with the help of small molecule inhibitors. Most of the commonly used inhibitors have been designed and synthesized by organic chemists or by Nature. To continue to develop new inhibitors and reagents for the proteasome, a rapid screening method is required that allows not only assessment of potency but also selectivity of inhibitors for each of the primary catalytic sites in the complex. This chapter outlines methods for the solid-phase synthesis of diverse peptide vinyl sulfone libraries and a rapid screen for potent and selective inhibitors that makes use of an active site label (Nazif and Bogyo, 2001). This assay can be performed with small quantities of total cellular extracts as a source of enzyme and can be used to rapidly screen virtually any potential inhibitor.