Identification of a Peptoid Inhibitor of the Proteasome 19S Regulatory Particle

Abstract

The 26S proteasome is an approximately 2.5 MDa multi-catalytic protease complex that is responsible for most non-lysosomal protein degradation in eukaryotic cells. It is composed of a barrel-like catalytic 20S core particle (CP) that consists of four stacked heteroheptameric rings capped on each side by a 19S regulatory particle (RP) (Figure 1), 1 which contains a putative hetero-hexameric ring of ATPases (Rpt1-6) as well as several other proteins. The RP binds polyubiquitylated proteins, unfolds them, and feeds the polypeptide chain into the interior of the barrel where the proteins are degraded into small peptides. 2 Inhibition of the 20S CP proteolytic activity has emerged as an attractive pharmacological target for cancer and inflammatory diseases 3,4 as well as useful as mechanistic probes of proteasome function in a variety of biological processes. 3 Recently, a number of pioneering studies have revealed that stimulating proteasome-mediated proteolysis is but one of several activities of the Rpt proteins. It is now clear that these ATPases play non-proteolytic roles in RNA polymerase II transcription, DNA repair, structural modification of chromatin and other nuclear processes. 5-10 Therefore, pharmacological inhibitors of the proteasomal ATPases (Rpts) would be of great interest. Here we report the isolation of the first compound of this type from a library of nucleoside-capped peptoids. We show that this peptoid derivative inhibits the protein unfolding activity of the Rpt proteins in vitro and inhibits the proteasome activity in living cells.A "one bead one compound" peptoid library was constructed by split and pool synthesis (see Supporting Information Figure S1). 11 Each peptoid molecule was capped with a purine analogue ( Figure 2A) in hope of biasing the library toward targeting one of the ATPases. The theoretical diversity of the library was 8 5 (32 768 compounds). Approximately 100 000 beads, representing about 3-fold coverage of the library, were used in the screen for compounds that bind to the proteasome. The screen employed a whole cell extract 12 prepared from a yeast strain that expressed a FLAG-tagged 4 proteasome subunit (one of the 20S proteins). The beads were exposed to the extract, washed thoroughly (see Supporting Information), probed with anti-FLAG monoclonal antibody, and then washed again. Finally, putative proteasomebinding peptoids were visualized by addition of a secondary antiIgG antibody labeled with red-emitting quantum dots. 13 Three beads evinced an obvious red halo, consistent with retention of the quantum-dot-conjugated secondary antibody ( Figure 2B). Note that the library had been prescreened to eliminate peptoids that bound directly to the antibodies or the quantum dot before exposing it to the yeast extract. Edman degradation revealed that all three hits were identical, indicating the presence of a single proteasome-binding peptoid in the library with sufficient affinity and specificity to register under these relatively demanding conditions. We called this compound ...

Non-proteolytic Regulation of p53-mediated Transcription through Destabilization of the Activator·Promoter Complex by the Proteasomal ATPases

“…This reagent is highly specific for its target and has been shown previously to block destabilization of Gal4⅐VP16 complexes (15,31). As shown in Fig.…”

“…The mammalian ␣-Rpt6 and ␣-Rpt4 antibodies were purchased from BIOMOL International and Abcam, respectively. The RIP-1 peptoid was synthesized according to a published protocol (31).…”

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

“…Taking advantage of the distinction in molecular properties, it is feasible to develop agents which target specific subpopulations of proteasomes (55). Recently, pharmacological agents aiming at 19S complexes have also emerged (56,57), which may enable selective adjustment of targeting ubiquitinated proteins for degradation. The comprehensive delineation of the heterogeneity in cardiac 19S subpopulations will aid in the engineering of a new generation of agents with enhanced specificity.…”

Peptoids: An Emerging Class of Peptidomimetics for Cancer Therapy and Diagnostics

“…These peptoid libraries are synthesized through the "split-pool" approach, and these split-pool cycles will lead to development of 'one-bead onecompound' (OBOC) libraries with huge diversity [14,15].For example, the Kodadek group developedseveral number of suchpeptoid librarieswith diversities varied up to millions of permutations [3,9,12,[16][17][18]. These peptoid libraries are developed in less than one week, using the very efficient and rapid microwave synthesis method.…”

“…It became clear very quickly that the most significant hurdle to compete in the drug discovery race was to access to large collections of compounds forhigh throughput screens. Large combinatorial libraries of peptoids (in millions) can be synthesized easily, inexpensively, and rapidly (less than one week) [8][9][10][11][12]. Peptoid sequences can be deduced sensitively by Edman degradation [9,10] or mass spectrometry [11][12][13].…”