Identification of a peptoid inhibitor of the proteasome 19S regulatory particle

Article abstract of DOI:10.1021/ja072027p

The first chemical inhibitor of the 19S regulatory particle (RP) of the proteasome is described. The molecule was identified by screening a library of nucleoside-capped peptoids for binding to the yeast 26S proteasome in a crude extract. The hit was resynthesized and shown to block 19S RP-mediated protein unfolding in vitro and proteasome-mediated turnover of p27 in HeLa cells. Copyright

Full text of DOI:10.1021/ja072027p

Published on Web 05/31/2007
Identification of a Peptoid Inhibitor of the Proteasome 19S Regulatory Particle
Hyun-Suk Lim, Chase T. Archer, and Thomas Kodadek*
DiVision of Translational Research, Departments of Internal Medicine and Molecular Biology, UniVersity of Texas
Southwestern Medical Center, 5323 Harry Hines BouleVard, Dallas, Texas 75390-9185
Received March 22, 2007; E-mail: thomas.kodadek@utsouthwestern.edu
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 hetero-
heptameric rings capped on each side by a 19S regulatory particle
1
(
RP) (Figure 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
Figure 1. A cartoon of the 26S proteasome. The black line represents a
substrate protein in the process of being degraded. The 19S RP (pink and
blue shapes) that would cap the other end of the 20S CP (gray) has been
omitted for clarity.
2
are degraded into small peptides. Inhibition of the 20S CP
proteolytic activity has emerged as an attractive pharmacological
target for cancer and inflammatory diseases3 as well as useful as
mechanistic probes of proteasome function in a variety of biological
processes.3
,4
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 Therefore, pharmacological inhibitors of the protea-
somal 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.
-10
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
2
A) in hope of biasing the library toward targeting one of the
5
ATPases. The theoretical diversity of the library was 8 (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 extract12 prepared from a yeast strain that expressed a
FLAG-tagged â4 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 proteasome-
binding peptoids were visualized by addition of a secondary anti-
IgG antibody labeled with red-emitting quantum dots.1
Figure 2. Isolation and characterization of RIP-1. (A) General structure
of the purine-capped peptoid library. (B) A fluorescence micrograph of a
bead scored as a “hit” (red halo) in the midst of a large number of beads
scored as negatives (blue). (C) The structure of the hit, RIP-1, as determined
by Edman sequencing and C2, a control peptoid not selected for binding to
the proteasome.
3
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
To examine the effect of the peptoid-purine conjugate RIP-1
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 condi-
tions. We called this compound RIP-1 (Regulatory Particle Inhibitor
Peptoid-1; Figure 2C).
on the unfolding activity of the 19S regulatory particle, we
employed an assay recently developed in our laboratory that
10
involves exposing Gal4-VP16 protein bound to an immobilized
DNA containing five Gal4 binding sites. The base of the 19S RP
7
binds to the VP16 activation domain and thus engages the fusion
protein as a substrate for unfolding, resulting in the ATP-dependent
1
0
disruption of the protein-DNA complex (Figure 3A). An excess
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J. AM. CHEM. SOC. 2007, 129, 7750-7751
10.1021/ja072027p CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. RIP-1 induces the accumulation of p27 in HeLa cells. (A) HeLa
cells were treated with C2 (100 µM), the indicated concentrations of RIP-
Figure 3. RIP-1 inhibits in vitro chaperonin activity. (A) A cartoon
describing the proteasome-mediated stripping of the Gal4-VP16 protein from
the immobilized DNA. (B) Inhibition of proteasome-mediated destabilization
of the activator-DNA complex by RIP-1. The graph shows the average
amount and standard error of the mean of GST-Gal4-VP16 remaining on
DNA for three replicates. Increasing activator remaining on the DNA
indicates decreased chaperonin activity of the ATPases.
1
, or MG132 (1 µM) for 6 h. Lysates were subjected to immunoblot analysis
with anti-p27 antibody. The results are representative of three independent
experiments. (B) Immunoblot intensity normalized to the DMSO control
for A. Error bars represent standard deviation from three independent
experiments.
specifically implicate one of the ATPases, Sug2/Rpt4, as the direct
receptor of the peptoid.
of soluble DNA is included in the assay to prevent reassociation
of Gal4-VP16 with the immobilized DNA, and the amount of
protein remaining on the bead-bound DNA thus reflects the rate of
ATPase-mediated “stripping” of the activator. A control lacking
the proteasome allows correction for the intrinsic rate of dissociation
of the complex. The proteolytic activity of the 20S CP is not
involved in this process, and in fact, purified 19S RP lacking the
Acknowledgment. This work was supported by a contract from
the National Heart, Lung, and Blood Institute (NO1-HV-28185)
and a grant from the Welch Foundation (I-1299). H.-S.L. was
partially supported by a Korea Research Foundation Grant (M01-
2004-000-20395-0).
2
0S core is quite active.10 As seen in Figure 3B, RIP-1 inhibited
the proteasome-mediated stripping of the Gal4-VP16 protein from
the immobilized DNA with an IC50 of approximately 3 µM. A
control peptoid, called C2 (Figure 1), did not affect the chaperonin
activity (see Supporting Information Figure S2). The purine cap
alone lacking a peptoid also had no effect. The peptoid lacking the
purine cap did show some activity, though it was a weaker inhibitor
than RIP-1 (not shown). A more complete structure activity analysis
of this system will be presented in a subsequent full paper.
Peptoids are generally more cell permeable than peptides, and
thus we hoped that RIP-1 might have activity against the proteasome
in cellular assays. To examine this point, the effect of RIP-1 on
steady-state levels of p27, which is turned over via the ubiquitin-
proteasome pathway,15 was examined. HeLa cells were incubated
with the indicated concentrations of RIP-1 (Figure 4) or, as controls,
the known proteasome inhibitor MG132, compound C2 or DMSO
carrier alone (Figure 4). RIP-1 induced increased accumulation of
p27 in a dose-responsive fashion. The effect of 100 µM RIP-1 was
similar to that of 1 µM MG132. Neither the control compound C2
nor the DMSO carrier affected p27 levels significantly.
Supporting Information Available: Detailed experimental pro-
cedures and supplementary figures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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