Chemistry & Biology
Small Molecule SENP Inhibitors and ABPs
concentration of Bio-EQTGG-VS, Bio-QQTGG-VME, Bio-VEA505, Bio-
VEA355, Cy5-VEA505, Cy5-VEA355) for varying time (30 min to 7 hr) at
37ꢀC. Proteins were separated by SDS-PAGE. Fluorescent gels were scanned
using a Typhoon 9400 flatbed laser scanner (GE-Healthcare). All other gels
were visualized by biotin affinity blotting using horseradish peroxidase-conju-
gated streptavidin.
production of SUMO precursor and deconjugation of SUMO
from substrates are fundamental for a number of biological
processes regulated by SUMOylation. Unfortunately, there
are no optimal methods to temporally regulate the activity
of SENPs. Current reagents for studying SENP activity
require a full-length protein with an electrophile for covalent
modification of the proteases. In this study, we present two
new small molecule scaffolds that can be used as inhibitors
and activity-based probes of the hSENPs. Our results
suggest that peptide aza-epoxides are relatively efficient
inhibitors of various SENPs, but these reagents show high
nonspecific labeling properties when used in complex
proteomes. Peptide AOMKs containing a large aromatic
O-acyl group, on the other hand, are covalent inhibitors
that also function as highly selective probes of SENP
activity. While further work is required to increase the
potency of these compounds, we believe that they are an
important new class of reagents for the study of SENP
function.
Labeling and Competition for Recombinant hSENP1 and 2 Activity
with Activity-Based Probes
Recombinant DhSENP1 or 2 or DhSENP2 C548S was diluted to 0.5 mM in
reaction buffer (50 mM Tris [pH 7.4], 20 mM NaCl, 5 mM DTT) and treated
with either DMSO, VEA260, VEA499 (both 50 mM final concentration) or N-eth-
ylmaleimide (20 mM final concentration) for 1 hr at 37ꢀC. Labeling was carried
out with 1 mM final probe concentration (Bio-EQTGG-VS, Bio-QQTGG-VME,
Bio-VEA505, Bio-VEA355, Cy5-VEA505, Cy5-VEA355) for
1
hr at 37ꢀC.
Proteins were separated by SDS-PAGE. Fluorescent gels were scanned using
a Typhoon 9400 flatbed laser scanner (GE-Healthcare). All other gels were
visualized by biotin affinity blotting using horseradish peroxidase-conjugated
streptavidin.
Labeling of Recombinant hSENP1 Spiked into HEK293 Cell Lysates
Recombinant DhSENP1 (25 ng) was added to HEK293 cell lysates (25 mg) in
reaction buffer (50 mM Tris [pH 7.4], 20 mM NaCl, 5 mM DTT) and treated
with serial dilution of probe (Bio-VEA505, Bio-VEA355, Cy5-VEA505, Cy5-
VEA355, HA-SUMO1-VME, HA-SUMO1-VS) for 1 hr at 37ꢀC. Proteins were
separated by SDS-PAGE. Fluorescent gels were scanned using a Typhoon
9400 flatbed laser scanner (GE-Healthcare). Biotinylated probes were visual-
ized by biotin affinity blotting using horseradish peroxidase-conjugated strep-
tavidin and full-length SUMO probes were visualized using an anti-HA
antibody.
EXPERIMENTAL PROCEDURES
Synthetic Protocols
See Supplemental Experimental Procedures.
Cloning and Expression of hSENP1, 2, 5, 6, and 7
All final constructs and primers are summarized in Tables S1 and S2. The
catalytic mutant of hSENP2 (C548S) was generated by PCR-SOE (primers
#1–4) and ligated into the pET28a vector for expression. All constructs were
expressed in BL21(DE3) Escherichia coli (OD600 = 0.6; 0.1 mM IPTG) for
3 hr at 30ꢀC, lysed in 50 mM HEPES (pH 7.4), 100 mM NaCl, 5% glycerol,
5% sucrose, immobilized on Ni2+-NTA agarose (QIAGEN), and eluted with
stepwise fractions of lysis buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 5%
glycerol, 5% sucrose) supplemented with 20–200 mM imidazole. The hSENPs
were prepared as previously described (Mikolajczyk et al., 2007).
Lysate Preparation
Lysates of HEK293 cells were prepared from cells grown as a 90% confluent
monolayer. The cells were washed with PBS before being scraped and
transferred to an Eppendorf tube. The cells were spun down at 2000 rpm for
4 min and the PBS was removed. An equivalent volume of hypotonic lysis buffer
(20 mM Tris [pH 7.4], 5 mM NaCl, and 5 mM DTT) was added to the pellet. The
mixture was allowed to sit on ice for 10 min before mechanical lysis through
a 26.5 gauge needle. The mixture was spun down at maximum speed for
30 min at 4ꢀC. The supernatant was transferred and quantified to be 8 mg/ml.
ProhSUMO Cleavage Assays
SUMO-pro cleavage assays were performed as previously described (Miko-
lajczyk et al., 2007). In brief, 100 nM hSENP1 in reaction buffer (50 mM Tris
[pH 7.4], 5 mM NaCl, 5 mM DTT) was pretreated with inhibitor (JCP665-8)
for 30 min at 37ꢀC and then allowed to cleave SUMO-pro protein for 1 hr at
37ꢀC. Proteins were separated by SDS-PAGE and visualized by Gelcode
Blue protein stain reagent (Pierce).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
Supplemental Compound Characterization, eight figures, and two tables and
Fluorogenic Substrate Assay
ACKNOWLEDGMENTS
The fluorogenic substrate library screen was performed as described previ-
ously (Drag et al., 2008). In brief, hSENP1 (final concentration 2 mM) in low
salt Tris buffer (50 mM Tris [pH 8.0], 20 mM NaCl, 10 mM DTT) was preincu-
bated for 1 hr at 37ꢀC with inhibitor (50 mM final concentration) followed by
addition of substrate (100 nM). Fluorophore release (AFC) was monitored for
30 min at 37ꢀC with an excitation of 405 nm and emission at 510 nm. Rates
of cleavage were determined as the slope of the linear line representing
RFU/min. The DMSO-treated sample was set as 100% activity, and the activity
of the other samples was determined relative to this value. All values are the
average of independent triplicates.
We thank P. Bowyer and A. Shen for creative discussion and L. Edgington, A.
Puri, and J. Valderramos for outstanding technical assistance. This work was
supported by NIH grants R01 EB005011, R01 AI 078947, and a New Investi-
gator in Pathogenesis Award from Burroughs Wellcome Fund (to M.B.).
Received: December 7, 2010
Revised: May 17, 2011
Accepted: May 20, 2011
Published: June 23, 2011
IC50 Determinations
REFERENCES
IC50’s for the inhibitors were determined by serial dilution of the compounds in
the fluorogenic assay described above. The assay was repeated three or four
times per compound and the IC50 determined using Graphfit software.
Arastu-Kapur, S., Ponder, E.L., Fonovic, U.P., Yeoh, S., Yuan, F., Fonovic, M.,
Grainger, M., Phillips, C.I., Powers, J.C., and Bogyo, M. (2008). Identification of
proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium
falciparum. Nat. Chem. Biol. 4, 203–213.
Time Course of Labeling of Recombinant hSENP1 and 2 with
Activity-Based Probes
Recombinant DhSENP1 or 2 was diluted to 0.5 mM in reaction buffer (50 mM
Tris [pH 7.4], 20 mM NaCl, 5 mM DTT) and treated with probe (1 mM final
Berger, A.B., Witte, M.D., Denault, J.B., Sadaghiani, A.M., Sexton, K.M.,
Salvesen, G.S., and Bogyo, M. (2006). Identification of early intermediates of
Chemistry & Biology 18, 722–732, June 24, 2011 ª2011 Elsevier Ltd All rights reserved 731