Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Article abstract of DOI:10.1074/jbc.M111.279984

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5′-O-sulfamoyl-N ⁶-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP_i exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.

Full text of DOI:10.1074/jbc.M111.279984

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2011/10/03/M111.279984.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 40867–40877, November 25, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Mechanistic Studies of Substrate-assisted Inhibition of
Ubiquitin-activating Enzyme by Adenosine Sulfamate
□
S
Analogues
Received for publication, July 6, 2011, and in revised form, September 21, 2011 Published, JBC Papers in Press, October 3, 2011, DOI 10.1074/jbc.M111.279984
Jesse J. Chen1, Christopher A. Tsu, James M. Gavin, Michael A. Milhollen, Frank J. Bruzzese, William D. Mallender,
Michael D. Sintchak, Nancy J. Bump, Xiaofeng Yang, Jingya Ma, Huay-Keng Loke, Qing Xu, Ping Li, Neil F. Bence,
James E. Brownell, and Lawrence R. Dick
From Discovery, Millennium Pharmaceuticals Inc., Cambridge, Massachusetts 02139
Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin
via an adenylate intermediate and catalyzes its transfer to a
ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine
sulfamate analogue that was identified as a selective, mecha-
nism-based inhibitor of NEDD8-activating enzyme (NAE),
another E1 enzyme, by forming a NEDD8-MLN4924 adduct
that tightly binds at the active site of NAE, a novel mechanism
termed substrate-assisted inhibition (Brownell, J. E., Sintchak,
M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy,
T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke,
H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis,
C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D.,
Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick,
L. R. (2010) Mol. Cell 37, 102–111). In the present study, sub-
strate-assisted inhibition of human UAE (Ube1) by another
adenosine sulfamate analogue, 5ꢀ-O-sulfamoyl-N6-[(1S)-2,3-di-
hydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective
E1 inhibitor, was characterized. Compound I inhibited UAE-de-
pendent ATP-PPi exchange activity, caused loss of UAE thioes-
ter, and inhibited E1-E2 transthiolation in a dose-dependent
manner. Mechanistic studies on Compound I and its purified
ubiquitin adduct demonstrate that the proposed substrate-as-
sisted inhibition via covalent adduct formation is entirely con-
sistent with the three-step ubiquitin activation process and that
the adduct is formed via nucleophilic attack of UAE thioester by
the sulfamate group of Compound I after completion of step 2.
Kinetic and affinity analysis of Compound I, MLN4924, and
their purified ubiquitin adducts suggest that both the rate of
adduct formation and the affinity between the adduct and E1
contribute to the overall potency. Because all E1s are thought to
use a similar mechanism to activate their cognate ubiquitin-like
proteins, the substrate-assisted inhibition by adenosine sulfa-
mate analogues represents a promising strategy to develop
potent and selective E1 inhibitors that can modulate diverse bio-
logical pathways.
intensively studied pathways is the ubiquitin-proteasome sys-
tem that attaches a polyubiquitin chain to a lysine residue on a
target protein and directs it to proteasome-mediated proteoly-
sis. The ubiquitin-proteasome system is a key system responsi-
ble for maintaining cellular protein homeostasis, an emerging
research area that could potentially transform our understand-
ing of human diseases (1–3). Bortezomib (VELCADEꢁ), a pro-
teasome inhibitor, is currently approved in the treatment of
patients with multiple myeloma and relapsed mantle cell lym-
phoma (4, 5). The clinical success of bortezomib suggests that
targeting other components in the ubiquitin-proteasome sys-
tem pathway might represent an opportunity to develop novel
anti-cancer therapeutics (6–9).
Conjugating ubiquitin to a protein substrate is mediated by
an enzymatic cascade initiated by ubiquitin-activating enzyme
(UAE)2 (or Ube1 in humans, also known as E1) (10). Previous
mechanistic studies show that, in vitro, UAE activates ubiquitin
by a three-step process using ATP as a cofactor (Fig. 1A) (11,
12). In step 1, UAE binds ATP and ubiquitin, catalyzes forma-
tion of ubiquitin adenylate intermediate, and releases inorganic
pyrophosphate (PPi). The ubiquitin adenylate activates the
C-terminal carboxyl group of ubiquitin for nucleophilic substi-
tution. In step 2, the catalytic cysteine residue in UAE attacks
the adenylate to form a thioester intermediate (UAE-Sϳubiq-
uitin, ϳ denotes the thioester bond between the C-terminal
carboxyl group of ubiquitin and Cys632 of human UAE) with
AMP as the by-product. In step 3, UAE-Sϳubiquitin binds
another equivalent of ATP and ubiquitin and in a second round
of adenylation, forms a UAE-Sϳubiquitinꢂubiquitin-adenylate
ternary complex. Although UAE-Sϳubiquitin is capable of
transferring ubiquitin to the conjugating enzyme (E2) via a
transthiolation reaction, E1-E2 transthiolation is greatly stim-
ulated by occupancy of the nucleotide binding site by either
ubiquitin adenylate or ATP alone (13).
2 The abbreviations used are: UAE, ubiquitin-activating enzyme; NEDD8, neu-
ral precursor cell expressed, developmentally down-regulated 8; NAE,
NEDD8-activating enzyme; Ubl, ubiquitin-like protein; SPR, surface plas-
mon resonance; RU, response unit; TLC, thin layer chromatography; ITC,
isothermal titration calorimetry; Compound I, 5Ј-O-sulfamoyl-N6-[(1S)-2,3-
dihydro-1H-inden-1-yl]-adenosine; Ub-I, ubiquitin-Compound I covalent
adduct; Ub-4924, ubiquitin-MLN4924 covalent adduct; UbcH2, human
ubiquitin-conjugating enzyme encoded by UBE2H gene; UbcH10, human
ubiquitin-conjugating enzyme encoded by UBE2C gene; DMSO, dimethyl
sulfoxide; SUMO, small ubiquitin-like modifier.
Post-translational modification by ubiquitin plays an essen-
tial role in a wide range of cellular processes. One of the most
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S10.
1 To whom correspondence should be addressed: 40 Landsdowne St., Cam-
bridge, MA 02139. Tel.: 617-444–3396; Fax: 617-551-8906; E-mail: jesse.
chen@mpi.com.
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Substrate-assisted E1 Inhibition
panel of E1s (21). In this study, we describe detailed mechanistic
studies of Compound I-mediated UAE inhibition and present
biochemical evidence to support the proposed substrate-as-
sisted inhibition mechanism using the purified Compound
I-ubiquitin adduct. In addition, pre-steady-state kinetic analy-
sis has been performed to directly obtain kinetic parameters of
the adduct-forming reaction catalyzed by UAE. Inhibition and
affinity studies were also conducted with MLN4924, a weak
UAE inhibitor, and its ubiquitin adduct. The results indicate
that both adduct formation and its affinity to E1 contribute to
potency of E1 inhibition mediated by adenosine sulfamate
analogues.
EXPERIMENTAL PROCEDURES
Materials—[32P]PPi (catalog number NEX019), [␣-32P]ATP
(catalog number BLU003H250UC), and [␥-32P]ATP (catalog
number BLU002250UC) were obtained from PerkinElmer Life
Sciences. [3H]AMP was purchased from American Radiola-
beled Chemicals, Inc. (St. Louis, MO, catalog number
ART1556). Bovine ubiquitin (catalog number U6253), rabbit
muscle pyruvate kinase Type III (catalog number P0072), and
rabbit muscle myokinase (catalog number M3003) were pur-
chased from Sigma. Rabbit muscle lactate dehydrogenase was
purchased from Calbiochem (catalog number 427217). N-ter-
minal FLAG-tagged ubiquitin with the sequence of
N-MDYKDDDDK-ubiquitin2–76 was generated by gene syn-
FIGURE 1. Proposed mechanisms of Ubl activation and E1 inhibition. A,
three-step Ubl activation leading to a ternary complex via a Ubl-adenylate
intermediate. B, structures of AMP and two adenosine sulfamate analogues,
MLN4924 and Compound I. C, proposed mechanism of E1 inhibition by aden-
osine sulfamates.
In humans, eight E1 enzymes and more than a dozen ubiqui- thesis and subcloning in a pDEST14 vector and expressed in
tin-like protein (Ubls) have been identified that play important Escherichia coli. Mouse monoclonal anti-FLAG M2 antibody
roles in regulating diverse biological pathways (10). Members of was purchased from Sigma (catalog number F1804). Mouse
the E1 enzyme class share common sequence and structural monoclonal anti-ubiquitin antibody (P4D1) and anti-tubulin
features and all use a similar mechanism involving adenylation antibody were purchased from Santa Cruz Biotechnology (cat-
and thioester formation (14). Besides ubiquitin, another exten- alog numbers sc-8017 and sc-23948, respectively). Rabbit poly-
sively studied Ubl-conjugation pathway involves NEDD8 (Rub1 clonal anti-UbcH10 antibody was purchased from Boston
in yeast), a Ubl that shares ϳ60% sequence similarity with ubiq- Biochem Inc., Cambridge, MA (catalog number A-650). Rabbit
uitin (15, 16). Like ubiquitin, NEDD8 is activated via an adeny- monoclonal anti-Compound
I antibody was generated
late intermediate by an E1 known as NEDD8-activating enzyme in-house using a method described in previous studies (18, 21).
(NAE), which then transfers NEDD8 to an E2 (Ubc12 or Other chemicals were purchased from Sigma. N-terminal His6-
Ube2M in humans) (17). Recently, we described MLN4924 (Fig. tagged human Ube1 (UAE, wild-type and C632A mutant) and
1B), a potent and selective inhibitor of NAE that is currently other E1s were expressed in Sf9 insect cells and purified as
being evaluated in Phase I clinical trials (18–20). MLN4924 is a described before (18, 22). N-terminal glutathione S-transferase
mechanism-based NAE inhibitor that forms a covalent (GST)-tagged UAE or UbcH2 fusion protein and untagged
NEDD8-MLN4924 adduct, a novel mechanism termed sub- UbcH10 were expressed in E. coli. Expressed proteins were
strate-assisted inhibition (21). Biochemical studies demon- purified by affinity or conventional chromatography using
strated that formation of the NEDD8-MLN4924 adduct is standard buffers.
strictly ATP-dependent and requires the catalytic cysteine,
Purification of Ubiquitin-Compound I (Ub-I) and Ubiquitin-
which leads to the hypothesis that the adduct is formed via MLN4924 (Ub-4924) Adduct—A typical reaction mixture of 1
nucleophilic attack of the NAE-SϳNEDD8 thioester by the sul- ml contained 12 ␮M UAE, 10 ␮M ubiqutitin, 50 ␮M Compound
famate group of MLN4924 after completion of step 2 (Fig. 1C). I or MLN4924, 250 ␮M ATP, and 10 mM MgCl2 in 50 mM Tris-
The covalent adduct mimics the NEDD8-adenylate intermedi- HCl, pH 7.5. The reaction mixture was incubated at room tem-
ate and binds at the adenylation site of NAE to form a tight perature for 30 min and quenched with addition of 100 ␮l of 0.5
binary complex, which inhibits the ability of NAE to bind M EDTA, pH 8.0. A reverse-phase HPLC system was developed
NEDD8 or ATP (21).
to purify the ubiquitin-inhibitor adduct from the crude reaction
Because all E1s are thought to utilize similar mechanisms to mixture using a Proto300 C4 (300 Å, 5 ␮m, 2.1 ϫ 100 mm,
activate their cognate Ubls (10, 14), the substrate-assisted NAE Higgins Analytical, Mountain View, CA, catalog number
inhibition demonstrated by MLN4924 could potentially repre- RS-1021-W045). The elution system utilized 0.1% formic acid/
sent a general strategy to develop selective inhibitors against water as Buffer A and 0.1% formic acid in 90:10 water/acetoni-
other E1s. Indeed, one such adenosine sulfamate analogue, trile as Buffer B. The elution was performed using a gradient of
Compound I (Fig. 1B), was shown to form Ubl-adducts with a 20% B from 0 to 5 min followed by 20% B to 60% B from 5 to 25
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Substrate-assisted E1 Inhibition
min with a flow rate of 0.3 ml/min (retention time: ubiquitin, PHERAstar plus instrument equipped with an HTRFꢁ optical
14.9 min; Ub-I, 15.7 min; Ub-4924, 15.7 min). The fractions module (BMG Labtech, Offenburg, Germany).
containing ubiquitin-inhibitor adducts were pooled and
The steady-state rate of E1-E2 transthiolation was measured
co-evaporated with water 3 times in vacuo to remove organic by quantitating AMP production using a coupled assay with an
solvent. The final samples were re-dissolved in 20 mM HEPES, ADP-ATP cycling system (24). A typical reaction mixture (2
pH 7.5. The concentration of ubiquitin adduct was determined ml) contained 0.5 nM UAE, 4 ␮M ubiquitin, 1 ␮M UbcH10, 100
using UV absorption at 280 nm with calculated extinction coef- ␮M ATP, 10 units/ml of rabbit muscle myokinanse, 20 units/ml
ficients based on ⑀280 values of ubiquitin and inhibitors (⑀280 for of rabbit muscle pyruvate kinase, 50 units/ml of rabbit muscle
Ϫ1
Ϫ1
Ub-I: 15.7 mM cmϪ1; for Ub-4924, 15.2 mM cmϪ1). The lactate dehydrogenase, 1 mM phosphoenolpyruvate, 3.4 ␮M
average overall yields were ϳ60–70%. The identity of the puri- NADH in 5 mM MgCl2, 25 mM NaCl, 50 mM HEPES, pH 7.5.
fied adduct samples was confirmed by LC/MS analysis (m/z for The reaction mixture was incubated at 37 °C and the loss of
ϩ
[M ϩ H] : Ub-I, calculated, 9009.38, observed, 9009.80; NADH fluorescence was monitored on a Cary Eclipse Fluorim-
Ub-4924, calculated, 8990.42, observed, 8991.31).
eter (Varian Inc., Mulgrave, Victoria, Australia), with the fol-
ATP-PPi Exchange Assay—The ATP-PPi exchange assay was lowing instrument settings: ␭ex, 350 nm; ␭em, 460 nm; slits, 20
performed using an improved protocol developed by Bruzzese nm; filter, auto; PMT, 650; cycle, 2 s; and read, 0.1 s. The fluo-
et al. (22). For potency measurement, inhibitors were serially rescence signal loss due to NADH reduction was converted to
diluted into a 96-well assay plate and a mixture containing 0.5 the amount of AMP produced in the reaction mixture using a
nM wild-type UAE or UAE mutant (C632A), 0.01, 0.1, or 1 mM standard curve. Time-dependent inhibition of E1-E2 transthio-
ATP, and 0.1 mM PPi (containing 50 cpm/pmol of [32P]PPi) in lation was measured in the presence of 50–300 nM Compound
1ϫ E1 buffer (50 mM HEPES (pH 7.5), 25 mM NaCl, 10 mM I. For each Compound I concentration, the observed rate of
MgCl2, 0.05% BSA, 0.01% Tween 20, and 1 mM DTT) was inhibition (kobs) was derived by fitting the progress curve with a
added. Reactions were initiated by adding ubiquitin (final con- slow, tight-binding kinetic model (23).
centration: 1 ␮M) and were incubated for 60 min at 37 °C before
UAE-SϳUbiquitin Thioester Inhibition Assay—The reaction
quenching with 5% (w/v) trichloroacetic acid (TCA) containing mixture (50 ␮l) contained 1 ␮M N-FLAG-ubiquitin, 50 nM UAE,
10 mM PPi. The quenched reaction mixtures were transferred to 0.1 or 1 mM ATP, 10 mM MgCl2 in 1ϫ E1 buffer with Com-
a Dot-Blot System (Whatman, catalog number 10447900) pound I in a 3ϫ dilution series (in 1 ␮l of DMSO, top concen-
loaded with activated charcoal filter paper, washed, and quan- tration of 100 ␮M, total 10 concentrations). The reaction mix-
titated on a phosphorimager (Fujifilm FLA-7000, GE Health- ture without UAE was first mixed with Compound I in a 96-well
care) as described previously (22). The spot intensities were plate and UAE was added to initiate the reaction. The plate was
converted to the amount of ATP using a standard curve gener- incubated at room temperature for 15 min and quenched with
ated with [␣-32P]ATP (22). Inhibition studies of other E1s by 4ϫ LDS sample loading buffer (Invitrogen, catalog number
Compound I were performed with their cognate Ubls using NP0007). The quenched samples were analyzed by SDS-PAGE
similar procedures as described above. Time-dependent inhi- under nonreducing conditions, transferred to 0.2-␮m Immo-
bition of the ATP-PPi exchange activity by UAE was performed bilon-P PVDF membrane (Millipore, catalog number
under similar conditions except that at each time point, an ali- ISEQ20200), and probed with either anti-FLAG or anti-Com-
quot of reaction mixture was quenched with 5% (w/v) TCA pound I antibody. Alexa Fluor 680-labeled secondary antibody
containing 10 mM PPi and was transferred onto charcoal filter (Invitrogen, catalog number A21109 for anti-rabbit and
paper for the quantitation of radioactive ATP produced in the A21057 for anti-mouse) was then used and quantitation of pro-
reaction. The data were fitted using the slow, tight-binding tein bands was performed on a Li-Cor Odyssey Imaging System
kinetic model described by Morrison and Walsh (23).
(Li-Cor Biosciences, Lincoln, NE).
E1-E2 Transthiolation Assays—Time-resolved fluorescence
Quantitation of AMP and PPi by Thin Layer Chromatogra-
resonance energy transfer was used to quantitate the amount of phy (TLC)—To quantitate the amount of ubiquitin adenylate or
UbcH2-Sϳubiquitin catalyzed by UAE following a similar pro- AMP generated in the UAE-catalyzed ubiquitin activation
tocol developed for NAE activity measurement (18). The inhib- reaction, 5 ␮M ubiquitin was mixed with 1 ␮M UAE in a mixture
itor potency assay mixture contained 0.35 nM UAE, 35 nM containing 25 ␮M ATP, [␣-32P]ATP (1 ␮Ci/nmol), 10 mM
N-FLAG-ubiquitin, 8 nM GST-UbcH2, 2.5 mM MgCl2, and 75 MgCl2, and 50 mM Tris, pH 7.5. The reaction mixture was incu-
nM ATP in 50 mM HEPES, pH 7.5, and 0.05% BSA with inhibi- bated at room temperature for 5 min and quenched by adding
tors in a 3ϫ dilution series. The reaction mixtures were incu- 0.5 M EDTA. The reaction mixture (1 ␮l) was then spotted on a
bated in a solid white 384-well microtiter plate at 37 °C for 90 PEI-TLC plate (J. T. Baker, Inc., Phillipsburg, NJ, catalog num-
min before being quenched with 20 mM EDTA. The quenched ber 4474-04) that was pre-run with water and eluted with 1 M
samples (50 ␮l) were then mixed with 25 ␮l of the detection LiCl. Under this condition, ubiquitin adenylate was shown to be
solution containing 0.6 nM anti-FLAG europium cryptate (Cis- completely hydrolyzed to ubiquitin and AMP (data not shown).
bio, Bedford, MA, catalog number 61FG2KLB), 8.1 ␮g/ml of To quantitate the amount of PPi generated in the reaction, a
anti-GST-allophycocyanin (Prozyme, Hayward, CA, catalog similar reaction mixture was prepared as described above
number PJ252p1), and 0.4 M potassium fluoride (in 50 mM except that [␥-32P]ATP (1 ␮Ci/nmol) was used instead of
HEPES, pH 7.5, and 0.05% BSA). The reaction mixtures were [␣-32P]ATP. The reaction mixture was quenched by adding 0.5
incubated for 2 h at room temperature and time-resolved fluo- M EDTA and eluted on a PEI-TLC plate with 0.75 M KH2PO4, 4
rescence resonance energy transfer was measured on a M urea, pH 3.5. The PEI-TLC plate was air dried and exposed to
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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Substrate-assisted E1 Inhibition
an imaging plate for 1 h before being visualized and analyzed ranging from 50 ms to 2 s, the reaction mixture was quenched
using the phosphorimager. The amount of AMP or PPi pro- with 1 N HCl and analyzed by Western blot on a Li-Cor Odyssey
duced in the reaction was determined from the ratios of spot Imaging System as described in the UAE thioester inhibition
intensities between AMP or PPi and the remaining ATP.
assay. Samples from each time point were analyzed in triplicate.
ATP-AMP Exchange Assay—The reaction mixture con-
Surface Plasmon Resonance (SPR) Experiments—All SPR
tained 1.2 ␮M ubiquitin, 1 mM ATP, 1 mM AMP, 0.2 ␮Ci/nmol experiments were performed on a Biacore S51 instrument (GE
of [3H]AMP, 10 ␮M PPi, 200 nM UAE in 10 mM MgCl2, 0.1 mM Healthcare). N-terminal GST-tagged UAE was immobilized on
DTT, and 50 mM Tris-HCl, pH 7.5. The reaction mixture with- a sensor chip surface using the anti-GST antibody capturing
out ubiquitin was first mixed with Compound I in a 3ϫ dilution method. Goat polyclonal anti-GST antibody was covalently
series (in 1 ␮l of DMSO, top concentration of 100 ␮M, total 10 attached to a carboxymethyl dextran-coated sensor chip sur-
concentrations) in a 96-well plate and ubiquitin was added to face (CM5 from GE Healthcare, catalog number BR-1006-68)
initiate the reaction. The reaction mixture was incubated at using the amine-coupling protocol provided by the manufac-
37 °C for 20 min before being quenched by 0.5 M EDTA, pH 8.0. turer (GE Healthcare, catalog number BR-1006-33). Briefly, the
The reaction mixture (1 ␮l) was then spotted on a PEI-TLC carboxymethyl groups on the blank CM5 chip surface were first
plate that was pre-run with water and eluted with 1 M LiCl. The reacted with N-ethyl-NЈ-(3-dimethylaminopropyl)carbodiim-
PEI-TLC plate was air dried and exposed to a high-sensitivity ide and N-hydroxysuccinimide. Goat polyclonal anti-GST anti-
imaging plate for 48 h before being visualized and analyzed body (30 ␮g/ml, catalog number BR-1002-23, GE Healthcare)
using the phosphorimager.
was then injected onto the activated sensor chip surface at a
Pre-steady-state Kinetic Studies—Pre-steady-state kinetic flow rate of 10 ␮l/min in 10 mM sodium acetate, pH 5.0, for 10
experiments were performed at 37 °C on a rapid chemical min. The sensor chip surface was then blocked with ethanola-
quench apparatus manufactured by Kintek (Model RQF-3, Kin- mine. The level of immobilization was typically ϳ12,000–
tek, State College, PA). To measure the rate of Ub-I formation, 13,000 response units (RU, 1 RU represents binding of ϳ1 pg of
pre-formed UAE-Sϳubiquitin was used. To prepare UAE- protein/mm2 on the sensor chip surface). N-GST-UAE (10
Sϳubiquitin, 13 ␮M UAE was mixed with 10 ␮M ubiquitin in a ␮g/ml) was then captured on the sample spot with a capture
total volume of 300 ␮l containing 50 ␮M ATP, 5 mM MgCl2, and level of ϳ900–1200 RU (flow rate: 30 ␮l/min). Purified recom-
20 mM HEPES, pH 7.5. The reaction mixture was incubated at binant GST (5 ␮g/ml) was captured on the control spot with a
room temperature for 10 min and loaded onto a PD MiniTrap capture level of ϳ1200 RU. The SPR data were collected at
G25 desalting column (GE Healthcare) pre-equilibrated with 25 °C with a flow rate of 90 ␮l/min in a sample running buffer
1ϫ E1 buffer. The final elution volume was 1.5 ml with an containing 10 mM HEPES (pH 7.5), 150 mM NaCl, 0.005% P-20
expected UAE-Sϳubiquitin concentration of 2 ␮M. To initiate (as surfactant), and 0.1 mg/ml of BSA. All data acquisition (in
the reaction, 20 ␮l of 2 ␮M UAE-Sϳubiquitin in Syringe A was duplicate) and subsequent analysis were performed with
rapidly mixed with 20 ␮l of 20, 40, 60, 80, 120, or 160 ␮M Com- recombinant GST as the control. The kinetics of association
pound I in Syringe B at 37 °C. At various time points ranging and dissociation data were fit with a single exponential rise or
from 50 ms to 1.5 s, the reaction was quenched with 1 N HCl. decay equation. The equilibrium affinity binding data were fit
The quenched reaction mixture was then analyzed by SDS- using a one-site binding model.
PAGE followed by Western blotting with anti-Compound I
Binding Affinity Measurement Using Isothermal Titration
antibody. The intensity of the adduct bands were quantitated Calorimetry (ITC)—The affinity between UAE and ubiquitin
on a Li-Cor Odyssey Imaging System using a standard curve was measured by ITC using an Auto-iTC200 microcalorimeter
obtained from purified Ub-I samples (3–400 fmol). Samples (Micro-Cal Inc., a subsidiary of GE Healthcare) controlled by
from each time point were analyzed in duplicate.
VPViewer software. All samples were prepared in the same
To measure the rate of ubiquitin adenylate formation (step buffer (20 mM NaCl, 1 mM tricarboxyethyl phosphine in 20 mM
1), 20 ␮l of a solution containing UAE, ATP, and [␣-32P]ATP in HEPES, pH 7.5) and were thoroughly degassed by being stirred
Syringe A was rapidly mixed with 20 ␮l of a solution containing under vacuum before use. ITC experiments were performed at
ubiquitin in Syringe B on the Kintek RQF-3 apparatus at 37 °C. 25 °C using a reference power of 10 ␮cal/s. The sample cell (0.2
The final reaction mixture contained 2.5 ␮M UAE, 2 ␮M ubiq- ml) contained 20–25 ␮M UAE, which was titrated against 500–
uitin, 12.5, 25, or 50 ␮M ATP in 10 mM MgCl2, and 50 mM 550 ␮M ubiquitin (injectant) in 1.4-␮l volumes. The syringe
HEPES, pH 7.5. At 10, 20, 30, 40, 50, 60, 80, 100, 150 ms, the speed was set at 1000 rpm. The affinity between UAE and Com-
reaction was quenched with 1 M HCl. The quenched solution pound I was measured in a similar procedure. Experiments
(10 ␮l) was then mixed with 40 ␮l of 0.5 M EDTA, from which 1 were either terminated after saturation or left to continue until
␮l was spotted on a PEI-TLC plate, eluted with 1 M LiCl, and all 28 programmed injections had occurred. The ITC data were
analyzed using the phosphorimager as described before. Sam- corrected for the heat of dilution of the injectant by subtracting
ples from each time point were analyzed in triplicate.
the control experiments (ubiquitin titrated against buffer) for
To measure the rate of UAE-Sϳubiquitin formation, 20 ␮l of each titration. The corrected data were analyzed with Microcal
a solution containing UAE and ATP in Syringe A was rapidly Origin 7.0, and the fitting curves were calculated according to a
mixed with 20 ␮l of a solution containing ubiquitin in Syringe B one-site binding model. Experiments were performed in tripli-
on the Kintek RQF-3 apparatus at 37 °C. The final reaction mix- cate to confirm constant calculations.
ture contained 1.5 ␮M UAE, 1 ␮M ubiquitin, 500 ␮M ATP in 10
UAE Activity Recovery Assay—The recovery of the activity of
mM MgCl2, and 50 mM HEPES, pH 7.5. At various time points UAE was monitored using a similar procedure as described for
40870 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

Substrate-assisted E1 Inhibition
the E1-E2 transthiolation assay using time-resolved fluores-
cence resonance energy transfer. UAEꢂinhibitor complexes (1
␮M), which were formed by incubating UAE with Compound
I/ubiquitin/ATP, were diluted 1:50,000 (ϳ20 pM UAE final con-
centration) into UbcH2 transthiolation reaction mixtures as
described above except that the concentration of ATP was 3
mM. The reaction mixtures were incubated at 37 °C. An aliquot
was removed at various time points and quenched with 20 mM
EDTA. The quenched samples were then transferred to a solid
white 384-well microtiter plate, mixed with the detection solu-
tion, and analyzed on a PHERAstar plus instrument as
described above.
Time Course Analysis of Ub-4924 Formation—The reaction
mixture contained 1 ␮M ubiquitin, 40 nM UAE, 250 ␮M ATP, 50
␮M MLN4924, 5 mM MgCl2, in 50 mM HEPES, pH 7.5. The
reaction mixture was incubated at 37 °C. An aliquot of 80 ␮l was
removed at 0, 0.5, 1, 2, 3, and 4 h, quenched with 5 ␮l of 0.5 M
EDTA and 20 ␮l of acetonitrile, and analyzed by reverse phase-
HPLC under similar conditions as described for adduct
purification.
Cellular Assays to Study Inhibition of the UAE Pathway—The
cellular studies to assess the pathway inhibition by Compound
I were performed using the HCT116 cell line as described
before (18, 21). Briefly, cells were treated with either 0.1%
DMSO (negative control) or 10 ␮M Compound I for 1 h. Cells
were then harvested and washed with cold PBS solution. The
cells were then lysed and the whole cell extracts were prepared
and normalized by total protein concentration. The samples
containing 40 ␮g of protein were then separated by SDS-PAGE
under nonreducing conditions and transferred to nitrocellulose
membrane as described above. The membrane was then probed
with anti-UbcH10, anti-ubiquitin, or anti-tubulin antibodies.
FIGURE 2. Inhibition of UAE by Compound I or Ub-I using the ATP-PPi
exchange assay. A, wild-type UAE. B, C632A UAE. The assay mixtures con-
tained 1 mM ATP. Results are summarized in Table 1.
TABLE 1
Summary of IC50 values for UAE inhibition by Compound I or Ub-I with
different ATP concentrations
Data with 1 mM ATP are derived from Fig. 2.
RESULTS
IC50
100 ␮M ATP
Inhibition of the ATP-PPi Exchange Activity of UAE—Com-
pound I was identified as an inhibitor against a panel of recom-
binant human E1s including UAE (21). Further mass spectrom-
etry analysis revealed a species in the E1 inhibition reaction
mixture that was consistent with a covalent, Ubl-Compound I
adduct, similar to the NEDD8-MLN4924 adduct observed pre-
viously (21). In the present study, Compound I was shown to be
a potent inhibitor against wild-type UAE in ATP-PPi exchange
assays (Fig. 2A, filled circles). The IC50 values ranged from 10.2
nM to 5 ␮M with an increasing ATP concentration from 10 ␮M
to 1 mM, suggesting that the inhibition by Compound I is ATP
competitive (Table 1). Compound I did not inhibit UAE with a
C632A mutation that prevents UAE-Sϳubiquitin thioester for-
mation (Fig. 2B). The absolute requirement of ATP and an
intact Cys632 residue for inhibition are consistent with the pro-
posed reaction between UAE-Sϳubiquitin and Compound I as
the necessary step leading to UAE inhibition (Fig. 1C). In addi-
tion, ATP is expected to compete with Compound I in binding
to UAE-Sϳubiquitin (step 3 in Fig. 1A), which is consistent
with the observed ATP competitiveness of UAE inhibition by
Compound I (Table 1). In addition to UAE, Compound I also
10 ␮M ATP
1 mM ATP
nM
Compound I
Ub-I
10.2 Ϯ 1.7
1.1 Ϯ 0.1
90.8 Ϯ 9.8
5.0 Ϯ 0.5
2.9 Ϯ 0.1
5.21 ϫ103
14.4 Ϯ 1.4
21.7 Ϯ 0.3
Ub-I (C632A UAE)
0.89 Ϯ 0.04
Table S1), suggesting that unlike MLN4924, Compound I is a
nonselective E1 inhibitor.
To confirm that the covalent, ubiquitin-Compound I adduct
(Ub-I) is the species directly responsible for UAE inhibition, we
attempted to isolate Ub-I from the enzymatic reaction mixture
containing UAE, Mg2ϩ-ATP, ubiquitin, and Compound I.
With an excess amount of UAE, ubiquitin was quantitatively
converted to Ub-I, which was purified from the reaction mix-
ture by reverse-phase HPLC (supplemental Fig. S1). Ub-I is a
potent inhibitor of UAE and its inhibition is also ATP-compet-
itive (Fig. 2A, open circles, and Table 1). With the same ATP
concentration, IC50 values for Ub-I were measured to be about
10–140-fold lower than those for Compound I (Table 1), sug-
gesting that the pre-formed adduct, Ub-I, can directly bind
UAE to form a tight, inactive complex and inhibit its ATP-PPi
demonstrated varying degrees of potency against other recom- exchange activity (Fig. 1C). The difference observed in IC50
binant human E1s in ATP-PPi exchange assays (supplemental values could result from different inhibition mechanisms
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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Substrate-assisted E1 Inhibition
FIGURE 3. Inhibition of UAE-Sϳubiquitin thioester by Compound I. A,
Western blots showing dose-dependent loss of UAE-Sϳubiquitin thioester
with 0.1 mM ATP (top panel) or 1 mM ATP (bottom panel) using N-FLAG-ubiq-
uitin as the substrate. Blots were probed with mouse anti-FLAG antibody. B,
quantitationanddataanalysisofWesternblotsshowninA. Theproteinbands
were visualized and quantitated using a fluorescently labeled secondary anti-
body (A.U., arbitrary fluorescence unit). IC50 values of 2.8 Ϯ 0.2 and 12.9 Ϯ 2.4
␮M were obtained with 0.1 mM ATP (filled circles) and 1 mM ATP (open circles),
respectively. The concentration range of Compound I is from 0.15 to 100 ␮M
in a 3-fold dilution series.
FIGURE 4. Ub-I adduct formation during the course of Compound I-medi-
ated inhibition of UAE-Sϳubiquitin thioester. A, Western blots showing
dose-dependent formation of Ub-I with 0.1 mM ATP (top panel) or 1 mM ATP
(bottom panel) using N-FLAG-ubiquitin as the substrate. Blots were probed
with rabbit anti-Compound I antibody. B, quantitation and data analysis of
Western blots shown in A. The protein bands were visualized and quantitated
using a fluorescently labeled secondary antibody (A.U., arbitrary fluorescence
unit). K1/2 values of 4.4 Ϯ 1.3 and 16.8 Ϯ 1.1 ␮M were obtained with 0.1 mM
ATP (filled circles) and 1 mM ATP (open circles), respectively. The concentration
range of Compound I is from 0.15 to 100 ␮M in a 3-fold dilution series.
(hence kinetics of inhibition) between Compound I and Ub-I ATP competiveness (Fig. 4A). The apparent K1/2 values
(see the results and discussions in later sections). In addition, obtained from the dose-dependent adduct formation plots,
the proposed inhibition mechanism predicts that UAE-Sϳub- 4.4 Ϯ 1.3 (0.1 mM ATP) and 16.1 Ϯ 1.1 ␮M (1 mM ATP) (Fig. 4B),
iquitin is no longer required for the pre-formed Ub-I adduct to agree well with the observed IC50 values obtained in the thioes-
bind and inhibit the ATP-PPi exchange activity of UAE. Con- ter inhibition analysis (Fig. 3B).
sistent with this hypothesis, Ub-I inhibited C632A UAE as well
In an attempt to further characterize the binding interaction
in an ATP-competitive fashion (Fig. 2B and Table 1). The ATP between Compound I and UAE-Sϳubiquitin, we performed
competitiveness of both Ub-I and Compound I inhibition ATP-AMP exchange assays. Unlike the ATP-PPi exchange
reflects the ability of ATP to bind either apo-UAE to initiate assay in which radioactive PPi reacts with the adenylate inter-
step 1 (competing with Ub-I) or UAE-Sϳubiquitin to initiate mediate in steps 1 or 3 (Fig. 1A), in ATP-AMP exchange assays,
step 3 (competing with Compound I) (Fig. 1A).
the UAE-Sϳubiquitin intermediate is directly interrogated by
Inhibition of UAE-SϳUbiquitin and E1-E2 Transthiolation monitoring its reaction with radiolabeled [3H]AMP to form
by Compound I—Because Compound I is thought to bind and ubiquitin adenylate (step 2) and subsequent conversion to ATP
react with UAE-Sϳubiquitin, loss of the thioester mediated by in the presence of PPi (step 1) (11). The IC50 value obtained by
Compound I was directly assessed by SDS-PAGE followed by titrating Compound I into the reaction mixture is thought to
Western blot analysis using N-FLAG-ubiquitin. As shown in reflect direct competition between Compound I and AMP in
Fig. 3A, Compound I causes dose-dependent suppression of binding to UAE-Sϳubiquitin. Consistent with the hypothesis,
UAE-Sϳubiquitin. Furthermore, the effect of Compound I on the IC50 value obtained using the ATP-AMP exchange assay,
the level of UAE-Sϳubiquitin appears to be ATP competitive, 11.5 Ϯ 1.4 ␮M (supplemental Fig. S2), agrees relatively well with
as increasing the ATP concentration from 0.1 to 1 mM raised the IC50 value of 12.9 Ϯ 2.4 ␮M measured in the UAE-Sϳubiq-
the IC50 value from 2.8 Ϯ 0.2 to 12.9 Ϯ 2.4 ␮M (Fig. 3B). This uitin inhibition assay by Western blot analysis with the same
result is consistent with observed competitiveness of ATP in ATP concentration (1 mM) (11).
ATP-PPi exchange assays. Consistent with the inhibition
To probe whether suppression of UAE-Sϳubiquitin by
mechanism, formation of the N-FLAG-Ub-I adduct was also Compound I also leads to inhibition of the subsequent E1-E2
detected in the same reaction mixture, which shows similar transthiolation step, we also developed a proximity-based,
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Substrate-assisted E1 Inhibition
TABLE 2
Summary of quantitation of AMP/adenylate and PPi release during
UAE-mediated ubiquitin activation
Ratio of AMP to UAE Ratio of PPi to UAE
WT UAE
1.96
2.04
WT UAE ϩ Compound I 0.88
1.20
WT UAE ϩ Ub-I
0.01
1.07
Ͻ0.01
1.06
C632A UAE
time-resolved fluorescence energy transfer assay to detect the
E2-Sϳubquitin thioester using a similar strategy as described
before with UbcH2 as the E2 (18). Both Compound I and Ub-I
are potent inhibitors in this assay, with IC50 of 1.4 Ϯ 0.2 and
0.39 Ϯ 0.1 nM, respectively (supplemental Fig. S3). Similar to
that observed in ATP-PPi exchange assays, Ub-I demonstrated
a higher potency than Compound I, reflecting the different
inhibition mechanisms and/or possible kinetics between the
two inhibitors.
Quantitation of ATP Hydrolysis during UAE-mediated Ubiq-
uitin Activation—To gain additional insight into the mecha-
nism of E1 inhibition, we quantitated the amount of AMP or PPi
release in the ubiquitin-activation reactions using [␣-32P]- or
[␥-32P]ATP, respectively. Consistent with the proposed three-
step mechanism (Fig. 1A) (11, 12), wild-type UAE was shown to
catalyze 2 eq of ATP hydrolysis, generating 2 eq of AMP and PPi
(Table 2, the ubiquitin adenylate intermediate was converted to
AMP during the analysis). C632A UAE, which is expected to
catalyze the first ubiquitin adenylate formation but not subse-
quent step 2 and 3 reactions, only generated 1 eq of AMP and
PPi (Table 2). In the presence of Compound I, UAE-Sϳubiqui-
tin is proposed to be converted to the Ub-I adduct after step 2
and is unable to proceed to step 3 (Fig. 1A). Consistent with the
mechanism, release of only 1 eq of AMP and PPi was observed
(Table 2). When Ub-I was included in the reaction mixture, no
AMP or PPi release was observed, suggesting an inactive UAE
complex was formed before the first ATP hydrolysis (Table 2).
Time-dependent Inhibition of UAE by Compound I—Time-
dependent inhibition of UAE by Compound I was assessed by
progress curve analysis of its ATP-PPi exchange activity. As
shown in Fig. 5A, an apparent slow, tight binding inhibition
pattern was observed for Compound I in the presence of 100 ␮M
ATP, which is similar to what was observed for MLN4924-me-
diated NAE inhibition (21). The observed rates of inactivation
(kobs) were obtained for each Compound I concentration by
fitting the progress curves using a slow, tight-binding inhibition
model (Fig. 5A) (23). By plotting kobs versus Compound I con-
centration ([I]), we estimated the apparent kobs/[I] value to be
FIGURE 5. Time-dependent UAE inhibition demonstrated by progress
curve analysis. A, time course of radioactive ATP production in ATP-PPi
exchange assays at various [Compound I]. B, time course of AMP production
during UAE activation/UAE-UbcH10 transthiolation monitored using an ADT-
ATP cycling system at various [Compound I]. For curves in A and B, the
observed rate of inactivation (kobs) was estimated by fitting the curves using a
slow, tight-bindinginhibitionmodel: Yϭvbkg ϫtϩA0 ϫ(1Ϫexp(Ϫkobs t)). C,
estimationofkinact/Ki bylinearfittingkobs versus[CompoundI]. Similarkinact/Ki
Ϫ1
values were obtained in these two assays (2.6 Ϯ 0.2 ϫ 104
M
sϪ1 from
ATP-PPi exchange assays and 2.5 Ϯ 0.2 ϫ 104 Ϫ1 sϪ1 from transthiolation).
M
The concentration of ATP in both assays is 0.1 mM.
UAE-Sϳubiquitin that is available to bind Compound I and
form a tight, inactive complex.
2.6 Ϯ 0.2 ϫ 104 Ϫ1 sϪ1 (Fig. 5C, filled circles). This apparent
M
second-order rate constant reflects the combined kinetic and
binding parameters involved in the sequence of events leading
to UAE inhibition, including the rate of UAE-Sϳubiquitin for-
mation, binding of Compound I to UAE-Sϳubiquitin, and the
rate of Ub-I formation. When a similar analysis was performed
with 1 mM ATP, the apparent kobs/[I] was determined to be
Time-dependent UAE inhibition was also studied in the
E1-E2 transthiolation reaction by continuously monitoring
AMP production using UbcH10 as the E2. This assay couples
steady-state AMP release to loss of the NADH fluorescence
signal due to its reduction using a myokinase-mediated ADP-
ATP cycling system together with pyruvate kinase/lactate
dehydrogenase (24). The rate of E1-E2 transthiolation can be
quantitatively measured using this coupled assay with an
3.3 ϫ 103 Ϫ1 sϪ1, ϳ8-fold slower than that with 100 ␮M ATP
M
(data not shown). The negative influence of ATP on the kinetics
of UAE inactivation is consistent with its competitive binding observed rate of 1.98 Ϯ 0.02 sϪ1 (supplemental Fig. S4). Com-
to UAE-Sϳubiquitin, which, in turn, reduces the fraction of pound I was demonstrated to inhibit E1-E2 transthiolation in a
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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Substrate-assisted E1 Inhibition
time-dependent fashion, similar to that observed in ATP-PPi
exchange assays (Fig. 5B). By plotting the observed rate of inac-
tivation (kobs) versus Compound I concentration, we obtained
an apparent kobs/[I] value of 2.5 Ϯ 0.2 ϫ 104 Ϫ1 sϪ1 (Fig. 5C,
M
open circles), which is very similar to the one determined in
ATP-PPi exchange assays (Fig. 5C, filled circles) with the same
ATP concentration (100 ␮M). This result suggests that binding
of UbcH10 to UAE and subsequent transthiolation does not
kinetically affect the rate-limiting step of UAE inactivation by
Compound I.
Pre-steady-state Kinetic Analysis to Determine the Rate of
Adduct Formation—To study binding of Compound I to UAE-
Sϳubiquitin and the rate of Ub-I formation directly, we per-
formed pre-steady-state single turnover kinetic analysis by
mixing pre-formed UAE-Sϳubiquitin with Compound I and
quantitating the rate of Ub-I formation on a millisecond time
scale. Pre-formed UAE-Sϳubiquitin was obtained by reacting
an excess amount of UAE with ubiquitin and Mg2ϩ-ATP and
FIGURE 6. Determination of the rate of Ub-I formation under single-turn-
over conditions. The observed rates (kobs)were plotted versus [CompoundI].
Ki (27 Ϯ 6 ␮M) and kinact (8.3 Ϯ 0.7 sϪ1) were obtained by fitting the data to a
hyperbolic equation: kobs ϭ kinact ϫ [Compound I]/([Compound I] ϩ Ki).
Ub-I Forms a Tight Complex with UAE—Crystallographic
the resulting mixture was desalted to remove small molecule studies revealed that the NEDD8-MLN4924 adduct forms a
components. Under such a condition, we expected that most of complex with NAE by binding at both the NEDD8- and nucle-
the UAE-Sϳubiquitin would not bind a second ubiquitin or otide-binding pockets and prevents NAE from binding either
ATP molecule because of the limiting amount of ubiquitin (13). ATP or NEDD8 (21). In the present study, the purified Ub-I
The desalted sample contained about 2 ␮M UAE-Sϳubiquitin adduct allowed us to examine its binding affinity with UAE
assuming 100% conversion. We expected the actual concentra- directly. SPR analysis showed that, in the absence of ATP, Ub-I
tion to be lower than 2 ␮M due to the efficiency of the reaction, binds UAE with an association rate (ka) of 1.9 ϫ 106 Ϫ1 sϪ1 and
M
Ϫ4 Ϫ1
degradation of UAE-Sϳubiquitin, and formation of the ternary a dissociation rate (kd) of Ͻ1 ϫ 10
s
(Fig. 7). This result
complex. Therefore, the final sample contained a mixed popu- suggests that the dissociation constant (KD or kd/ka) is less than
lation of UAE consisting of unreacted UAE, UAE-Sϳubiquitin, 50 pM for the UAEꢂUb-I complex. The ka values also decreased
and the ternary complex. However, the exact concentration of with an increasing ATP concentration (Table 3), which is con-
pre-formed UAE-Sϳubiquitin is not critical to the subsequent sistent with ATP-competitive inhibition by Ub-I (Table 1). The
Ϫ1
kinetic analysis because other UAE species were not expected fast association rate of Ub-I (1.09 ϫ 106
M
s
Ϫ1) compared
to interact with Compound I based on previous biochemical with the observed rate of inactivation for Compound I (kobs/[I]:
and biophysical studies (21, 22). In addition, once formed, Ub-I 2.6 ϫ 104
M
s
Ϫ1) under similar conditions (100 ␮M ATP)
Ϫ1
binds tightly to UAE, which renders the reaction between UAE- could explain the observed potency difference between Ub-I
Sϳubiquitin and Compound I inherently a single turnover. The and Compound I in ATP-PPi exchange assays (5.0 and 91 nM for
UAE-Sϳubiquitin sample (with a calculated concentration of 1 Ub-I and Compound I, respectively). As a comparison, ubiqui-
␮M after mixing) was then rapidly mixed with Compound I tin alone showed weak affinity to UAE by SPR and ITC studies
using a chemical quench apparatus and the amounts of Ub-I (supplemental Fig. S6). One-site model analysis of the equilib-
formed were determined by quantitative Western analysis rium binding signals of SPR suggests that the KD for ubiquitin is
using a standard curve generated with the purified Ub-I adduct. 10.3 Ϯ 4.7 ␮M (supplemental Fig. S6, A and B), which is consist-
Under this condition, a first-order kinetic profile was observed ent with the result from ITC studies (KD, 12.0 Ϯ 4.6 ␮M; N,
for each Compound I concentration (supplemental Fig. S5). 1.01 Ϯ 0.05) (supplemental Fig. S6C).
The apparent rate constant (kobs) was then plotted versus Com-
To assess the effect of the slow dissociation rate on the enzy-
pound I concentration and the data were fitted to obtain the matic activity of UAE, we performed UAE activity recovery
maximum rate of inactivation (kinact) and the inhibition con- assays. The UAEꢂUb-I complex was pre-formed by mixing UAE
stant (Ki) using the equation: kobs ϭ kinact ϫ [Compound with ubiquitin, ATP, and Compound I. The resulting mixture
I]/([Compound I] ϩ Ki). Fig. 6 shows that formation of Ub-I was desalted and diluted so that the complex concentration was
occurs with kinact of 8.3 Ϯ 0.7 sϪ1 and Ki of 27 Ϯ 6 ␮M. Based on below the apparent IC50 (ϳ20 pM). The recovery of UAE activ-
the amount of Ub-I formed at the end of the reaction, we esti- ity was assessed by measuring the E1-E2 transthiolation activity
mated that about 50% of the total UAE species existed as UAE- of UAE using the fluorescence energy transfer assay as
Sϳubiquitin in the final reaction mixture (ϳ0.5 ␮M). The kin
act/Ki value determined under this condition in the absence of presence of 3 mM ATP, about 10% UAE activity was recovered
ATP, 3.1 ϫ 105 Ϫ1 sϪ1, is about 12-fold higher than the kobs/[I] in 4 h, suggesting that kd is ϳ1.6 ϫ 10Ϫ4 sϪ1. The recovery assay
-
described above. As shown under supplemental Fig. S7, in the
M
value obtained from the progress curve analysis with 100 ␮M result agrees with the one obtained in SPR studies and further
ATP (2.6 ϫ 104
M
Ϫ1 sϪ1) or 90-fold higher than the one with 1 supports stable complex formation between UAE and Ub-I.
Ϫ1
mM ATP (3.3 ϫ 103
M
s
Ϫ1). Therefore, the single-turnover
Inhibition of UAE by MLN4924 and Ub-4924 Adduct—
analysis using a pre-formed UAE thioester allowed us to study MLN4924, which shows potent and selective inhibition against
the key step of Ub-I formation without competition from ATP. NAE, is a relatively weak inhibitor of UAE (IC50, 12.0 Ϯ 1.6 nM
40874 JOURNAL OF BIOLOGICAL CHEMISTRY
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Substrate-assisted E1 Inhibition
FIGURE 8. Steady-state formation of the UAE-catalyzed ubiquitin-
MLN4924 (Ub-4924) adduct analyzed by reverse-phase HPLC. Peaks cor-
responding to Ub-4924 are highlighted in red. The area under the Ub-4924
peaks were integrated and converted to the amount of Ub-4924 using a puri-
fied sample as a standard, which yielded a steady-state rate of 1.4 ϫ 10Ϫ3 sϪ1
or ϳ5 hϪ1
.
E1-selective inhibition mediated by a Ubl-adenosine sulfamate
adduct.
Fast Dissociation Rate of Ub-4924 Contributes to Its Weak
Inhibition against UAE—To assess the affinity of Ub-4924 and
UAE directly, we performed SPR analysis on the purified
Ub-4924 adduct as described for Ub-I. The Ub-4924 adduct
Ϫ2
showed a dissociation rate of 1.1 ϫ 10
s
Ϫ1, more than 100
times faster than Ub-I (Ͻ1 ϫ 10Ϫ4 sϪ1) (supplemental Fig. S9).
In addition, the UAE activity recovery assay showed that the
pre-formed Ub-4924ꢂUAE complex regained activity at a simi-
lar rate as the DMSO control (supplemental Fig. S7). These
results suggest that Ub-4924 does not form a tight complex
with UAE and that dissociation of Ub-4924 allows rapid recov-
ery of UAE activity in the presence of ATP and ubiquitin. Con-
sistent with these results, we found that UAE catalyzed multiple
FIGURE 7. Binding interaction between UAE and Ub-I studied by surface
plasmon resonance. A, sensorgrams of Ub-I (0.1, 0.2, and 0.4 ␮M, black, red,
andgreentraces, respectively)bindingtoUAEimmobilizedonthesensorchip
surface. The binding phase of the data were fit to a single-exponential rise to
derive apparent association rates (kobs). No measurable dissociation was
observed during the course of experiments. B, kobs was plotted against [Ub-I]
to obtain the association rate (ka) of 1.9 ϫ 106 Ϫ1 sϪ1
M .
TABLE 3
Association rates of Ub-I binding to UAE with different ATP concentra-
tions determined by SPR
cycles of Ub-4924 production with an estimated steady-state
0 ␮M ATP 50 ␮M ATP 100 ␮M ATP 200 ␮M ATP
rate of 5 hϪ1, or 1.4 ϫ 10
s
(Fig. 8). From this aspect,
Ϫ3 Ϫ1
Ϫ1
Ϫ1s
ka (ϫ106
M
)
1.85 Ϯ 0.06 1.79 Ϯ 0.07 1.09 Ϯ 0.03
0.73 Ϯ 0.01
MLN4924 acts as a substrate for UAE. The steady-state rate of
Ub-4924 formation reflects the rate-limiting step of the cata-
lytic cycle, which could be the rate of Ub-4924 formation or the
rate of adduct release under the assay conditions. Therefore,
the potency of adenosine sulfamate analogues in inhibition of
E1s is affected not only by the rate of adduct formation, but also
by the affinity between the adduct and E1.
for NAE and 22.1 Ϯ 5.8 ␮M for UAE in the ATP-PPi exchange
assay with 0.1 mM ATP) (supplemental Fig. S8A). In addition,
the progress curve analysis showed that the observed rate of
UAE inhibition was less than 1 ϫ 10Ϫ4 sϪ1 with 5 ␮M MLN4924
Ϫ1
(kobs/[MLN4924], Ͻ20 MϪ1 sϪ1, compared with 3.3 ϫ 103
M
Inhibition of Ubiquitination in HCT116 Cells by Compound I—
Compound I also inhibits UAE-mediated polyubiquitination in
a cellular setting. When HCT116 cells (a human colon cancer
cell line) were treated with 10 ␮M Compound I for 1 h, loss of
polyubiquitinated protein substrates was observed by Western
blot analysis compared with DMSO-treated cells (Fig. 9, middle
panel). Furthermore, loss of UbcH10-Sϳubiquitin thioester
was also detected (top panel), which is entirely consistent with
the inhibition of UAE-UbcH10 transthiolation observed in pro-
gress curve analysis (Fig. 5, B and C). Because Compound I also
inhibited other E1s in biochemical assays (supplemental Table
S1), the cellular outcomes of Compound I treatment are
expected to be pleiotropic due to its potential impact on diverse
biological pathways regulated by different E1s. Detailed studies
Ϫ1
s
for Compound I with 1 mM ATP) (data not shown), sug-
gesting that weak compound binding and/or slow adduct for-
mation might be responsible for the observed weak potency.
Consistent with this hypothesis, the purified ubiquitin-
MLN4924 adduct (Ub-4924), which could be obtained by incu-
bating UAE, ubiquitin, and ATP with MLN4924, demonstrated
higher potency against UAE (IC50, 1.6 Ϯ 0.8 ␮M) than the free
compound (IC50, 22.1 Ϯ 5.8 ␮M) (supplemental Fig. S8B). How-
ever, Ub-4924 (IC50, 1.6 Ϯ 0.8 ␮M) was still much less potent
than Ub-I (IC50, 5.0 Ϯ 0.5 nM) under similar assay conditions
(supplemental Fig. S8B). Furthermore, no inhibitory effect was
observed for Ub-4924 (up to 0.8 ␮M) when NAE was used in the
E1 activity assay (supplemental Fig. S8C, open circles), suggest-
ing that both Ubl and the compound moieties are critical to are underway to understand cellular responses caused by Com-
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Substrate-assisted E1 Inhibition
(KD Ͻ8 pM) (25). These results strongly suggest that Ub-I mim-
ics the ubiquitin adenylate intermediate and binds at the active
site of UAE to prevent further recruitment of substrates.
The covalent adduct-forming mechanism we hypothesize for
E1 inhibition by adenosine sulfamates is entirely consistent
with the three-step ubiquitin activation process first proposed
for UAE by Haas et al. (11, 12). Inhibition of the enzymatic
activity of UAE by Compound I is strictly dependent on the
catalytic cysteine residue (Cys632), suggesting that UAE-Sϳub-
iquitin is the required intermediate leading to Ub-I formation
(Fig. 2B). Consistent with this observation, the purified Ub-I
adduct, which circumvents the adduct forming step, inhibits
both wild-type and C632A UAE (Fig. 2). To further support the
proposed mechanism of UAE inhibition via Ub-I adduct forma-
tion, we quantitated the amount of ATP hydrolyzed by UAE
during ubiquitin activation in the absence and presence of
inhibitors. As proposed by Haas et al. (12) (Fig. 1A), without
ubiquitin-conjugating enzymes (E2), UAE is thought to
undergo one cycle of the three-step ubiquitin activation, con-
suming 2 eq of ATP and generating 2 eq of AMP-containing
species (1 eq of free AMP and 1 eq of ubiquitin adenylate),
concomitantly releasing 2 eq of PPi (Fig. 1A). In the presence of
Compound I, hydrolysis of only 1 eq of ATP was observed,
compared with no ATP hydrolysis with Ub-I (Table 2). These
results are consistent with Compound I reacting with UAE-
Sϳubiquitin, which occurs after the first ATP hydrolysis,
whereas Ub-I is capable of binding apo-E1, thereby inhibiting
FIGURE 9. Western blot analysis demonstrating the ubiquitination path-
way inhibition in Compound I-treated cells. HCT116 cells were treated
with DMSO or 10 ␮M Compound I for 1 h. The cells were then harvested and
whole cell extracts were subjected to SDS-PAGE and Western blot analysis ATP binding and hydrolysis all together.
under nonreducing conditions. Upper panel, anti-UbcH10, middle panel, anti-
Two distinct conformations, open and closed, have been pro-
ubiquitin; bottom panel, anti-tubulin.
posed for E1s during the Ubl-activation cycle. Most structural
studies on E1s with a distinct catalytic cysteine domain revealed
a distance of ϳ25–30 Å between the catalytic cysteine residue
and the nucleotide binding pocket, which represents an “open”
conformation (26–29). The open conformation is thought to be
required for binding Ubl and Mg2ϩ-ATP in steps 1 and 3 (28,
pound I and other more selective adenosine sulfamate ana-
logues and will be presented in future communications.
DISCUSSION
UAE and other E1s activate their cognate Ubls via a Ubl- 29). However, in step 2, the long distance in the open confor-
adenylate intermediate and initiate an enzymatic cascade that mation has to be overcome in order for the catalytic Cys to
ultimately conjugates ubiquitin and Ubls to targeted substrates. initiate nucleophilic attack of the ubiquitin adenylate located in
Given the conserved Ubl-activation process exhibited by all E1s the nucleotide binding pocket. Recent x-ray crystallographic
(10, 14), the mechanism-based E1 inhibition mediated by aden- studies of small ubiquitin-like modifier (SUMO)-activating
osine sulfamate analogues first discovered for MLN4924 and enzyme revealed a “closed” conformation in which the catalytic
NAE could also be observed in other E1 systems (21). In the cysteine residue formed a covalent, tetrahedral intermediate
current study, we presented biochemical studies that demon- with a SUMO 5Ј-vinylsulfonylaminodeoxy adenylate analogue
strated potent inhibition of UAE by Compound I, an adenosine in the nucleotide binding pocket (30). Interestingly, in our
sulfamate analogue (Fig. 1B). Compound I inhibited UAE-de- study, no apparent affinity was observed between Compound I
pendent ATP-PPi exchange activity (Fig. 2), caused loss of and apo-UAE (data not shown), which is presumably in the
UAE-Sϳubiquitin thioester (Fig. 3), and inhibited E1-E2 tran- open conformation based on previous structural studies (26).
sthiolation (Fig. 5B and supplemental Fig. S3) in a dose-depen- We believe that Compound I binds to UAE-Sϳubiquitin in a
dent manner. The species that was proposed to be responsible conformation that either mimics or can transition to a closed
for UAE inhibition, a covalent ubiquitin-Compound I adduct conformation in which the nucleophilic attack of UAE-Sϳub-
(Ub-I) similar to the NEDD8-MLN4924 adduct, was identified iquitin by Compound I likely occurs (Fig. 1C), a conformation
by mass spectroscopic analysis of the inhibition reaction mix- that probably resembles what was observed for SUMO-activat-
ture (21) and was isolated for biochemical characterization ing enzyme with a covalently trapped intermediate (30). This
(supplemental Fig. S1). Ub-I potently inhibited the ATP-PPi process is similar to the reverse reaction of step 2 where AMP
exchange activity of UAE and E1-E2 transthiolation (Fig. 2 and serves as the nucleophile (Fig. 1A). Furthermore, our results
supplemental Fig. S3). In addition, Ub-I was shown to form a suggest that UAE-Sϳubiquitin most likely undergoes rapid
tight complex for UAE with KD Ͻ50 pM in SPR studies (Fig. 7), equilibrium among different conformations, because ATP,
similar to what has been estimated for the ubiquitin adenylate which is thought to bind selectively to the open conformation
40876 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

Substrate-assisted E1 Inhibition
4. Kane, R. C., Bross, P. F., Farrell, A. T., and Pazdur, R. (2003) Oncologist 8,
508–513
(30), competes effectively with Compound I-mediated inhibi-
tion and adduct formation (Table 1 and Figs. 3 and 4).
5. Kane, R. C., Dagher, R., Farrell, A., Ko, C. W., Sridhara, R., Justice, R., and
Pazdur, R. (2007) Clin. Cancer Res. 13, 5291–5294
Pre-steady-state kinetic studies under single-turnover condi-
tions provide additional insight into the adduct forming reac-
tion between Compound I and UAE-Sϳubiquitin. The rate
constant (kinact) obtained in this study, 8.3 Ϯ 0.7 sϪ1, reflects a
rate-limiting step leading to Ub-I formation. It could be the
conformational change that allows the catalytic cysteine
domain to position ubiquitin thioester to the proximity of
Compound I or the actual chemical step of nucleophilic attack
by the amino group in Compound I. To place this rate constant
in the context of the three-step ubiquitin-activation process, we
also measured the rates of ubiquitin-adenylate formation (step
1) and UAE-Sϳubiquitin formation (step 2) directly by rapid
chemical quench on a millisecond to second time scale. In the
absence of E2, UAE can only catalyze one cycle of ATP hydrol-
ysis/adenylate formation under ubiquitin-limiting conditions,
resulting in a single-turnover kinetic process. The observed
adenylation rate was ϳ18.8 Ϯ 2.2 sϪ1, which was about 4 times
faster than the rate of UAE-Sϳubiquitin formation (4.3 Ϯ 0.8
6. Nalepa, G., Rolfe, M., and Harper, J. W. (2006) Nat. Rev. Drug Discov. 5,
596–613
7. Hoeller, D., Hecker, C. M., and Dikic, I. (2006) Nat. Rev. Cancer 6,
776–788
8. Hoeller, D., and Dikic, I. (2009) Nature 458, 438–444
9. Bedford, L., Lowe, J., Dick, L. R., Mayer, R. J., and Brownell, J. E. (2011) Nat.
Rev. Drug Discov. 10, 29–46
10. Schulman, B. A., and Harper, J. W. (2009) Nat. Rev. Mol. Cell Biol. 10,
319–331
11. Haas, A. L., and Rose, I. A. (1982) J. Biol. Chem. 257, 10329–10337
12. Haas, A. L., Warms, J. V., Hershko, A., and Rose, I. A. (1982) J. Biol. Chem.
257, 2543–2548
13. Pickart, C. M., Kasperek, E. M., Beal, R., and Kim, A. (1994) J. Biol. Chem.
269, 7115–7123
14. Streich, F. C., Jr., and Haas, A. L. (2010) Subcell. Biochem. 54, 1–16
15. Liakopoulos, D., Doenges, G., Matuschewski, K., and Jentsch, S. (1998)
EMBO J. 17, 2208–2214
16. Osaka, F., Kawasaki, H., Aida, N., Saeki, M., Chiba, T., Kawashima, S.,
Tanaka, K., and Kato, S. (1998) Genes Dev. 12, 2263–2268
17. Bohnsack, R. N., and Haas, A. L. (2003) J. Biol. Chem. 278, 26823–26830
18. Soucy, T. A., Smith, P. G., Milhollen, M. A., Berger, A. J., Gavin, J. M.,
Adhikari, S., Brownell, J. E., Burke, K. E., Cardin, D. P., Critchley, S., Cullis,
C. A., Doucette, A., Garnsey, J. J., Gaulin, J. L., Gershman, R. E., Lublinsky,
A. R., McDonald, A., Mizutani, H., Narayanan, U., Olhava, E. J., Peluso, S.,
Rezaei, M., Sintchak, M. D., Talreja, T., Thomas, M. P., Traore, T., Vysko-
cil, S., Weatherhead, G. S., Yu, J., Zhang, J., Dick, L. R., Claiborne, C. F.,
Rolfe, M., Bolen, J. B., and Langston, S. P. (2009) Nature 458, 732–736
19. Soucy, T. A., Smith, P. G., and Rolfe, M. (2009) Clin. Cancer Res. 15,
3912–3916
s
Ϫ1) under similar single turnover conditions (supplemental
Fig. S10). These observed rates agree well with previous studies
(11, 31) and suggest that UAE-Sϳubiquitin formation is the
rate-limiting step in ubiquitin activation (31). Although the
intrinsic rate of Ub-I formation, 8.3 Ϯ 0.7 sϪ1, is about twice as
fast as the rate of UAE-Sϳubiquitin formation, under physio-
logical conditions of ϳ1 mM ATP, the rate was estimated to
decrease significantly to ϳ0.1 sϪ1, making adduct formation
the rate-limiting step in the entire UAE inhibition process.
Human E1 family members share structural similarity and
are proposed to use a common mechanism to activate Ubls.
However, many residues in their nucleotide binding domain are
not conserved, which leads to distinct spatial arrangement of
amino acid side chains that are in close proximity to ATP (26–
29, 32). The striking selectivity of MLN4924 toward NAE sug-
gests that the adenosine sulfamate scaffold can be tailored to
specifically inhibit individual E1 enzymes. The current work
shows that both the rate of adduct formation and the affinity
between Ubl-inhibitor adduct and E1 contribute to the overall
potency of inhibition. Thus, a complex structure-activity rela-
tionship must be considered in developing novel therapeutics
that selectively target each E1 pathway.
20. Milhollen, M. A., Traore, T., Adams-Duffy, J., Thomas, M. P., Berger, A. J.,
Dang, L., Dick, L. R., Garnsey, J. J., Koenig, E., Langston, S. P., Manfredi,
M., Narayanan, U., Rolfe, M., Staudt, L. M., Soucy, T. A., Yu, J., Zhang, J.,
Bolen, J. B., and Smith, P. G. (2010) Blood 116, 1515–1523
21. Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump,
N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke,
H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A.,
Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P.,
Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37,
102–111
22. Bruzzese, F. J., Tsu, C. A., Ma, J., Loke, H. K., Wu, D., Li, Z., Tayber, O., and
Dick, L. R. (2009) Anal. Biochem. 394, 24–29
23. Morrison, J. F., and Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol.
Biol. 61, 201–301
24. Valero, E., Varón, R., and García-Carmona, F. (2000) Biochem. J. 350,
237–243
25. Burch, T. J., and Haas, A. L. (1994) Biochemistry 33, 7300–7308
26. Lee, I., and Schindelin, H. (2008) Cell 134, 268–278
27. Lois, L. M., and Lima, C. D. (2005) EMBO J. 24, 439–451
28. Walden, H., Podgorski, M. S., Huang, D. T., Miller, D. W., Howard, R. J.,
Minor, D. L., Jr., Holton, J. M., and Schulman, B. A. (2003) Mol. Cell 12,
1427–1437
Acknowledgments—We thank M. Bembenek and J. Yan for technical
help with adduct and E1 thioester quantitation, A. Burkhardt for
developing compound-specific antibodies, T. Soucy, H. Liao, B. Ami-
don, and F. Melandri for helpful discussions, and J. Bolen for support
and guidance.
29. Walden, H., Podgorski, M. S., and Schulman, B. A. (2003) Nature 422,
330–334
REFERENCES
30. Olsen, S. K., Capili, A. D., Lu, X., Tan, D. S., and Lima, C. D. (2010) Nature
463, 906–912
1. Balch, W. E., Morimoto, R. I., Dillin, A., and Kelly, J. W. (2008) Science 319,
31. Tokgöz, Z., Bohnsack, R. N., and Haas, A. L. (2006) J. Biol. Chem. 281,
14729–14737
916–919
2. Cohen, F. E., and Kelly, J. W. (2003) Nature 426, 905–909
3. Mu, T. W., Ong, D. S., Wang, Y. J., Balch, W. E., Yates, J. R., 3rd, Segatori,
L., and Kelly, J. W. (2008) Cell 134, 769–781
32. Bacik, J. P., Walker, J. R., Ali, M., Schimmer, A. D., and Dhe-Paganon, S.
(2010) J. Biol. Chem. 285, 20273–20280
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