Design of High-Throughput Screening Assays and Identification of a SUMO1-Specific Small Molecule Chemotype Targeting the SUMO-Interacting Motif-Binding Surface

Article abstract of DOI:10.1021/co500181b

Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small

Full text of DOI:10.1021/co500181b

Research Article
pubs.acs.org/acscombsci
Design of High-Throughput Screening Assays and Identification of a
SUMO1-Specific Small Molecule Chemotype Targeting the SUMO-
Interacting Motif-Binding Surface
Aileen Y. Alontaga,† Yifei Li,† Chih-Hong Chen,† Chen-Ting Ma,‡ Siobhan Malany,§ Danielle E. Key,§
Eduard Sergienko,‡ Qing Sun,‡ David A. Whipple,∥ Daljit S. Matharu,∥ Baozong Li,† Ramir Vega,†
Yi-Jia Li,† Frank J. Schoenen,∥ Brian S. J. Blagg,∥ Thomas D.Y. Chung,‡ and Yuan Chen*,†
†Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, California 91010, United States
‡Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Institute, La Jolla, California 92037, United States
§Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Institute at Lake Nona, Orlando, Florida 32827, United States
∥University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66049, United States
S
* Supporting Information
ABSTRACT: Protein−protein interactions are generally
challenging to target by small molecules. To address the
challenge, we have used a multidisciplinary approach to
identify small-molecule disruptors of protein−protein inter-
actions that are mediated by SUMO (small ubiquitin-like
modifier) proteins. SUMO modifications have emerged as a
target with importance in treating cancer, neurodegenerative
disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein−protein interactions can sensitize
cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence
resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS)
to identify inhibitors for SUMO-dependent protein−protein interactions. Using these assays, we have identified a
nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation
studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein−protein interaction,
despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.
KEYWORDS: small ubiquitin-like modifier (SUMO), TR-FRET, fluorescence polarization, SUMO-interacting motif (SIM)
development of neurodegenerative disorders, such as Hunting-
ton’s disease3 and pathogen infection.4 SUMO conjugation
does not lead to protein degradation directly, but enables
protein−protein interactions through SUMO-interacting motifs
(SIM).5
INTRODUCTION
■
Despite the important roles of protein−protein interactions
(PPI) in cellular regulation, it has been difficult to modulate
these interactions with small molecules. With increased studies
in this area, we can begin to identify and develop effective
approaches to discover small molecule PPI modulators. In this
study, we used a multidisciplinary approach including both
biochemical and biophysical methods to address PPI mediated
by small ubiquitin-like modifiers (SUMO).
SUMO proteins are ubiquitin homologues that can conjugate
to other cellular proteins through a biochemical mechanism
similar to ubiquitination. Emerging data indicate that
SUMOylation is a potential target for developing therapies
for cancer and other life-threatening diseases. SUMOylation is
important for major oncogenesis pathways, such as those driven
by c-Myc,1 and thus could be targeted for anticancer therapy
when these oncogenes are activated. In addition, SUMOylation
regulates the DNA-damage response that is important to cancer
therapy, allowing it to be targeted to either enhance cancer cell
sensitivity to DNA-damaging chemo and radiation therapy, or
to inhibit the aberrant DNA repair pathways to induce cell
death.2 Besides cancer, SUMOylation is involved in the
At least three SUMO paralogs, SUMO1, 2, and 3, are
expressed in human cells and conjugate to other proteins.6
SUMO1 shares less than 50% sequence identity to other
SUMO isoforms. However, SUMO2 and 3 are nearly identical
in sequence and cannot be distinguished by antibodies. In
particular, the surfaces of SUMO2 or 3 that bind SIM have
identical amino acid residues. However, the various SUMO
paralogs appear to have distinct functions. For example,
SUMO2 and SUMO3 conjugations respond strongly to
environmental stress, such as heat shock, while SUMO1
conjugation does not.7 In addition, the cellular localization
patterns of the SUMO paralogs do not completely overlap.
SUMO-1 primarily localizes to the nuclear pore and nucleolus,
Received: December 1, 2014
Revised: February 25, 2015
Published: February 26, 2015
© 2015 American Chemical Society
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Figure 1. FRET assay development for SUMO1−S1. (A) Titration of N-terminal fluorescein-tagged (F-S1) or C-terminal fluorescein-tagged (S1F)
S1 peptide with 2 nM Tb-anti-GST and 10 nM GST-SUMO1. Signal was background-subtracted. EC50 were determined to be 1.0 0.067 and 5.5
0.038 μM for F-S1 and S1F, respectively. Assay windows were 33000 1100 units (FRET Ratio 520 nm/490 nm × 10000, unless otherwise noted)
and 81000 360 units, respectively. (B) Corresponding signal/background (S/B) plot for panel A. (C) Background fluorescence without SUMO1.
Linear increase of background decreases S/B. (D) Donor optimization. Two nanomolar Tb-anti-GST and 10 nM GST-SUMO1 were confirmed to
be the optimal condition. (E) Displacement dosage response curve. Unlabeled S1 competed effectively with F-S1 for GSTSUMO1 binding. IC50 was
determined to be 11
0.6 μM. (F) DMSO effect (32 wells per each condition). No signal change for 0.5% DMSO or lower. (G and H)
Corresponding S/B plot and Z′ factor at various DMSO concentrations. (I) FP Assay development for SUMO1-S1. Titration of GST-SUMO1
against 7 nM N-terminal tagged (F-S1) or C-terminal tagged (S1F) S1. Assay window were 99 1.2 and 150 5.0 mP, respectively. EC50 were 940
27 and 5400 350 nM, respectively.
but SUMO2 and 3 primarily localize in the nucleoplasm and
nuclear bodies.8 Furthermore, SUMO1 and SUMO2 con-
jugation to the same protein could have opposing functions.9
Small-molecule modulators of SUMO-SIM interactions with
paralog-specificity would be very helpful for elucidating the
functions of the different SUMO paralogs. Because the SIM
sequence is short, it was not surprising that we previously found
peptidomimetics that have low affinity to bind all SUMO
proteins (Kd ≈ 1 mM). Conjugation of these molecules to
nanoparticles achieved high affinity for binding poly-SUMO
chains due to the multivalency effect.10 Recently, non-
peptidomimetic small molecule modulators of SUMO-SIM
interaction were described, but these molecules bind all SUMO
paralogs without specificity.11 Small-molecule modulators that
bind to a SUMO protein with paralog-specificity have not been
reported.
In this study, we developed biochemical HTS assays that
were used to detect SUMO1 interaction with a SUMO1-
specific-SIM peptide using time-resolved fluorescence reso-
nance energy transfer (TR-FRET) and fluorescence polar-
ization (FP) assay formats. Using these assays, we screened a
∼365 000 compound library and identified a SUMO1-specific
nonpeptidomimetic compound that targets the SIM-binding
surface. Analogs of the initial hits have revealed the conforma-
tional flexibility of the compounds as important for binding
affinity to SUMO1. This study not only provides the method
for identification of SUMO paralog-specific SUMO-SIM
interaction inhibitors, but also demonstrates that it is feasible
to identify such small molecules, which are not peptidomi-
metics, despite the high sequence identity of the SIM-binding
surfaces of the different SUMO paralogs.
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Figure 2. Primary high-throughput screen of a large library (∼365 000 compounds) with a SUMO1-S1 TR-FRET assay. (A) Schematic of primary
assay. (B) Frequency distribution of inhibitory activity of the chemical library. (C) Scatterplot of individual inhibitory activity by plate ID.
(Figure 1F, 1G, and 1H). Therefore, this assay is suitable for
high-throughput screening (HTS).
RESULTS AND DISCUSSION
■
Development of the HTS Assays. TR-FRET assays were
An FP assay was developed in parallel with the FRET assay
that can serve as a secondary assay using an “orthogonal”
detection technology to eliminate false-positive hits in HTS.
For this assay, initial assay development involved titration of
small amounts of the F-S1 or S1F peptide with GST-SUMO1.
Similar to TR-FRET assay results, F-S1 offered a smaller assay-
response window but much better binding affinity, with EC50
near 1 μM (Figure 1I), consistent with the reported affinity of
the SUMO-SIM interaction.8c S1F may have a better assay-
response window but the binding affinity was lower, which may
reflect interference of fluorescein on the interaction, and
restricted assay sensitivity to higher compound concentrations.
Despite the narrow assay-response window of the F-S1 peptide,
the standard deviation among quadruplicates was very low and
2 μM SUMO1 appears sufficient to generate a good Z′-factor in
this assay.
Primary HTS. Using the TR-FRET-based biochemical assay
(Figure 2A) using GST-labeled SUMO1 PPI and FITC-labeled
FS-1 peptide was used to screen the entire MLSMR of
∼365 000 compounds at 20 μM in a 1536-well format
(PubChem Summary AID 602467). The assay performed
robustly with a Z′ average over 274 plates of 0.88 and a signal-
to background of 4.0. As shown in Figure 2B, the binned data
shown in blue as % inhibition shows a Gaussian distribution
centered over the negative control shown in pink (wells
receiving SUMO1 protein, peptide, and antibody). The positive
control is shown in red (well receiving peptide and antibody
only). A scatterplot (Figure 2C) of % inhibition vs plate ID
showed no well position-specific effects that would have
otherwise suggested evaporation or dispenser-related artifacts.
After data normalization, 1206 compounds (0.33% hit rate)
were selected as initial hits (≥40%I). One known advantage of
the TR-FRET assay is its inherent ability to separate real hits
from optical artifacts through simple data analysis. Compounds
developed for the binding of SUMO1 to a SUMO1-specific
binding peptide referred to as S1 (sequence DNEIECIIVW-
EKK) below.8c The S1 peptide has more than 10-fold higher
affinity for SUMO1 than other tested SIMs for binding either
SUMO1 or SUMO2 or 3, and thus enabled the detection of
SUMO1-S1 interaction at low concentrations. For GST-tagged
SUMO1 and S1 peptide, several detection approaches were
tested, including Amplified Luminisecense Proximity Homoge-
nous Assay (ALPHA) Screen, TR-FRET, and FP. The TR-
FRET-based assay platform showed the best assay-response
window. For the TR-FRET assay, initial steps in assay
development involved titration of low-nanomolar concentra-
tions of GST-SUMO1 and Cisbio Tb-anti-GST (Tb) with the
fluorescein-tagged S1 peptide. Fluorescein is attached to either
the N-terminus (referred to as F-S1) or the C-terminus
(referred to as S1F). F-S1 provides better signal-to-background
ratio (S/B) than S1F, and it gives near-maximal S/B values at
500 nM (Figure 1A and 1B). Of note, a decrease in the S/B
value above 1000 nM, known as a “hook effect”, is a result from
capping in the specific TR-FRET signal corresponding to
SUMO1-S1 binding and concurrent proportional growth of the
background (Figure 1C). F-S1 exhibited a smaller assay-
response window but greater binding affinity (Figure 1B and
1C). Concentrations of SUMO1 and Tb were further
optimized to maximize S/B value using the F-S1 peptide
(Figure 1D). Effective displacement of F-S1 by unlabeled S1
confirmed that the fluorescein tag did not significantly alter the
binding mode of the S1 peptide and the TR-FRET signal was
derived from the desired protein−protein interaction (Figure
1E). DMSO study showed ≤10% signal decline from 1−4%
DMSO but no signal change for ≤0.5% DMSO. The data
scatter was also reduced by lowering DMSO concentrations,
leading to an improved Z′ factor and excellent S/B value
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that either absorb at 337 or 490 nm, or fluoresce at 490−520
nm and appear as initial positives judged by TR-FRET signal,
are easily detected though their fluorescent intensity in the
reference (490 nm) channel. Using the F-ratio parameter
(defined as the ratio of fluorescence in a compound well
normalized to an average value of fluorescence observed in
control wells), we were able to identify hits that have no optical
interference issues. Furthermore, we applied additional
cheminformatic filtering to eliminate known pan assay
interference compounds (PAINS) and promiscuous com-
pounds (PubChem and internal).
Hit Identification. Hits were reconfirmed (36%) in the
primary assay. Of these, 176 had potencies (IC50) better than
10 μM, while 24 also had better than 10 μM IC50 in the
orthogonal FP assay. The final validated hits were analyzed by
nuclear magnetic resonance (NMR) spectroscopy to detect the
interaction of these compounds with SUMO1. Control
experiments using SUMO1 titrated with deuterated DMSO
were first acquired to identify solvent effect12 on NMR
chemical shifts. Compounds were titrated to SUMO1 at
0.5:1, 1:1, and 2:1 of compound:SUMO1 stoichiometry to a
sample containing 20 μM SUMO1. Similar titration experi-
ments were also conducted for SUMO2 to determine the
specificity of these compounds.
suggest that the compound causes chemical shift perturbation
(CSP) and that the exchange rate between the free and
complex states is in the intermediate regime relative to NMR
chemical shift time scale. The residues of SUMO1 that showed
line-broadening effects are 22, 35, 37, 43, and 46 upon binding
to SIMI-4. These residues are colored in red in Figure 3B. The
residues that showed CSP larger than 0.03 ppm (twice the
average CSP) upon binding the compound are 38, 42, and 45.
These residues are colored in pink in Figure 3B. The chemical
shift of a nucleus is sensitive to the changes of its local
environment due to a complex formation. Any small additional
conformation changes near the direct contacting surfaces will
cause additional chemical shift perturbation. Thus, the surface
mapped by chemical shift perturbation contains but usually
extends beyond the direct binding surface. The data indicate
that the compound binds to the surface of SUMO1 that is
required for binding SIM, but near one end of the surface
including part of the loop connecting the β-strand and α-helix.
This compound does not bind to SUMO2, as indicated by
the lack of line broadening effects on SUMO2 upon titration to
the SUMO2 sample (Figure 3C). Although CSP was observed
on a few residues on the surface opposite from the SIM-binding
surface, these residues showed similar CSP when titrating with
other analogs, indicating nonspecific effects (Supplementary
Figure 1, Supporting Information). The amino acid sequences
between SUMO1 and SUMO2 or SUMO3 are nearly identical
within the SIM-binding surface, but a significant difference is in
the loop connecting the α-helix and β-sheet, which contains a
Pro in SUMO2 or 3, but is a His (residue 43, Figure 3B) in
SUMO1.8c Pro may restrict or alter the conformation of the
SIM-binding surface due to the restricted backbone dihedral
angles. We tested whether substitution of His-43 of SUMO1 by
a Pro affects the compound binding. H43P mutant of SUMO1
is properly folded as shown by the similar chemical shifts as the
wild type protein (Figure 3D). However, it no longer binds to
the compound, as indicated by the lack of CSP at compound to
protein ratio of approximately 4:1, which is twice of that (2:1)
when CSP was observed with the wild type SUMO1 (Figure
3A). This result further indicates that the compound binds to
the SIM-binding surface of SUMO1 near or including the loop
connecting the α-helix and β-strand.
We identified a compound with specificity for binding to
SUMO1 (Pubmed SID 152137659, CID 3598), which will be
referred as SIMI-4 in subsequent discussions (Table 1). It
causes significant line-broadening effects on SUMO1 (Figure
3A). The line broadening effects on the SUMO1 resonances
Table 1. Summary of the SUMO-Binding Activities of the
SIMI-4 Analogs Estimated by ITC (SIMI-4 and 4-1) and
NMR (Other Compounds)
Purchase and Synthesis of SIMI-4 Analogs. A handful of
SIMI-4 compound analogues was purchased or synthesized to
explore the SAR for this chemotype. The compounds 4-1, 4-2,
4-3, and 4-4 (Table 1) were purchased from commercial
suppliers, and, after analysis for identity and purity, were used
as purchased or purified as necessary. The compounds 4-5 and
4-6 (Table 1) were prepared by halogenation of commercially
available 2,2′-oxidiphenol as shown in Scheme 1 (and
Supporting Information).
Structure−Activity Relationship. Analogs of this com-
pound were tested for binding SUMO1 by NMR studies.
Replacement of the carbon atom in the linker by the sulfur
atom (4-1) did not significantly change the ability to bind
SUMO1, as shown by similar line-broadening effects and CSP
in NMR spectra (Supplementary Figure 1, Supporting
Information). Removal of the two meta-chlorine atoms
(meta- compared to the phenol group) greatly lowered binding
affinity, as indicated by the loss of CSP and line-broadening
effects (compounds 4-2 to 4-6) (Supplementary Figure 2,
Supporting Information). This is unlikely due to electronic
effects, because different Cl and Br substitutions on the
benzene rings did not lead to recovery of the activity, nor did
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Figure 3. SIMI-4 specifically binds to SUMO1 and not to SUMO2. (A) Expanded view of a region of the superimposed 1H−15N HSQC spectra of
SUMO1, free (red) and in complex with SIMI-4 (yellow). The assignments of the cross peaks are indicated with their amino acid residue type and
number. (B) The structure of SUMO1 with residues that showed severe line-broadening effects indicated in red and significant CSP shown in pink.
1
Residue H43, which showed severe line-broadening effect, is indicated with its side chain. (C) Overlay of the H−15N HSQC spectra of SUMO2,
free (blue) and in complex with SIMI-4 at SUMO:SIMI-4 of 1:2 stoichiometry (red). (D) Overlay of the 1H−15N HSQC spectra of SUMO1_H43P
mutant, free (red) and in complex with SIMI-4 at different SUMO:SIMI-4 stoichiometry, 1:0.5 (orange), 1:1 (yellow), 1:2 (green), and 4:1 (blue).
a
CSP, the 4-1 analog also binds SUMO with a similar, but
slightly lower affinity according to ITC measurements
(Supplementary Figure 3, Supporting Information). The
interaction is enthalpy driven, with ΔH of −12.8 1.0 kcal/
mol for SIMI-4. Similarly, the interaction of the 4-1 analog with
SUMO1 is also enthalpy driven, with ΔH of −23.4 3.3 kcal/
mol and a larger entropy cost (−60 cal/mol/deg) than SIMI-4
(−24 cal/mol/deg) (Supplementary Figure 3, Supporting
Information). The larger entropy cost could reflect the higher
flexibility of the thioether linkage than the alkyl linkage.
Because NMR can detect interactions with Kd up to 1 mM, the
absence of CSP of the other analogs of SIMI-4 indicates that
their binding affinities to SUMO1 or SUMO2 have Kd greater
than 1 mM.
Scheme 1. Synthesis of SIMI-4 Analogs
a
Reagents and conditions: (a) NBS or NCS, PTSA, CH3CN, rt, 3 h,
58% or 38%, respectively
the replacement of the linker C atom with an O atom
(compounds 4-5 and 4-6). The structure−activity relationship
suggests that the steric effect provided by the two meta-
chlorines is important for the recognition with SUMO1.
The binding affinity of SIMI-4 and 4-1 to SUMO1 was
characterized by isothermal titration calorimetry (ITC), which
was used to estimate the Kd of 107 13 μM (Figure 4A). Kd
was also estimated from NMR chemical shift perturbation and
line-shape analysis to be 84 40 μM.13 Consistent with NMR
The affinity of these compounds was not sufficient to inhibit
SUMO-SIM interactions required for the E3-ligase activity of
RanBP2 (data not shown). Development of protein−protein
interaction inhibitors is challenging due to the large surface of
the interactions that needs to be blocked by a small molecule.
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Figure 4. Measurement of the binding affinity between SUMO1 and SIMI-4. (A) Isothermal titration calorimetry (ITC) measurements of
compound−SUMO1 interaction. SUMO1 (300 μM in 10 μL increment) was injected to a total volume of 290 μL into a sample cell containing 1.4
mL of 20 μM SIMI-4. Same concentrations of DMSO were added to both the compound and protein solutions in both the experiments. The heat of
dilution was subtracted for baseline correction and analyzed with Microcal ORIGIN 5.0 software to extract binding thermodynamic parameters. (B)
1
The 1D slices were extracted with LineShapeKin SPARKY extension from the proton dimension in H−15N HSQC spectra (dotted lines) at the
SIMI-4:SUMO1 molar ratios of 0, 0.5, 1, 1.5, 2, 3, 4. Kd values were obtained from fitting line shape changes to the Bloch−McConnel equation for a
2-site exchange model using LineShapeKin13 (solid lines).
Further studies are needed to determine whether modifications
of this chemotype to limit the flexibility of the molecule or to
increase its size would significantly improve the binding affinity
to SUMO1.
Small-molecule inhibitors with specificity to SUMO1 could
have therapeutic potential. In a previous study,8c we found that
expression of S1 peptide in the MCF-7 breast cancer cells was
toxic to the cells, but expression of a SUMO2-specific SIM
peptide was not, because the SUMO2-specific SIM peptide
could be stably expressed in MCF-7 cells, but stable cell lines
expressing the S1 peptide could not be established. This
suggests that SUMO1 interaction with its SIM is critical for the
MCF-7 breast cancer cells.
In summary, the studies show the effectiveness of a
multidisciplinary approach to tackle the challenging problem
of developing PPI modulators. Further development of such
molecules will help to determine the affinities needed to
achieve a cellular effect and produce a useful tool to dissect the
roles of different SUMO paralogs in regulating cellular
functions and validate the specific SUMO paralogs as novel
innovative therapeutic targets.
(PerkinElmer) using their TR-FRET protocol (em340 and
ex520/485). Full inhibition controls contained no SUMO1.
The assay was robust over the entire screen of 274 plates with a
Z′ of 0.88.
NMR Studies. Uniformly 15N-labeled mature SUMO1 was
expressed and purified as described previously.14 All 15N-
SUMO1 samples (20 μM) used in the NMR screening
experiments were in phosphate buffer (20 mM, 1 mM DTT,
90% H2O, 10% D2O, pH 6.8). For inhibitor screening, 15N-
SUMO1 samples were added with 0.5, 1.0, and 2.0 mol
equivalence of the compounds identified from the high
throughput screen and transferred to the 96-well SampleJet
tubes 5.0 mm (5.0 × 103.5 mm). 1H−15N-HSQC spectra of the
samples were collected on Bruker Avance III spectrometer
1
equipped with a cryoprobe operating at 700.243 MHz H
frequency using the Bruker SampleJet automated sample
changer. The weighted proton and nitrogen chemical shifts
were calculated and quantified. The final concentrations of
deuterated DMSO in all the samples after addition of the
compounds were below 2% (v/v). All experiments were carried
out at 298 K. NMR data were processed using NMRPipe and
analyzed with the program Sparky.15
EXPERIMENTAL PROCEDURES
Line shape analysis was carried out by using the software
■
package LineShapeKin.13 The 1D slices from the proton
HTS Assays. The initial primary TR-FRET assay was
developed and conducted on 363 827 unique compounds in a
1536-well format and is described in PubChem AID 602429.
Briefly, 10 nM GST-SUMO1 and 2 nM Tb-anti-GST in 50 mM
HEPES pH 7.5, 0.005% Tween 20, and 1 mM DTT were
preincubated at room temperature with 20 μM compounds for
an hour, followed by addition of 500 nM FITC-labeled S1
peptide (F-S1). Following an additional hour of incubation, the
plates were read on a Pherastar (BMG LabTech) with
Lanthascreen optical module (ex337/em520, with emission at
490 nm as an internal reference) or with an Envision
1
dimension in H−15N HSQC spectra were obtained at the
SIMI-4:SUMO1 molar ratios of 0, 0.5, 1, 1.5, 2, 3, 4 from
extraction using LineShapeKin SPARKY extension. Kd values
were obtained from fitting line shape changes to the Bloch−
McConnel equation for a 2-site exchange model.
Isothermal Titration Calorimetry (ITC) Measurements.
Microcal (Amherst, MA) VP-ITC calorimeter was used to
perform ITC measurements for the binding interaction of
SUMO1 with different compounds at 30 °C. SUMO1 and
compounds were dissolved in the same buffer (20 mM
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phosphate buffer, pH 6.8). SUMO1 (300 μM in 10 μL
increment) was injected at intervals of 180 s up to a total
volume of 290 μL into a sample cell containing 1.4 mL of 20
μM inhibitor. The heat of dilution was measured after each
experiment by performing buffer injections into compound
using the same experimental concentrations and volumes. In
addition, injection of SUMO1 to buffer and to an inactive
compound (4−2) was also measured for heat of dilution and
control. Same concentrations of DMSO were used in both
experiments. The heat of dilution was subtracted for baseline
correction and analyzed with Microcal ORIGIN 5.0 software to
extract binding thermodynamic parameters.
mixture was stirred at room temperature for 3 h, whereupon
the mixture was quenched by the addition of 10% Na2S2O3 (15
mL), and the layers were separated. The organic layer was
washed once with 10% Na2S2O3 (15 mL) and twice with water
(10 mL), dried (MgSO4), filtered, and concentrated. The
residue was purified by flash chromatography (0−20% EtOAc/
hexanes gradient) to afford 83 mg (58%) of the compound 4−5
as a colorless solid. 1H NMR (400 MHz, CDCl3): δ 7.21 (dd, J
= 8.8 and 2.0 Hz, 2H), 6.99 (d, J = 2.0 Hz, 2H), 6.95 (d, J = 8.8
Hz, 2H), 5.60 (br s, 2H). 13C NMR (126 MHz, CDCl3): δ
146.22, 143.55, 128.53, 121.29, 118.12, 112.18. LC-MS: tR =
3.38 min, purity =96%. HRMS (m/z): calcd for C12H9O3Br2
(M + H)+ 358.8913; found 358.8785.
Chemistry General Procedures. All solvents and reagents
were used as received from commercial suppliers, unless noted
otherwise. 1H and 13C NMR spectra were recorded on a Bruker
AM 400 spectrometer (operating at 400 and 101 MHz
respectively) or a Bruker AVIII spectrometer (operating at
500 and 126 MHz, respectively) in CDCl3 with 0.03% TMS as
an internal standard. The chemical shifts (δ) reported are given
in parts per million (ppm) and the coupling constants (J) are in
Hertz (Hz). The spin multiplicities are reported as s = singlet, d
= doublet, t = triplet, q = quartet, dd = doublet of doublet, ddd
= doublet of doublet of doublet, dt = doublet of triplet, td =
triplet of doublet, and m = multiplet. Column chromatography
separations were performed using the Teledyne Isco Combi-
Flash Rf using RediSep Rf silica gel columns. The analytical
RPLC method used an Agilent 1200 RRLC system with UV
detection (Agilent 1200 DAD SL) and mass detection (Agilent
6224 TOF). The analytical method conditions included a
Waters Aquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and
elution with a linear gradient of 5% acetonitrile in pH 9.8
buffered aqueous ammonium formate to 100% acetonitrile at
0.4 mL/min flow rate. Automated preparative RP HPLC
purification was performed using an Agilent 1200 Mass-
Directed Fractionation system (Prep Pump G1361 with
gradient extension, makeup pump G1311A, pH modification
pump G1311A, HTS PAL autosampler, UV-DAD detection
G1315D, fraction collector G1364B, and Agilent 6120
quadrapole spectrometer G6120A). The preparative chroma-
tography conditions included a Waters X-Bridge C18 column
(19 × 150 mm, 5 um, with 19 × 10 mm guard column), elution
with a water and acetonitrile gradient, which increases 20% in
acetonitrile content over 4 min at a flow rate of 20 mL/min
(modified to pH 9.8 through addition of NH4OH by auxiliary
pump), and sample dilution in DMSO. The preparative
gradient, triggering thresholds, and UV wavelength were
selected according to the analytical RP HPLC analysis of
each crude sample. Compound purity was measured on the
basis of peak integration (area under the curve) from UV−vis
absorbance at 214 nm, and compound identity was determined
on the basis of mass spectral and NMR analyses. All
compounds had >95% purity as determined using the HPLC
methods described above.
2,2-Oxybis(4-chlorophenol) (4-6). Following the same
procedure used to synthesize 4-5, N-chlorosuccinimide was
1
used to produce 4-6 (41 mg, 38%) as an off-white solid. H
NMR (400 MHz, CDCl3): δ 7.06 (dd, J = 8.8 and 2.4 Hz, 2H),
6.99 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 2.4 Hz, 2H), 5.58 (br s,
2H). 13C NMR (126 MHz, CDCl3): δ 145.67, 143.20, 125.56,
125.44, 118.49, 117.60. LC-MS: tR = 3.30 min, purity =99%.
HRMS (m/z): calcd for C12H7O3Cl2 (M − H)− 268.9778;
found 268.9819.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H−15N HSQC spectra, isothermal titration calorimetry
1
measurements, H NMR spectra, and 13C NMR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ychen@coh.org. Phone: 626-930-5408.
Author Contributions
The manuscript was written through contributions of all
authors.
■
Notes
The authors declare the following competing financial
interest(s): Dr. Yuan Chen holds equity in SUMO Biosciences
LLC.
ACKNOWLEDGMENTS
■
The chemistry effort at the University of Kansas Specialized
Chemistry Center was supported by a grant from the NIH
Molecular Libraries Probe Production Centers Network to
́
Jeffrey Aube (PI) (U54HG005031). Support for University of
Kansas NMR instrumentation was provided by NIH Shared
Instrumentation Grant S10RR024664 and NSF Major Research
Instrumentation Grant 0320648. The authors thank Patrick
Porubsky (University of Kansas) for compound management
and Ben Neuenswander (University of Kansas) for compound
purification and high-resolution mass determination. The assay
development and HTS efforts were supported by the Molecular
Libraries grant U54 HG005033 to John C. Reed at Sanford-
Burham Medical Research Institute and by grant
1R21NS066498-01, R01GM086717, and R01GM102538 to
Yuan Chen.
Compounds SIMI-4 (Princeton Biomolecular, Inc.), 4-1
(Princeton Biomolecular, Inc.), 4-2 (Sigma-Aldrich, Inc.), and
4-3 (Sigma-Aldrich, Inc.) were purchased from commercial
sources, and, after analysis for identity and purity, were used as
purchased. Compound 4-4 was purchased from Sigma-Aldrich,
Inc., and was purified by RP HPLC to 100% purity prior to use.
2,2-Oxybis(4-bromophenol) (4-5). To a solution of 2,2-
oxidiphenol (75 mg, 0.396 mmol) and PTSA hydrate (80 mg,
0.396 mmol) in acetonitrile (8 mL) was added N-
bromosuccinimide (141 mg, 0.791 mmol). The resulting
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