Supplemental Material can be found at:

http://www.jbc.org/content/suppl/2012/05/29/M112.359505.DC1.html

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 34, pp. 28840 –28851, August 17, 2012
Published in the U.S.A.

Down-regulation of NF-␬B Transcriptional Activity in

HIV-associated Kidney Disease by BRD4 Inhibition*□ S

Received for publication, March 6, 2012, and in revised form, April 20, 2012 Published, JBC Papers in Press, May 29, 2012, DOI 10.1074/jbc.M112.359505
Guangtao Zhang‡1, Ruijie Liu§¶1, Yifei Zhong储1, Alexander N. Plotnikov‡, Weijia Zhang§, Lei Zeng‡, Elena Rusinova‡,
Guillermo Gerona-Nevarro‡, Natasha Moshkina‡, Jennifer Joshua‡, Peter Y. Chuang§, Michael Ohlmeyer‡,
John Cijiang He§¶2, and Ming-Ming Zhou‡3
From the ‡Department of Structural and Chemical Biology and §Department of Medicine, Mount Sinai School of Medicine, New
York, New York 10029, the ¶Department of Medicine, James J. Peters Veterans Affairs Medical Center, Bronx, New York 10486, and
the 储Department of Nephrology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 201203 Shanghai, China
Background: NF-␬B and BRD4 control proinflammatory gene activation in HIV-associated kidney disease.
Results: Small molecule inhibition of BRD4 binding to NF-␬B blocks target gene activation.
Conclusion: Targeting the proinflammatory activity of NF-␬B may be a new therapeutic approach.

Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
Significance: This study has broad implications as NF-␬B-mediated inflammation represents the major pathology in chronic
kidney and non-kidney diseases.

NF-␬B-mediated inflammation is the major pathology in
chronic kidney diseases, including HIV-associated nephrop-
athy (HIVAN) that ultimately progresses to end stage renal dis-
ease. HIV infection in the kidney induces NF-␬B activation,
leading to the production of proinflammatory chemokines,
cytokines, and adhesion molecules. In this study, we explored
selective inhibition of NF-␬B transcriptional activity by small
molecule blocking NF-␬B binding to the transcriptional cofac-
tor BRD4, which is required for the assembly of the productive
transcriptional complex comprising positive transcription elon-
gation factor b and RNA polymerase II. We showed that our BET
(Bromodomain and Extra-Terminal domain)-specific bro-
modomain inhibitor MS417, designed to block BRD4 binding to
the acetylated NF-␬B, effectively attenuates NF-␬B transcrip-
tional activation of proinflammatory genes in kidney cells
treated with TNF␣ or infected by HIV. MS417 ameliorates
inflammation and kidney injury in HIV-1 transgenic mice, an
animal model for HIVAN. Our study suggests that BET bro-
modomain inhibition, targeting at the proinflammatory activity
of NF-␬B, represents a new therapeutic approach for treating
NF-␬B-mediated inflammation and kidney injury in HIVAN.
Despite the decreased incidence of HIV-associated nephrop-
athy (HIVAN)4 in the antiretroviral therapy era, the prevalence
* This work was supported, in whole or in part, by National Institutes of Health
Grants HG004508 (to M.-M. Z.) and DK088541 (to J. C. H.). This work was
also supported by Veterans Affairs Merit Award 1I01BX000345 (to J. C. H.).
The atomic coordinates and structure factors (codes 2LSP and 4F3I) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The chemical shifts were deposited in BioMagResBank under code 18439.
□S
This article contains supplemental Materials and Methods, Tables 1– 4,
Figs. 1– 6, and Schemes 1 and 2.
1
These authors contributed equally to this work.
2
To whom correspondence may be addressed: 1425 Madison Ave., Box 1677,
New York, NY 10029. Fax: 212-831-0114; E-mail: cijiang.he@mssm.edu.
3
To whom correspondence may be addressed: 1425 Madison Ave., Box 1677,
New York, NY 10029. Fax: 212-849-2456; E-mail: ming-ming.zhou@mssm.edu.
4
The abbreviations used are: HIVAN, HIV-associated nephropathy; RTEC,
renal tubular epithelial cell; BET, Bromodomain and Extra-Terminal
remains high due to aging of HIV-seropositive patients (1, 2).
Many patients with HIVAN ultimately progress to end stage
renal disease despite the antiretroviral therapy treatment (3).
Other forms of renal disease are also increased in HIV-seropos-
itive patients (4). Although it remains unclear how HIV induces
a variety of kidney diseases, inflammation is the major pathol-
ogy seen in HIVAN. Tubulointerstitial inflammation, tubular
dilatation with formation of microcysts, and apoptosis of renal
tubular epithelial cells (RTECs) are prominent findings in the
kidney of HIVAN (5).
Infection of RTECs by HIV induces inflammatory response
(6, 7). Using gene expression analysis, Ross et al. (8) identified
differentially expressed genes in RTECs isolated from a HIVAN
patient after infection with vesicular stomatitis virus-pseu-
dotyped gag/pol-deleted HIV-1. The most prominent response
of these cells to HIV-1 infection was the production of proin-
flammatory mediators, including chemokines, cytokines, and
adhesion molecules. Many of these genes, as suggested by path-
way analysis of the microarray data, are mediated by transcrip-
tional activation of nuclear factor ␬B (NF-␬B).
As a master transcription factor, NF-␬B regulates expression
of a host of cellular genes in immune response to infection (9,
10). Its proinflammatory activity contributes to the etiology and
progression of many human diseases, including cancer, inflam-
matory and autoimmune diseases, and viral infection (11, 12).
NF-␬B-mediated proinflammatory response has been shown to
play a key role in the pathogenesis of kidney disease, including
diabetic nephropathy (13, 14) as well as in HIV-induced kidney
cell injury (15–17). NF-␬B also interacts with HIV-1 long ter-
minal repeat (LTR) in the kidney (18). Thus, inhibition of the
proinflammatory activity of NF-␬B may attenuate HIV-in-
domain; BrD, bromodomain; BrDi, bromodomain inhibitor; p-TEFb, posi-
tive transcription elongation factor b; CBP, CREB-binding protein; CREB,
cAMP-response element-binding protein; RMA, robust multiarray analysis;
ITC, isothermal titration calorimetry; ISRE, interferon-sensitive response
element; PCAF, p300/CBP-associated factor; K310ac, lysine 310-acetylated;
H3K9ac and H3K18ac, acetylation at histone H3 lysine 9 and 18, respec-
tively; H3K4me3 and H3K27me3, trimethylation at H3 lysine 4 and 27,
respectively.

28840 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 34 • AUGUST 17, 2012

duced inflammation and kidney injury. Given its protective role
in cell immune response to infection, however, a complete inhi-
bition of NF-␬B might lead to adverse effects. A selective and
temporal inhibition of NF-␬B activity would maximize its ben-
eficial effects.
It has been shown that NF-␬B transcriptional activity is
dependent upon its acetylation at lysine 310 and that Lys-310-
acetylated NF-␬B recruits the BET protein BRD4 in complex
with positive transcription elongation factor b (p-TEFb) (con-
sisting of CDK9 and cyclin T1) and RNA polymerase II that
together form a productive transcriptional machinery complex
(9). A recent study has reported that pharmacological treat-
ment of bone marrow-derived macrophages with a BET bro-
modomain inhibitor (BrDi) blocks the innate immune response
gene expression induced by lipopolysaccharide (LPS) (19). In
this study, we sought to determine how a BET-specific BrD
inhibitor might influence NF-␬B transcriptional activity in con-
trol of the expression of proinflammatory genes in HIV-in-
fected kidney cells and whether and how BrDi might attenuate
kidney injury in HIV-1 transgenic mice (Tg26), which is an
established animal model for HIVAN. Our study shows that
BET bromodomain inhibition could represent an attractive
therapeutic strategy for treating patients with HIVAN.
EXPERIMENTAL PROCEDURES
Sample Preparation—Expression and purification of the
recombinant bromodomains from ASH1L, ATAD2, BAZ1B,
BAZ2B, BPTF, BRD3, BRD4, BRD7, CBP, PCAF, PHIP-1,
TAF1, TAF1L, and SMARCA4 in poly-His tag form were per-
formed using a procedure described previously (20). Isotope-
labeled protein samples of the BrDs were prepared from cells
grown on a minimal medium containing 15NH4Cl with or with-
out 13C6-glucose in either H2O or 75% 2H2O. The protein was
purified by affinity chromatography on a nickel-IDA column
(Invitrogen), followed by the removal of poly-His tag by throm-
bin cleavage.
Protein Structure Determination by NMR—The NMR data
collection, analysis, and structure determination of the BRD4-
BD2䡠NF-␬B-K310ac peptide complex were performed using
the procedures previously reported (20). Briefly, NMR samples
contained a protein䡠ligand complex of 0.5 mM in a 100 mM
phosphate buffer, pH 6.5, that contains 5 mM perdeuterated
DTT and 0.5 mM EDTA in H2O/2H2O (9:1) or 2H2O. All NMR
spectra were collected at 30 °C on NMR spectrometers of 800,
600, or 500 MHz. The 1H, 13C, and 15N resonances of a protein
of the complex were assigned by triple-resonance NMR spectra
collected with a 13C/15N-labeled and 75% deuterated protein
bound to an unlabeled ligand (21). The distance restraints were
obtained in three-dimensional 13C or 15N NOESY spectra.
Slowly exchanging amides, identified in two-dimensional 15N
HSQC spectra recorded after an H2O buffer was changed to a
2
H2O buffer, were used with structures calculated with only
NOE distance restraints to generate hydrogen bond restraints
for final structure calculations. The intermolecular NOEs were
detected in the 13C-edited (F1), 13C/15N-filtered (F3) three-di-
mensional NOESY spectrum. Protein structures were calcu-
lated with a distance geometry-simulated annealing protocol
with X-PLOR (22). Initial structure calculations were per-
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
Transcriptional Inhibition of NF-␬B in HIVAN
formed with manually assigned NOE-derived distance re-
straints. Hydrogen bond distance restraints, generated from the
hydrogen/deuterium exchange data, were added at a later stage
of structure calculations for residues with characteristic NOEs.
The converged structures were used for iterative automated
NOE assignment by ARIA for refinement (23). Structure qual-
ity was assessed by Procheck-NMR (24). The structure of the
protein䡠ligand complex was determined using intermolecular
NOE-derived distance restraints.
Isothermal Titration Calorimetry—Experiments were car-
ried out on a GE MicroCal auto-ITC200 instrument at 15 °C
while stirring at 1,000 rpm in the isothermal titration calorim-
etry (ITC) buffer, pH 7.4, consisting of 50 mM sodium phos-
phate and 150 mM sodium chloride. Compound concentration
was determined by NMR, and protein concentrations were
determined by A280 measurements. The compound sample
Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
(40 –50 ␮M) was placed in the cell, whereas the microsyringe
(130 ␮l) was loaded with a solution of the correspondent pro-
tein sample (0.6 ␮M) in the ITC buffer. Injections were per-
formed using protein in the syringe rather than the compound
to overcome limitations of compound solubility. All titrations
were conducted using 20 identical injections of 2 ␮l with a dura-
tion of 4 s/injection and a spacing of 150 s between injections.
The heat of dilution was determined by independent titrations
(protein into buffer) and was subtracted from the experimental
data. The collected data were implicated in the MicroCalTM
Origin software supplied with the instrument to yield enthalp-
ies of binding (⌬H) and binding constants (KB). Thermody-
namic parameters were calculated (⌬G ⫽ ⌬H ⫺ T⌬S ⫽
⫺RTlnKB, where ⌬G, ⌬H, and ⌬S are the changes in free
energy, enthalpy, and entropy of binding, respectively). In all
cases, a single binding site model was employed.
Fluorescence Anisotropy Binding Assay—The dissociation
constant, Kd, for various BrD proteins to a fluorescein isothio-
cyanate (FITC)-labeled MS417 (named as MS574, or com-
pound 9; see Schemes 1 and 2 in supplemental Materials and
Methods) was measured in direct binding experiments using
fluorescence anisotropy as a readout. The fluorescence anisot-
ropy assay was carried out in polypropylene 96-well plates
(Costar) with 80 nM FITC-MS417 and varying concentrations
of a BrD protein in a PBS buffer (pH 7.4) in a total volume of 80
␮l. Measurements were obtained after a 1-h incubation of the
fluorescent ligand and the protein at 25 °C with a Safire 2
microplate reader (Tecan). The dissociation constant was
determined by using the following one-site model equation
using Prism software,
共 K d ⫹ P T ⫹ L T 兲 ⫺ 冑共 K d ⫹ P T ⫹ L T 兲 2 ⫺ 4P T L T
Fb ⫽
2L T
(Eq. 1)
where Fb represents the fraction of bound labeled ligand, Kd is
the dissociation constant, PT is total protein concentration, and
LT is total fluorescent ligand concentration. Competition
experiments were performed with a BrD protein (0.25–1 ␮M)
and the fluorescent probe (80 nM) and increasing concentra-
tions of unlabeled competing ligand. In a competition-binding
assay, fluorescent ligand concentration was ⱕ2Kd, and protein
JOURNAL OF BIOLOGICAL CHEMISTRY 28841
Transcriptional Inhibition of NF-␬B in HIVAN
concentration was set at which 50 – 80% of fluorescent ligand is
bound. Dissociation constant of a competing ligand was calcu-
lated with the correction to the Cheng-Prussoff equation intro-
duced by Nicolovska-Coleska et al. (25). Assuming a one-site
competitive binding model, the following equation was used to
calculate Ki values from IC50 values recovered from fitting data
using Prism,
关 I 50 兴
冉 冊
Ki ⫽ (Eq. 2)
关 L 50 兴 关 P 0 兴
⫹ ⫹1
Kd Kd
where [I50] represents the concentration of free inhibitor at 50%
inhibition, [L50] is the concentration of free labeled ligand at
50% inhibition, and [P0] is the concentration of free protein at
0% inhibition. Note that Kd for each protein-probe pair is the
limit of resolvable Ki in a competition assay.
Protein Crystallization and X-ray Diffraction Data Collection—
Purified BRD4-BD1 protein (16 mg/ml) was mixed with MS417
at a 1:10 molar ratio of protein/ligand. The complex was crys-
tallized using the sitting drop vapor diffusion method at 20 °C
by mixing 1 ␮l of protein solution with 1 ␮l of the reservoir
solution that contains 25% PEG 3350, 0.2 M Li2SO4, and 0.1 M
Tris-HCl, pH 8.5. Crystals were soaked in the corresponding
mother liquor supplemented with 20% ethylene glycerol as
cryoprotectant before freezing in liquid nitrogen. X-ray diffrac-
tion data were collected at 100 K at beamline X6A of the
National Synchrotron Light Source at Brookhaven National
Laboratory. Data were processed using the HKL-2000 suite
(26). The BRD4-BD1 structure was solved by molecular
replacement using the program MOLREP (27), and the struc-
ture refinement was done using the program Refmac (28). The
graphics program COOT (29) was used for model building and
visualization. Crystal diffraction data and refinement statistics
for the structure are displayed in supplemental Table 2.
Cell Cultures—Primary human RTECs were obtained from
Promocell and cultured according to the manufacturer’s proto-
col. RTECs were incubated with either MS417 or DMSO (0.1%)
as the control. Cells were also stimulated with TNF␣ (10 ng/ml)
to determine NF-␬B activation. Cells were also infected with
HIV or control vector as described below.
HIV Infection—For HIV infection, pNL4 –3⌬G/P-EGFP, a
gag/pol-deleted HIV-1 construct that contains EGFP in the gag
open reading frame (HIV vector), and pHR⬘-IRES-EGFP (GFP
control vector) were used to generate vesicular stomatitis virus-
G-pseudotyped virus for infection of RTECs as described pre-
viously (8).
Western Blot—RTECs or kidney tissues were lysed with a
buffer containing 1% Nonidet P-40, a protease inhibitor mix-
ture (Fisher 78443), and acetylation inhibitors of 10 ␮M tricho-
statin A (Sigma T1952) and 5 mM nicotinamide (Sigma 72340).
After protein concentration determination, protein lysates
were subjected to Western blot analysis using specific antibod-
ies for acetylated p65 or total p65 (Cell Signaling 3045). ␤-Actin
(Sigma) was used as the control. Western blot was also per-
formed for HIV Nef (Abcam 42355) in RTECs and kidney tis-
sues of mice.
28842 JOURNAL OF BIOLOGICAL CHEMISTRY
Reporter Gene Assay—The PGL-2 reporter vector containing
the promoter of NF-␬B or ISRE (gifts from Dr. Huabao Xiong)
was used. 293T cells were transfected with luciferase expression
plasmids (1 ␮g) using Lipofectamine 2000 (Invitrogen). 48 h
after transfection, the cells were rinsed and cultured in serum-
free medium containing TNF␣ (10 ng/ml) for 24 h. The cells
were then lysed in luciferase assay buffer (Promega luciferase
assay system). Luciferase was measured in a luminometer, and
the data were analyzed after normalization for protein amount.
PCR Array and Microarray Studies—The PCR array for the
Inflammatory Cytokines & Receptors PCR Array (PAHS-011,
Qiagen) was used to examine the expression of NF-␬B target
genes. Human RETCs were treated with MS417 at the indicated
concentrations for 24 h, and total RNA was extracted. PCR
arrays were performed according to the manufacturer’s
instructions. -Fold changes for each gene were calculated by the
Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
2⫺⌬⌬CT method compared with GADPH. Human RTECs were
infected with HIV pseudotyped virus or control vector with
GFP for 4 days and then treated with either DMSO or MS417 at
1.0 ␮M for an additional 24 h. Total RNA was extracted from
cells using the RNeasy minikit (Qiagen). Affymetrix gene
expression microarrays were performed at the Mount Sinai
Microarray Core Facility. Data were analyzed by t test con-
trolled with false discovery rate, n ⫽ 3. Data were expressed as
mean ⫾ S.D. The unpaired t test was used to analyze data
between two groups. Statistical significance was considered to
be when p was ⬍0.05.
Bioinformatics Analysis of Microarray Data—The raw inten-
sity data were normalized across the chips by the log scale
robust multiarray analysis (RMA) method (30, 31), and the
quality control of each chip data was performed by investigat-
ing overall intensity values for all probes and negative and pos-
itive controls. The probe sets with low intensity values or out-
liers due to poor RNA quality or hybridization were excluded
for further analysis. Significance Analysis of Microarray (SAM,
version 2.1) (32) was used to identify significantly differentially
expressed genes between groups (i.e. untreated versus BrD
inhibitor-treated groups). SAM, a widely used microarray dif-
ferential analysis tool, correlates gene expression data to a wide
variety of clinical parameters and uses permutation to estimate
the false discovery rate for multiple testing. The cut-off of
the false discovery rate q value of 0.05 was applied to determine
differentially expressed genes. The differentially expressed
genes were queried against a well curated and experimentally
verified NF-␬B target database (see the Boston University Biol-
ogy Web site) to identify NF-␬B targets whose transcriptional
expression was altered by HIV infection or the BrD inhibitor.
The enrichment significance of NF-␬B targets was calculated
by comparing the percentage of NF-␬B targets in differentially
expressed gene with that of NF-␬B targets in the whole gene list
using Fisher’s exact test. To understand how other genes were
regulated by NF-␬B targets upon HIV infection and BrDi, the
genes that were up-regulated by HIV and subsequently down-
regulated by BrDi or vice versa were identified and then sub-
jected to association network analysis with the NF-␬B target by
the Ingenuity System (available on the World Wide Web). The
significant biological functions of these genes were also deter-
mined by DAVID (33).
VOLUME 287 • NUMBER 34 • AUGUST 17, 2012

Chromatin Immunoprecipitation (ChIP)—Human RTECs
were infected with HIV or GFP control vector for 72 h. The cells
were then starved for 6 h and treated with DMSO or MS417 at
1 ␮M for 24 h in serum-free medium. Genomic DNA was
precipitated, and ChIP was performed according to the man-
ufacturer’s protocol (Millipore). The following antibodies
from Cell Signaling were used: anti-H3K9ac, anti-H3K18ac,
anti-H3K4me3, and anti-H3K27me3 antibodies. Immuno-
precipitated DNA was quantified by real-time PCR using the
following primer sequences: CCL2-F, 5⬘-CCGGCCAA-
AGCTTGAGAGCTCC-3⬘; CCL2-R, 5⬘-GTCAGGCTCGCT-
GCCAGCTTAC-3⬘; CCL20-F, 5⬘-CCCCATGTGGCAACAC-
GCCTT-3⬘; CCL20-R, 5⬘-GTACACAGAAGGCGTGTTGCC-
ACAT-3⬘; CXCL5-F, 5⬘-CACCCTCCCCACCAGTTCCCA-
3⬘; CXCL5-R, 5⬘-CTCATCTCTCCCCACTGACAGGAA-3⬘;
TGM2-F, 5⬘-GCCGCCGTCCCTCCCTCGGG-3⬘; TGM2-R,
5⬘-AGCCCGCTTTGGGGCGG-3⬘.
All experiments were repeated three times. -Fold changes to
the control are shown.
In Vivo Animal Model Study—As a well established trans-
genic model of HIV-associated nephropathy, the HIV-1 trans-
genic mouse (Tg26) bears a replication-defective form of the
HIV-1 provirus lacking 3 kilobases of sequence overlapping
the gag and pol sequences. The transgene is under the control
of the viral LTR promoter and is expressed in most tissues (34).
The Tg26 line is maintained as a heterozygous line as homozy-
gous mice die prior to the age of breeding. Heterozygous mice
appeared normal at birth but then develop wasting, heavy pro-
teinuria, edema, hypoalbuminemia, and hypercholesterolemia
between 4 and 8 months of age based on different penetration.
Blood urea nitrogen is significantly elevated in these mice, and
mice die probably from uremia (35). Pathologically, kidneys
from animals with renal failure demonstrate diffuse segmental
and global glomerulosclerosis, microcystic tubular dilatation
with a monocytic interstitial infiltrate, and minimal interstitial
fibrosis remarkably similar to HIVAN (35). Apart from kidney
disease, these animals develop significant skin edema and cat-
aract. However, these animals do not exhibit any other organ
damage. For the in vivo animal model study, Tg26 mice and
their corresponding littermates (n ⫽ 6/group, including three
male and three female mice in each group) were fed with either
control vehicle (DMSO) or MS417 by daily gavage at a concen-
tration of 0.08 mg/kg. The mice were fed with this compound
daily from the age of 4 weeks to 8 weeks. Unrestricted food and
water were provided throughout the duration of the experi-
ment. The mice were euthanized at 8 weeks of age for blood,
urine, and tissue collection by exposure to carbon monoxide.
Body weight was recorded. All animal studies were performed
according to the protocols approved by the Institutional Ani-
mal Care and Use Committee at the Mount Sinai School of
Medicine.
Measurement of Urine Protein and Creatinine—Urine albu-
min was quantified by ELISA using a kit from Bethyl Laboratory
Inc. (Houston, TX). Urine creatinine levels were measured in
the same samples using the urinary Creatinine Assay Kit (Cay-
man Chemical, Ann Arbor MI) according to the manufacturer’s
instructions. The urine albumin excretion rate was expressed as
the ratio of albumin to creatinine.
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
Transcriptional Inhibition of NF-␬B in HIVAN
Quantitative Histopathology—Mice were perfused with PBS
containing 4% paraformaldehyde, and kidneys were further
fixed in 4% paraformaldehyde for 2 h. Kidney tissue was embed-
ded into paraffin by American Histolabs, Inc. (Gaithersburg,
MD). Kidney histology was examined after periodic acid-Schiff
staining. Glomerulosclerosis was scored as described previ-
ously by Dr. D’Agati (36). Briefly, each specimen received a
score for three parameters: percentage of collapsing glomerular
sclerosis, percentage of tubular cysts or casts, and podocyte
hypertrophy. The percentage of collapsing glomerulosclerosis
was obtained by identifying the total number of glomeruli with
any sclerosis and dividing this number by the total number of
glomeruli seen. The percentage of tubular cysts or casts score
was obtained by the number of tubules with either microcystic
dilatation or filled with casts divided by the total number of
tubular cross-sections in a representative area. Finally, the
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degree of podocyte hypertrophy was scored as 0 (absent), 1⫹
(podocyte hypertrophy observed in ⬍25% of all glomeruli), 2⫹
(podocyte hypertrophy observed in 25–50% of all glomeruli),
and 3⫹ (podocyte hypertrophy in ⬎50% of all glomeruli).
Real-time PCR—Total RNA was isolated from human
RTECs or kidney cortex of mice using TRIzol (Invitrogen).
Real-time PCR was performed with a Roche Applied
Science Lightcycler and Qiagen QuantiTect One Step RT-
PCR SYBR Green kit (Qiagen) according to the manufactur-
er’s instructions. Predesigned primer sets were obtained
from Qiagen (GeneGlobe). The sequences of RT-PCR primers
were as follows: HsFAS-F, 5⬘-TATCACCACTATTGC-
TGGAGTCA-3⬘; HsFAS-R, 5⬘-GCTGTGTCTTGGACATTG-
TCA-3⬘; HsTRAF1-F, 5⬘-CCGGCCCCTGATGAGAATG-
3⬘; HsTRAF1-R, 5⬘-TTCCTGGGCTTATAGACTGGAG-
3⬘; MmCcl2-F, 5⬘-TTAAAAACCTGGATCGGAACCAA-
3⬘; MmCcl2-R, 5⬘-GCATTAGCTTCAGATTTACG-
GGT-3⬘; MmCcl5-F, 5⬘-GCTGCTTTGCCTACCTC-
TCC-3⬘; MmCcl5-R, 5⬘-TCGAGTGACAAACACGAC-
TGC-3⬘; MmCcl20-F, 5⬘-GCCTCTCGTACATACAGA-
CGC-3⬘; MmCcl20-R, 5⬘-CCAGTTCTGCTTTGGATC-
AGC-3⬘; MmTlr2-F, 5⬘-GCAAACGCTGTTCTGCT-
CAG-3⬘; MmTlr2-R, 5⬘-AGGCGTCTCCCTCTATTGT-
ATT-3⬘; MmTlr9-F, 5⬘-ATGGTTCTCCGTCGAAGG-
ACT-3⬘; MnTlr9-R, 5⬘-GAGGCTTCAGCTCACAGGG-
3⬘; HsGADPH-F, 5⬘-AATTGAGCCCCGCAGCCT-
CCC-3⬘; HsGADPH-R, 5⬘-CCAGGCGCCCAATACGACCA-
3⬘; MmGadph-F, 5⬘-GCCATCCAACGAACCCCTT-
CAT-3⬘; MmGadph-R, 5⬘-ATGATGACCCGTTTGG-
CTCC-3⬘.
Light cycler analysis software was used to determine crossing
points using the second derivative method. Data were normal-
ized to housekeeping genes (tubulin) and presented as -fold
increase compared with RNA isolated from WT animals using
the 2⫺⌬⌬CT method.
RESULTS
Molecular Basis of BRD4 Binding to Lysine 310-acetylated
NF-␬B—To determine the molecular basis of lysine acetyla-
tion-dependent NF-␬B association with BRD4, we first charac-
terized binding of the two bromodomains of BRD4 to a Lys-
310-acetylated NF-␬B peptide (NF-␬B-K310ac) by NMR
JOURNAL OF BIOLOGICAL CHEMISTRY 28843
Transcriptional Inhibition of NF-␬B in HIVAN
FIGURE 1. Structural basis of K310ac NF-␬B recognition by BRD4. A, two-dimensional 1H-15N HSQC spectra of BrDs of BRD4, CBP, and PCAF in the free form
(black) and in the presence of a K310ac NF-␬B peptide (red). Shown are ribbon (B) and surface-filled representations (C) of the three-dimensional solution
structure of the BRD4-BD2 bound to the NF-␬B-K310ac peptide. Side chains of key residues involved in intermolecular interactions are highlighted and
color-coded by atom types.
titration. As shown in two-dimensional 1H-15N HSQC spectra
(Fig. 1A), the second bromodomain of BRD4 (BRD4-BD2)
exhibited much more extended chemical shift perturbations
upon binding to the NF-␬B-K310ac peptide than that by the
BRD4-BD1 or the BrDs of transcriptional co-activators CBP
and PCAF, indicating a stronger interaction with the former.
The differential NF-␬B-K310ac binding by the bromodomains
of BRD4 was further confirmed in a fluorescence anisotropy
competition binding study using a fluorescein-labeled bro-
modomain inhibitor as an assay probe (see below), yielding a Ki
of 274 ␮M and ⬎2,000 ␮M for BRD4-BD2 and BRD4-BD1,
respectively. We further observed that BRD4-BD1 or BD2
binds only very weakly if at all to other reported acetylation sites
in NF-␬B, including Lys-314, Lys-315, Lys-122, Lys-123, Lys-
218, and Lys-221 in RelA-p65 and Lys-431, Lys-440, and Lys-
441 in p50.
We next solved the three-dimensional structure of BRD4-
BD2 bound to the NF-␬B-K310ac peptide by using NMR spec-
troscopy (supplemental Fig. 1 and Table 1), which explains the
molecular basis of this selective interaction (Fig. 1, B and C). As
shown in the structure of the BRD4-BD2䡠NF-␬B-K310ac com-
plex, the NF-␬B-K310ac peptide is bound in the protein across
an elongated cavity formed between the ZA and BC loops of this
left-handed four-helical bundle structure. Specifically, the
acetylated Lys-310 forms a hydrogen bond between its carbonyl
oxygen and the side-chain nitrogen of the conserved Asn-433.
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In addition, Tyr-306 of NF-␬B establishes aromatic and hydro-
phobic interactions with side-chain methyl groups of Ile-393,
whereas Phe-309 binds to His-437, and Ile-312 is sandwiched
between His-437 and Trp-374. Notably, in the conserved acetyl-
lysine binding pocket, His-437 in BRD4-BD2 is unique, which cor-
responds to Asp-144 in BRD4-BD1. The latter cannot favorably
interact with Phe-309 or Ile-312 of the NF-␬B peptide, explaining
markedly reduced binding of BRD4-BD1 to the K310ac site. The
observation of direct recognition of Phe-309 (Kac⫺1) and Ile-312
(Kac⫹2) by the BRD4-BD2 explains why the K310ac site is
selected over the other acetylation sites in NF-␬B.
Blocking BRD4/NF-␬B Association by a BET-specific BrD
Inhibitor—To characterize the functional importance of acety-
lation-mediated BRD4/NF-␬B binding in cells, we synthesized
a small molecule bromodomain inhibitor MS417 (see supple-
mental Materials and Methods), which belongs to a group of
thienodiazepine-based compounds that was first reported for
their binding activity for BRD4 by the Mitsubishi Tanabe
Pharma Corporation (37). MS417 shares the same thieno-tria-
zolo-1,4-diazepine scaffold as a recently reported BET BrDi
(⫹)-JQ1 (38) and is structurally related to I-BET (19, 39). The
main difference between MS417 and (⫹)-JQ1 is that the former
consists of a methyl ester, whereas the latter is substituted with
a t-butyl ester moiety. Our 1.4 Å resolution crystal structure of
the BRD4-BD1䡠MS417 complex (Fig. 2A and supplemental Fig.
2A and Table 2) reveals that MS417 is embedded in the acetyl-
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FIGURE 2. Molecular basis of MS417 binding to the BRD4 bromodomains. A, x-ray crystal structure of MS417 bound to BRD4-BD1, depicted in a stereo view
(left) and surface-filled representation (right). Side chains of key protein residues involved in ligand recognition are color-coded by atom types. The chemical
structures of MS417 and its enantiomer MS566 are shown. B, ITC measurement of BRD4-BD1 binding MS417. C, two-dimensional 1H-15N HSQC spectra of
BRD4-BD1 in the free form (black) and in the presence of the BrDi MS417 with a protein/ligand molar ratio of 1:0.5 (green) and 1:1 (red). D, affinity measurements
of MS417 or its enantiomer MS566 binding to the BrDs of BRD4, BRD3, and CBP, as assessed in a fluorescence anisotropy competition assay using an
FITC-labeled MS417 (i.e. MS574) as an assay probe.

lysine binding pocket, forming a hydrogen bond between the
triazole ring and the conserved Asn-140 in BRD4-BD1. Nota-
bly, the methyl ester interacts with Leu-95, Tyr-97, and Tyr-139
in a small hydrophobic cavity formed between the ZA and BC
loops. The corresponding t-butyl group in (⫹)-JQ1 is too big to
fit in this cavity such that it is rotated 180° with respect to the
methyl ester in MS417, projecting outward on the protein sur-
face (supplemental Fig. 2B). Similar to (⫹)-JQ1, MS417 is not in
contact with Asp-144, which is unique in BRD4-BD1 (corre-
sponding to His-437 in BRD4-BD2). This explains why MS417
binds BRD4-BD1 and BRD4-BD2 with similar affinity of Kd ⫽
36.1 ⫾ 7.8 and 25.4 ⫾ 3.4 nM, respectively, as determined by
isothermal titration calorimetry measurement (Fig. 2B). Nota-
bly, the tight binding of MS417 by the BRD4 bromodomains
was confirmed by simultaneous observation of both the free
and MS417-bound NMR resonances of the protein in the pres-
ence of substoichiometry concentrations of the ligand as those
illustrated in two-dimensional 1H-15N HSQC spectra of 15N-
labeled BRD4-BD1 (Fig. 2C). This is due to slow exchange of the
protein conformations between the free and ligand-bound
states on the NMR time scale, which is characteristic of submi-
cromolar affinity of the binding ligand. Further, the structure
explains that the BRD4-BD1 prefers MS417 over its enantiomer
(i.e. MS566, with R-configuration of the methyl ester as sup-
posed to S-configuration in MS417) (Fig. 2D and supplemental
Fig. 6 and Table 4) because the methyl ester moiety in the latter
would have steric clash with the protein residues, such as Leu-
92, Leu-95, and Tyr-139. Finally, we demonstrated in a fluores-
cence anisotropy binding assay using a fluorescein-conjugated-
MS417 as an assay probe that MS417 is highly specific for the
BET bromodomains, and it binds to CBP, BRD7, or BPTF with
a 100 –200-fold weaker affinity or does not bind at all to many
other bromodomains tested, including BAZ1B, BAZ2B,
ATAD2, ASH1L, SMARCA4, PCAF, PHIP-1, TAF1L, and
TAF1 (Fig. 2D and supplemental Fig. 3).
We next evaluated the ability of MS417 to modulate NF-␬B
transcription activity in cells. Using a luciferase reporter gene
assay, we found that MS417 suppressed TNF␣-induced NF-␬B
transcription activation in human embryonic kidney 293T cells
in a dose-dependent manner, and a nearly complete suppres-
sion of the NF-␬B activation was observed at 1.0 ␮M MS417
(Fig. 3A). Little effect was seen on ISRE as a negative control.
The inhibitory effect of MS417 on NF-␬B activity correlates
with a dose-dependent reduction of NF-␬B acetylation (Fig.

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Transcriptional Inhibition of NF-␬B in HIVAN

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FIGURE 3. Modulation of BRD4 binding to K310ac NF-␬B. A, effects of MS417 on TNF␣-induced NF-␬B activation in 293T cells, as measured by a NF-␬B
luciferase reporter assay. 293T cells were transfected with a NF-␬B luciferase reporter vector for 2 days and then incubated with MS417 at the indicated doses
in serum-free medium with or without TNF␣ for 24 h. As a negative control, the cells were also transfected with an ISRE luciferase reporter vector and treated
with MS417 and TNF␣ in the same condition as above. The cells were harvested for determination of luciferase activity (n ⫽ 5; **, p ⬍ 0.01). B, Western blot
analysis assessing effects of MS417 on acetylated p65 in 293T cells. Representative Western blots of three independent experiments are shown. C, effects of
MS417 on NF-␬B activation by HIV pseudovirus infection in human RTECs. RTECs were infected with HIV pseudovirus or control GFP vector for 3 days and then
incubated with MS417 at the indicated doses in serum-free medium for 24 h. The cells were harvested for determination of acetylated p65 (p65-Kac), p65, and
␤-actin as well as HIV Nef protein by Western blot. Representative Western blots of four independent experiments are shown. The densitometric analysis of the
Western blots in B and C was performed and the ratio of acetylated p65 to total p65 was calculated, *, p ⬍ 0.05; n ⫽ 4 (lower panels of Fig. 3, B and C).

3B). We next evaluated the NF-␬B-inhibitory effects of MS417
in human primary RTECs that were infected with HIV pseudo-
virus or control GFP vector for 3 days followed by stimulation
with MS417 in a serum-free medium for an additional 24 h. The
Western blot analysis showed a similar inhibitory effect of
MS417 on reducing NF-␬B p65 acetylation in the HIV-infected
RTECs as compared with TNF␣-treated RTECs (Fig. 3C).
These results suggest that MS417 can effectively suppress the
NF-␬B function through inhibition of its acetylation and tran-
scriptional activation.
MS417 Down-regulates Genes That Were Up-regulated in
HIV-infected RTECs—To gain mechanistic insights into the
role of NF-␬B/BRD4 binding in HIV-induced chronic inflam-
mation in the kidney cells, we infected human RTECs with HIV
pseudovirus for 4 days and then treated the cells with MS417
(1.0 ␮M) or DMSO for an additional 24 h. We then performed
Affymatrix microarrays to characterize the effects of MS417 on
genome-wide gene expression profiles of RTECs. Of 33,298
probe sets on the Human Gene 1.0 ST Array chip, gene expres-
sion significance analysis between groups by SAM showed that
MS417 treatment significantly altered the transcriptional
expression of 326 and 537 genes in the RTECs that were
infected with either HIV or control GFP vector, respectively
(with 2-fold cut-off, q ⬍ 0.05). To understand the functional
relevance of these differentially expressed genes, we performed
Ingenuity pathway analysis, which revealed several gene net-
works enriched in MS417-treated RTECs with or without HIV
infection (Fig. 4A). In particular, genes related to inflammation,
oxidative stress, and IL-17 proinflammatory pathways were
among those highly influenced by MS417. Notably, of a set of
134 genes that were up-regulated by HIV infection and then
down-regulated by MS417 are 11 NF-␬B target genes, which
were significantly enriched in this differentially expressed gene
set over those in the whole gene list (see below; Fig. 4B).
To understand how MS417 modulates NF-␬B transcrip-
tional activity in the RTECs, we analyzed these NF-␬B target
genes. As shown in supplemental Fig. 4A, NF-␬B target genes
were significantly enriched among MS417 down-regulated
genes in GFP or HIV-infected RTECs and HIV-up-regulated
genes. As illustrated by Venn diagram (supplemental Fig. 4A),
MS417 down-regulated 49 NF-␬B target genes in the HIV-in-
fected RTECs and 69 in the control, of which 38 are common.
Most notably, MS417 down-regulated 11 of the 30 NF-␬B genes
in RTECs that were up-regulated by HIV infection. On the
other hand, of the 14 and 15 genes up-regulated by MS417 in
the RTECs infected with or without HIV, respectively, 13 are
common, and none of the 18 HIV down-regulated NF-␬B tar-
gets was up-regulated by MS417 in the HIV-infected RTECs.
Given the low enrichment fold of these latter gene sets, we
focused our study on these 11 NF-␬B target genes, which were

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FIGURE 4. MS417 Modulation of gene transcription in HIV-infected human primary renal tubular epithelial cells. A, Ingenuity pathway analysis revealing
the top gene networks enriched in the down-regulated genes by MS417 treatment in the HIV-infected human RTECs. The RTECs were infected with control GFP
vector or HIV vector for 4 days and then treated with DMSO or MS417 (1.0 ␮M) for an additional 24 h. RNA was isolated from these cells for microarray studies.
B, gene heat-map of 11 NF-␬B targets that were both up-regulated by HIV infection and down-regulated by the MS417 treatment. C, MS417 inhibits NF-␬B
target gene activation by HIV infection in human RTECs. RTECs were infected with GFP or HIV for 72 h, and then the cells were starved for 6 h and treated with
DMSO control or MS417 (1.0 ␮M) for an additional 24 h. The cells were harvested for real-time PCR analysis of cytokine and chemokine (n ⫽ 3; *, p ⬍ 0.01 when
compared between HIV-infected cells treated with DMSO and MS417). D, some of the NF-␬B target genes by HIV infection in the RTECs were not inhibited by
MS417. E, chromatin immunoprecipitation analysis of the MS417-sensitive and non-sensitive genes probing changes of the corresponding histone modifica-
tions, such as H3K9ac, H3K18ac, H3K4me3, and H3K27me3, at the promoter sites of these genes (n ⫽ 3; *, p ⬍ 0.01 when compared between HIV-infected cells
treated with DMSO and MS417).

AUGUST 17, 2012 • VOLUME 287 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 28847
Transcriptional Inhibition of NF-␬B in HIVAN
up-regulated by HIV and down-regulated by MS417 treatment,
as illustrated in a gene expression heat map (Fig. 4B). Among
these genes are well known NF-␬B-dependent cytokine and
chemokine genes, such as CCL2, CCL20, and IL-8, which are
known to be up-regulated in RTECs by HIV infection as shown
previously (8). These genes are at the core of a well interlinked
expression network of genes centered on NF-␬B that were
mostly up-regulated upon HIV infection and reversely affected
by subsequent MS417 treatment (supplemental Fig. 4B). Col-
lectively, we concluded from our microarray data that MS417
inhibits the expression of the inflammation-related genes in
human RTECs, induced by HIV infection.
MS417 Suppresses NF-␬B-targeted Cytokines and Che-
mokines—We confirmed with RT-PCR analysis that MS417
suppressed the HIV-induced transcriptional activation of
NF-␬B-targeted proinflammatory chemokine genes, such as
CCL2 and CCL20, in human RTECs (Fig. 4C). Notably, several
genes, such as CXCL5 and TGM2, that underwent HIV-in-
duced transcriptional activation were not affected significantly
by the MS417 treatment in the RTECs (Fig. 4D). We further
performed a ChIP study to determine effects of MS417 on chro-
matin modifications at the promoter site of these cytokine and
chemokine genes. For the MS417-sensitive genes (i.e. CCL2 and
CCL20), we observed a markedly increase of acetylation at his-
tone H3 lysine 9 (H3K9ac) and lysine 18 (H3K18ac) and trim-
ethylation at H3 lysine 4 (H3K4me3) and a concomitant
decrease in trimethylation at H3 lysine 27 (H3K27me3) at their
promoter site due to HIV infection and then a dramatic
decrease of H3K9ac, H3K18ac, and H3K4me3 as well as an
increase of H3K27me3 upon the MS417 treatment (Fig. 4E). On
the other hand, for the MS417-insensitive genes CXCL5 and
TGM2, we detected similar histone modification changes upon
HIV infection (i.e. an increase of activation marks (i.e. H3K9ac,
H3K18ac, and H3K4me3) and a decrease of repression marks
(H3K27me3). However, very small changes if any, particularly
for H3K4me3 and H3K27me3, were observed upon the MS417
treatment. These results are consistent with the effects of
MS417 on transcriptional activation of these genes. In addition,
by PCR array, we observed that MS417 suppressed TNF␣-in-
duced transcriptional activation of proinflammatory cytokines
and chemokines in human RTECs, including IL1A, IL1B, LTA,
LTB, CCL2, CCL3, CCL20, CXCL3, and CXCL11 (supplemental
Fig. 5A). Interestingly, several genes, including CXCL5 and
CXCL6, were not affected significantly by the MS417 treatment
(supplemental Fig. 5B). Furthermore, we found that MS417
inhibited expression of proapoptotic genes targeted by NF-␬B
in the RTECs, such as BCL2 (Fig. 4B) and FAS and TRAF1
(supplemental Fig. 5C). Because RTEC apoptosis and inflam-
mation are the major pathogenic mechanisms in HIVAN as
well as in other kidney disease (16, 40 – 42), our findings suggest
that the BRD4 BrD inhibitor may function as an anti-inflamma-
tory agent to reduce inflammation and/or RTEC apoptosis in
kidney disease.
MS417 Attenuates Proteinuria and Kidney Injury in HIV-1
Transgenic Mice (Tg26)—Given that MS417 can block the
expression of both primary response genes (i.e. CCL2 and
CCL3) and secondary response genes (i.e. CXCL11, IL1A, and
IL1B), which are proinflammatory genes that are regulated by
28848 JOURNAL OF BIOLOGICAL CHEMISTRY
NF-␬B, we postulated that BET BrD inhibition may represent a
therapeutic strategy for HIVAN treatment. To test this hypoth-
esis, we carried out an in vivo study to evaluate anti-inflamma-
tory effects of MS417 in HIV-1 transgenic mice, an established
mouse model of human HIVAN with kidney tubulointerstitial
inflammation and RTEC apoptosis (43). The Tg26 mice are
known to develop proteinuria starting at the age of 4 weeks and
peaking at the age of 8 weeks associated with glomerular and
tubular injuries (44). Accordingly, we treated a group of the
Tg26 mice with MS417 or vehicle (0.1% DMSO) and compared
them with their age- and sex-matched littermates in a control
group starting at the age of 4 weeks for a total of 4 weeks. No
significant side effects were noticed in these mice treated with
MS417 at a dose of 0.08 mg/kg daily. Instead, we found that
MS417 markedly improved renal function in the Tg26 mice as
assessed by the measurement of blood urea nitrogen (BUN)
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without substantial changes of liver function and body weight
(Fig. 5A and supplemental Table 3). MS417 treatment also
resulted in reduced proteinuria in the Tg26 mice as compared
with the DMSO-treated mice (Fig. 5B), and histology analysis
showed significantly reduced glomerular and tubular injury
(supplemental Table 3). The infiltration of inflammatory cells
was also drastically reduced in the kidney of the Tg26 mice
treated with MS417 as compared with the Tg26 mice treated
with DMSO (Fig. 5C). NF-␬B acetylation level (Fig. 5D) as well
as the expression of NF-␬B target genes, including CCL2, CCL5,
CCL20, TLR-2, and TLR-9 (Fig. 5E) were reduced in kidney
cortices of the MS417-treated Tg26 mice as compared with the
control group. Finally, it appears that both mRNA and protein
levels of HIV Nef in the MS417-treated mice were reduced by
over 20% as compared with the control group, but this did not
reach statistical significance (Fig. 5, D and F). Taken together,
these data indicate that MS417 is capable of attenuating kidney
injury in the Tg26 mice, probably through inhibition of NF-␬B-
mediated inflammation.
DISCUSSION
Several lines of evidence suggest that HIV directly infects
renal tubular epithelial cells, leading to the development of
HIV-associated nephropathy. The effect of HIV expression on
the development of HIVAN was first revealed by kidney trans-
plantation between normal and HIV transgenic mice. This
study demonstrated that HIVAN was developed in HIV trans-
genic kidneys when transplanted into nontransgenic litter-
mates, whereas normal kidneys remained disease-free when
transplanted into HIV transgenic littermates, suggesting that
the renal disease in the HIV transgenic murine model is intrin-
sic to the kidney (45). HIV can also infect directly kidney glo-
merular and tubular epithelial cells, as shown by in situ PCR or
hybridization or by PCR of laser capture microdissected kidney
biopsy samples from HIV-positive patients (6, 46, 47). Recently,
the mechanisms of HIV entry into kidney epithelial cells have
been described (48, 49). Taken together, these studies suggest
that HIV infection probably occurs locally in kidney cells, lead-
ing to the development of HIVAN.
In this study, we have shown that the BET bromodomain-
specific inhibitor MS417 down-regulates HIV infection-in-
duced and NF-␬B-mediated transcriptional activation of pro-
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FIGURE 5. In vivo study of MS417 effects in HIV-1 transgenic mice (Tg26). A, MS417 improves renal function in Tg26 mice as measured by blood urea
nitrogen (BUN) (n ⫽ 6; *, p ⬍ 0.05 as compared with DMSO-treated Tg26 mice). B, MS417 reduces proteinuria in the Tg26 mice as determined by urinary
albuminuria/creatinine ratio (n ⫽ 6; *, p ⬍ 0.05 as compared with DMSO-treated Tg26 mice). C, MS417 reduces glomerulosclerosis, tubular injury, and
infiltration of inflammatory cells in the kidney of Tg26 mice. Tg26 mice were treated with vehicle (0.1% DMSO) or MS417 at 0.08 mg/kg from the age of 4 weeks
for a total duration of 4 weeks. The mice were sacrificed at the age of 8 weeks. The kidney sections from these mice were stained for periodic acid-Schiff, and
the representative pictures from six mice in each group are shown here. D, MS417 inhibits acetylation of p65 in the kidney of the Tg26 mice. The kidney cortices
from these mice were used for Western blot analysis of acetylated and total p65. The expression of HIV Nef and ␤-actin was also assessed by Western blot. The
densitometric analysis of these Western blots is shown in the lower panels (n ⫽ 6; *, p ⬍ 0.05 as compared with DMSO-treated Tg26 mice). E, NF-␬B target genes
were suppressed in kidneys of the Tg26 mice treated with MS417. The kidney cortices from these mice were used for total RNA isolation and real-time PCR
analysis for NF-␬B target genes (n ⫽ 6; *, p ⬍ 0.05 as compared with DMSO-treated Tg26 mice). F, effects of MS417 on the expression of HIV Nef in the kidneys
of Tg26 mice. All mRNA levels of the testing genes were normalized to that of GAPDH, a housekeeping gene.

inflammatory cytokine and chemokine genes in human renal
tubular epithelial cells. MS417 reduces proteinuria, improves
renal function, and attenuates NF-␬B acetylation and expres-
sion of its target genes in the kidney of the Tg26 mouse, a model
for HIVAN. Our findings, therefore, strongly suggest that BET
bromodomain inhibition, targeting NF-␬B proinflammatory
activity in the pathogenesis of HIVAN, represents a new thera-
peutic strategy for treating patients with HIVAN. This is the
first study that demonstrates BET bromodomain inhibitors as
potentially new therapeutic agents in a kidney disease model.
Transcription factor NF-␬B/Rel proteins govern cellular
immune and inflammatory responses in the form of cell apo-
ptosis, proliferation, or differentiation through regulation of
gene expression (9, 10). The prototypical NF-␬B is a het-
erodimer of p50 and RelA and is sequestered in the cytoplasm
when bound to inhibitor I␬B␣ in resting cells. Stimulation of
the cells activates IKKs, phosphorylation, and degradation of
I␬B␣, leading to nuclear translocation and activation of NF-␬B.
Transcriptional activity of NF-␬B requires its association with
the lysine acetyltransferase p300/CBP and PCAF and transcrip-
tional protein BRD4. The cofactor binding results in phosphor-
ylation and acetylation of NF-␬B, promoting the formation of
the transcriptional complexes that bridges the sequence-spe-
cific activators to the basal transcription machinery, thereby
facilitating its target gene activation.
BRD4 belongs to the BET protein family, which consists of
four members, BRD2, BRD3, BRD4, and BRDT, each of which
contains two bromodomains in tandem (50). Recent studies
show that BET proteins play an important role in coordinating
gene transcriptional activation in a highly ordered fashion in
chromatin through their ability to facilitate chromatin target-
ing and assembly of a productive transcriptional machinery
complex at a target gene transcriptional start site in an acetyla-
tion-sensitive manner. In the case of NF-␬B, BRD4 recruitment

AUGUST 17, 2012 • VOLUME 287 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 28849
Transcriptional Inhibition of NF-␬B in HIVAN
by the transcription factor to a target gene represents a preini-
tiation commitment step in transcriptional activation, which
involves chromatin structure opening through histone lysine
acetylation as well as BRD4 interaction with acetylated histone
H3 and H4 at the H3K14ac, H4K5ac, H4K8ac, H4K12ac, and
H4K16ac sites. In the next step of transcriptional initiation/
postinitiation, BRD4 functions in the recruitment of the initia-
tion factor mediator, which leads to Ser-5 phosphorylation of
the carboxyl-terminal domain of the RNA polymerase II (9).
Subsequently, BRD4 facilitates the recruitment of the elonga-
tion cofactor p-TEFb, consisting of CDK9 and cyclin T1, to
paused RNA polymerase II, which results in Ser-2 phosphory-
lation of the carboxyl-terminal domain, thereby enabling
polymerase II to resume transcriptional elongation.
The mechanism of action of our BET BrDi MS417 probably
involves its modulation of BRD4 functions in multiple steps
during gene transcriptional activation. As shown by the current
study, BRD4 binding to K310ac NF-␬B is achieved by its second
bromodomain (BRD4-BD2); blocking of this interaction by
MS417 resulted in a reduced acetylation level of NF-␬B at Lys-
310 and down-regulation of NF-␬B target gene transcriptional
activation. Further, inhibition of both bromodomains of BRD4
by MS417 may blunt the ability of BRD4 to interact with lysine-
acetylated H3 and H4 for chromatin targeting at the NF-␬B
target gene transcriptional start site or to promote the forma-
tion of a productive transcriptional machinery with p-TEFb
and RNA polymerase II, which is important for transcriptional
elongation (9).
NF-␬B activity is known to be elevated in HIV-infected renal
tubular epithelial cells and podocytes as well as in kidneys of
HIV transgenic mice, mediating HIV-induced kidney injury (8,
15, 16). NF-␬B has also been shown to be recruited to interact
with HIV-1 LTR to facilitate HIV transcription and replication
in the kidney (18). Targeting NF-␬B would be a logical
approach to attenuate both HIV-induced inflammation and
kidney injury. Indeed, we have shown that both primary
response and secondary response genes directed by NF-␬B acti-
vation that is triggered by HIV infection were down-regulated
by the treatment of MS417. Furthermore, the changes of their
transcriptional expression are consistently correlated to the
alteration of histone modifications at the transcriptional start
sites of these genes. Interestingly, we have also noticed that the
transcriptional expression of a subset of NF-␬B-inducible genes
encoding cytokines or chemokines, such as CXCL5 and CXCL6,
was not affected by MS417, suggesting that BRD4 is not
involved in their gene transcriptional activation. Further inves-
tigation is warranted to dissect the molecular mechanistic
details of BRD4 functions during transcriptional activation of
these genes.
Although suppression of viral replication with antiretroviral
therapy alters the course of kidney disease, many patients with
HIVAN still unfortunately progress to end stage renal disease
(3). No drugs are available to specifically stop or reverse this
disease process. Because NF-␬B is a key pathway in the patho-
genesis of HIVAN, MS417 could be developed as a new class of
drugs to treat patients with HIVAN. In addition, NF-␬B-medi-
ated inflammation is also a major pathology in other kidney
disease, such as diabetic nephropathy and lupus nephritis (51),
28850 JOURNAL OF BIOLOGICAL CHEMISTRY
as well as other non-kidney inflammatory diseases, such as
rheumatoid arthritis (52). Because of the protective role of
NF-␬B in acute inflammatory response, complete inhibition of
NF-␬B may lead to harmful side effects. Small molecules for
selective and transient inhibition of NF-␬B proinflammatory
activity may allow us to maximize their therapeutic index.
Therefore, we believe that BET bromodomain-specific inhibi-
tors could be developed as more effective and safer treatments
for many inflammatory diseases.
Acknowledgments—We acknowledge the staff at the X6A beamline of
the National Synchrotron Light Source at the Brookhaven National
Laboratory for facilitating x-ray data collection. We thank the Rock-
efeller University High-Throughput Screening Resource Center for the
ITC data collection.
Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
REFERENCES
1. Wyatt, C. M., and Klotman, P. E. (2007) HIV-1 and HIV-associated ne-
phropathy 25 years later. Clin. J. Am. Soc. Nephrol. 2, Suppl. 1, S20 –S24
2. Atta, M. G., Fine, D. M., Kirk, G. D., Mehta, S. H., Moore, R. D., and Lucas,
G. M. (2007) Survival during renal replacement therapy among African
Americans infected with HIV type 1 in urban Baltimore, Maryland. Clin.
Infect. Dis. 45, 1625–1632
3. Lucas, G. M., Eustace, J. A., Sozio, S., Mentari, E. K., Appiah, K. A., and
Moore, R. D. (2004) Highly active antiretroviral therapy and the incidence
of HIV-1-associated nephropathy. A 12-year cohort study. AIDS 18,
541–546
4. Wyatt, C. M., Winston, J. A., Malvestutto, C. D., Fishbein, D. A., Barash, I.,
Cohen, A. J., Klotman, M. E., and Klotman, P. E. (2007) Chronic kidney
disease in HIV infection. An urban epidemic. AIDS 21, 2101–2103
5. D’Agati, V., Suh, J. I., Carbone, L., Cheng, J. T., and Appel, G. (1989)
Pathology of HIV-associated nephropathy. A detailed morphologic and
comparative study. Kidney Int. 35, 1358 –1370
6. Bruggeman, L. A., Ross, M. D., Tanji, N., Cara, A., Dikman, S., Gordon,
R. E., Burns, G. C., D’Agati, V. D., Winston, J. A., Klotman, M. E., and
Klotman, P. E. (2000) Renal epithelium is a previously unrecognized site of
HIV-1 infection. J. Am. Soc. Nephrol. 11, 2079 –2087
7. Mikula, M., Fuchs, E., Huber, H., Beug, H., Schulte-Hermann, R., and
Mikulits, W. (2004) Immortalized p19ARF null hepatocytes restore liver
injury and generate hepatic progenitors after transplantation. Hepatology
39, 628 – 634
8. Ross, M. J., Fan, C., Ross, M. D., Chu, T. H., Shi, Y., Kaufman, L., Zhang,
W., Klotman, M. E., and Klotman, P. E. (2006) HIV-1 infection initiates an
inflammatory cascade in human renal tubular epithelial cells. J. Acquir.
Immune Defic. Syndr. 42, 1–11
9. Huang, B., Yang, X. D., Zhou, M. M., Ozato, K., and Chen, L. F. (2009) Brd4
coactivates transcriptional activation of NF-␬B via specific binding to
acetylated RelA. Mol. Cell. Biol. 29, 1375–1387
10. Lin, Y., Bai, L., Chen, W., and Xu, S. (2010) The NF-␬B activation path-
ways, emerging molecular targets for cancer prevention and therapy. Ex-
pert Opin. Ther. Targets 14, 45–55
11. Pasparakis, M. (2009) Regulation of tissue homeostasis by NF-␬B signal-
ing. Implications for inflammatory diseases. Nat. Rev. Immunol. 9,
778 –788
12. Chen, L. F., and Greene, W. C. (2004) Shaping the nuclear action of NF-
␬B. Nat. Rev. Mol. Cell Biol. 5, 392– 401
13. Rangan, G., Wang, Y., and Harris, D. (2009) NF-␬B signaling in chronic
kidney disease. Front. Biosci. 14, 3496 –3522
14. Schmid, H., Boucherot, A., Yasuda, Y., Henger, A., Brunner, B., Eichinger,
F., Nitsche, A., Kiss, E., Bleich, M., Gröne, H. J., Nelson, P. J., Schlöndorff,
D., Cohen, C. D., Kretzler, M., and European Renal cDNA Bank (ERCB)
Consortium (2006) Modular activation of nuclear factor-␬B transcrip-
tional programs in human diabetic nephropathy. Diabetes 55, 2993–3003
15. Martinka, S., and Bruggeman, L. A. (2006) Persistent NF-␬B activation in
VOLUME 287 • NUMBER 34 • AUGUST 17, 2012

renal epithelial cells in a mouse model of HIV-associated nephropathy.
Am. J. Physiol. Renal Physiol. 290, F657–F665
16. Ross, M. J., Martinka, S., D’Agati, V. D., and Bruggeman, L. A. (2005)
NF-␬B regulates Fas-mediated apoptosis in HIV-associated nephropathy.
J. Am. Soc. Nephrol. 16, 2403–2411
17. Ross, M. J., Wosnitzer, M. S., Ross, M. D., Granelli, B., Gusella, G. L.,
Husain, M., Kaufman, L., Vasievich, M., D’Agati, V. D., Wilson, P. D.,
Klotman, M. E., and Klotman, P. E. (2006) Role of ubiquitin-like protein
FAT10 in epithelial apoptosis in renal disease. J. Am. Soc. Nephrol. 17,
996 –1004
18. Bruggeman, L. A., Adler, S. H., and Klotman, P. E. (2001) Nuclear fac-
tor-␬B binding to the HIV-1 LTR in kidney. Implications for HIV-associ-
ated nephropathy. Kidney Int. 59, 2174 –2181
19. Nicodeme, E., Jeffrey, K. L., Schaefer, U., Beinke, S., Dewell, S., Chung,
C. W., Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kiril-
ovsky, J., Rice, C. M., Lora, J. M., Prinjha, R. K., Lee, K., and Tarakhovsky,
A. (2010) Suppression of inflammation by a synthetic histone mimic. Na-
ture 468, 1119 –1123
20. Zeng, L., Zhang, Q., Gerona-Navarro, G., Moshkina, N., and Zhou, M. M.
(2008) Structural basis of site-specific histone recognition by the bro-
modomains of human coactivators PCAF and CBP/p300. Structure 16,
643– 652
21. Clore, G. M., and Gronenborn, A. M. (1994) Multidimensional heteronu-
clear nuclear magnetic resonance of proteins. Methods Enzymol. 239,
349 –363
22. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P.,
Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S.,
Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Crystallog-
raphy & NMR system. A new software suite for macromolecular structure
determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921
23. Nilges, M., and O’Donoghue, S. (1998) Ambiguous NOEs and automated
NOE assignment. Prog. NMR Spectrosc. 32, 107–139
24. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and
Thornton, J. M. (1996) AQUA and PROCHECK-NMR. Programs for
checking the quality of protein structures solved by NMR. J. Biomol. NMR
8, 477– 486
25. Nikolovska-Coleska, Z., Wang, R., Fang, X., Pan, H., Tomita, Y., Li, P.,
Roller, P. P., Krajewski, K., Saito, N. G., Stuckey, J. A., and Wang, S. (2004)
Development and optimization of a binding assay for the XIAP BIR3 do-
main using fluorescence polarization. Anal. Biochem. 332, 261–273
26. Otwinowski, Z., Minor, W. (1997) Processing of x-ray diffraction data
collected in oscillation mode. Methods Enzymol. 276, 307–326
27. Vagin, A., and Teplyakov, A. (1997) MOLREP. An automated program for
molecular replacement. J. Appl. Crystallogr. 30, 1022–1025
28. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of
macromolecular structures by the maximum likelihood method. Acta
Crystallogr. D Biol. Crystallogr. 53, 240 –255
29. Emsley, P., and Cowtan K. (2004) Coot. Model-building tools for molec-
ular graphics. Acta Crystallogr. D 60, 2126 –2132
30. Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B., and Speed,
T. P. (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic
Acids Res. 31, e15
31. Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J.,
Scherf, U., and Speed, T. P. (2003) Exploration, normalization, and sum-
maries of high density oligonucleotide array probe level data. Biostatistics
4, 249 –264
32. Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Significance analysis of
microarrays applied to the ionizing radiation response. Proc. Natl. Acad.
Sci. U.S.A. 98, 5116 –5121
33. Dennis, G., Jr., Sherman, B. T., Hosack, D. A., Yang, J., Gao, W., Lane,
H. C., and Lempicki, R. A. (2003) DAVID. Database for annotation, visu-
alization, and integrated discovery. Genome Biol. 4, P3
34. Dickie, P., Felser, J., Eckhaus, M., Bryant, J., Silver, J., Marinos, N., and
Notkins, A. L. (1991) HIV-associated nephropathy in transgenic mice ex-
pressing HIV-1 genes. Virology 185, 109 –119
35. Kopp, J. B., Klotman, M. E., Adler, S. H., Bruggeman, L. A., Dickie, P.,
Marinos, N. J., Eckhaus, M., Bryant, J. L., Notkins, A. L., and Klotman, P. E.
(1992) Progressive glomerulosclerosis and enhanced renal accumulation
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
Transcriptional Inhibition of NF-␬B in HIVAN
of basement membrane components in mice transgenic for human im-
munodeficiency virus type 1 genes. Proc. Natl. Acad. Sci. U.S.A. 89,
1577–1581
36. D’Agati, V. (2003) Pathologic classification of focal segmental glomerulo-
sclerosis. Semin. Nephrol. 23, 117–134
37. Miyoshi, S., Ooike, S., Iwata, K., Hikawa, H., and Sugaraha, K. (December
26, 2008) International Patent PCT/JP2008/073864 (US2010/0286127 A1)
38. Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O.,
Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro,
S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J.,
Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O.,
Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET
bromodomains. Nature 468, 1067–1073
39. Dawson, M. A., Prinjha, R. K., Dittmann, A., Giotopoulos, G., Bantscheff,
M., Chan, W. I., Robson, S. C., Chung, C. W., Hopf, C., Savitski, M. M.,
Huthmacher, C., Gudgin, E., Lugo, D., Beinke, S., Chapman, T. D., Rob-
erts, E. J., Soden, P. E., Auger, K. R., Mirguet, O., Doehner, K., Delwel, R.,
Burnett, A. K., Jeffrey, P., Drewes, G., Lee, K., Huntly, B. J., and Kouzarides,
Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
T. (2011) Inhibition of BET recruitment to chromatin as an effective treat-
ment for MLL fusion leukemia. Nature 478, 529 –533
40. Snyder, A., Alsauskas, Z. C., Leventhal, J. S., Rosenstiel, P. E., Gong, P.,
Chan, J. J., Barley, K., He, J. C., Klotman, M. E., Ross, M. J., and Klotman,
P. E. (2010) HIV-1 viral protein r induces ERK and caspase-8-dependent
apoptosis in renal tubular epithelial cells. AIDS 24, 1107–1119
41. Havasi, A., and Borkan, S. C. (2011) Apoptosis and acute kidney injury.
Kidney Int. 80, 29 – 40
42. Susztak, K., Raff, A. C., Schiffer, M., and Böttinger, E. P. (2006) Glucose-
induced reactive oxygen species cause apoptosis of podocytes and podo-
cyte depletion at the onset of diabetic nephropathy. Diabetes 55, 225–233
43. Lu, T. C., He, J. C., and Klotman, P. (2006) Animal models of HIV-associ-
ated nephropathy. Curr. Opin. Nephrol. Hypertens. 15, 233–237
44. Ratnam, K. K., Feng, X., Chuang, P. Y., Verma, V., Lu, T. C., Wang, J., Jin,
Y., Farias, E. F., Napoli, J. L., Chen, N., Kaufman, L., Takano, T., D’Agati,
V. D., Klotman, P. E., and He, J. C. (2011) Role of the retinoic acid recep-
tor-␣ in HIV-associated nephropathy. Kidney Int. 79, 624 – 634
45. Bruggeman, L. A., Dikman, S., Meng, C., Quaggin, S. E., Coffman, T. M.,
and Klotman, P. E. (1997) Nephropathy in human immunodeficiency vi-
rus-1 transgenic mice is due to renal transgene expression. J. Clin. Invest.
100, 84 –92
46. Marras, D., Bruggeman, L. A., Gao, F., Tanji, N., Mansukhani, M. M., Cara,
A., Ross, M. D., Gusella, G. L., Benson, G., D’Agati, V. D., Hahn, B. H.,
Klotman, M. E., and Klotman, P. E. (2002) Replication and compartmen-
talization of HIV-1 in kidney epithelium of patients with HIV-associated
nephropathy. Nat. Med. 8, 522–526
47. Tanji, N., Ross, M. D., Tanji, K., Bruggeman, L. A., Markowitz, G. S.,
Klotman, P. E., and D’Agati, V. D. (2006) Detection and localization of
HIV-1 DNA in renal tissues by in situ polymerase chain reaction. Histol.
Histopathol. 21, 393– 401
48. Chen, P., Chen, B. K., Mosoian, A., Hays, T., Ross, M. J., Klotman, P. E., and
Klotman, M. E. (2011) Virological synapses allow HIV-1 uptake and gene
expression in renal tubular epithelial cells. J. Am. Soc. Nephrol. 22,
496 –507
49. Mikulak, J., Teichberg, S., Arora, S., Kumar, D., Yadav, A., Salhan, D.,
Pullagura, S., Mathieson, P. W., Saleem, M. A., and Singhal, P. C. (2010)
DC-specific ICAM-3-grabbing nonintegrin mediates internalization of
HIV-1 into human podocytes. Am. J. Physiol. Renal Physiol. 299,
F664 –F673
50. Chiang, C. M. (2009) Brd4 engagement from chromatin targeting to tran-
scriptional regulation. Selective contact with acetylated histone H3 and
H4. F1000 Biol. Rep. 1, 98
51. Zheng, L., Sinniah, R., and Hsu, S. I. (2006) Renal cell apoptosis and pro-
liferation may be linked to nuclear factor-␬B activation and expression of
inducible nitric-oxide synthase in patients with lupus nephritis. Hum.
Pathol. 37, 637– 647
52. Criswell, L. A. (2010) Gene discovery in rheumatoid arthritis highlights
the CD40/NF-␬B signaling pathway in disease pathogenesis. Immunol.
Rev. 233, 55– 61
JOURNAL OF BIOLOGICAL CHEMISTRY 28851
Supplemental Materials and Methods
Zhang, G.T. et al.

Chemicals and General Procedure

Unless otherwise stated, all commercially available materials were obtained from Acros
Organics, Aldrich, Alfa Aesar, EMD, J. T. Baker, Novabiochem, or TCI, and were used without
any further purification. When necessary, solvents and reagents were dried prior to use, using
standard protocols. All non-aqueous reactions were carried out in oven-dried glassware under
an atmosphere of argon. Automatic chromatography was performed on a Biotage Isolera
system equipped with a variable wavelength detector and a fraction collector, using Biotage
SNAP cartridge KP-Sil 50 g. Analytical thin layer chromatography (TLC) was performed
employing Sigma-Aldrich 250 µm 60F-254 silica plates. The plates were visualized either by
exposure to UV light, staining with iodine impregnated silica gel, or by staining with ceric

Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
ammonium molybdate (CAM). Preparative TLC was performed employing Silicycle 1000 µm
SiliaPlate Prep silica plates. LC/MS analysis was conducted on an Agilent Technologies high-
resolution ESI-TOF mass spectrometer attached to an Agilent Technologies 1200 HPLC
system. Samples were ionized by electrospray ionization (ESI) in positive mode.
Chromatography was performed on a 2.1 × 150 mm Zorbax 300SB-C18 5 µm column with
water containing 0.1% formic as solvent A and acetonitrile containing 0.1% formic acid as
solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0-1 min), 1-
99% B (1-4 min), 99% B (4-7 min). The temperature of the column was held at 50 °C for the
entire analysis. HPLC purification was performed on a 9.4 mm × 250 mm Eclipse XDB-C18 5
µm column coupled with an Agilent Technologies 1200 preparative HPLC system. The gradient
program was as follows: 10% B (0-4 min), 10-90% B (4-14 min), 90% B (14-18 min). Chiral
purification was performed on a 4.6 mm × 250 mm (R,R)-Whelk-O1 5100 Kromasil 5 µm chiral
column from Regis Technologies, Inc., which is coupled with an Agilent Technologies 1200
semi-preparative HPLC system. The samples were eluted with 1:1 methanol/ ethanol at a flow
rate of 1.0 mL/min. NMR spectra were acquired on a Bruker DRX-600 spectrometer at 600 MHz
for 1H and 125 MHz for 13C. Chemical shifts are expressed in parts per million downfield from
tetramethylsilane (TMS), using either TMS or the solvent resonance as an internal standard
(TMS, 1H: 0 ppm; chloroform, 13C: 77.0 ppm; DMSO-d6, 1H: 2.50 ppm; 13C: 39.5 ppm; methanol-
d4, 1H: 3.31 ppm; 13C: 49.0 ppm). Data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q =quartet, m = multiplet, br = broad), integration, and coupling
constant. IR spectra were obtained on a Bruker TENSOR 27 series FT-IR spectrometer
equipped with a Diamond ATR. Finally, for optical activity analysis of enantiomers, optical
rotations were recorded on a Jasco P-2000 multi-option polarimeter that is equipped with a
sodium lamp as radiation source (589 nm).

Chemical Synthesis
The synthesis of stereospecific thioenotriazolodiazepine (5), also named as MS417, was carried
out by following the reaction steps as outlined in Scheme 1.

(2-amino-4,5-dimethylthiophen-3-yl)(4-chlorophenyl)methanone (1). A 100 mL pressure

vessel was charged with 4-chlorobenzoylacetonitrile (2.88 g, 15.71 mmol), 2-butanone (1.14 g,
15.71 mmol, 1.41 mL), and sulfur (0.51 g, 15.71 mmol) sequentially. This mixture was dissolved
in absolute ethanol (35.0 mL) and allowed to stir vigorously. To this solution was added
piperidine (0.90 g, 15.71 mmol, 1.04 mL) dropwise. The solution was heated up to 110˚C for 10
h under argon in an oil bath. The organic solvent was removed in vacuo. Purification by
automatic chromatography (4:1 hexane in ethyl acetate, Rf = 0.36) provided the title product as
a beautiful yellow crystal (2.65 g, 63%). 1H NMR (CDCl3) δ 7.47 (d, J = 7.4, 2H), 7.39 (d, J =

  1  
7.4, 2H), 6.53 (br s, 2H), 2.14 (s, 3H), 1.56 (s, 3H); 13C NMR (CDCl3) δ 191.5, 163.1, 140.1,
136.7, 129.4, 128.4, 117.1, 15.5, 12.5. FTIR (neat, cm-1) 3344 (s, NH2), 3242 (s, NH2) 1085 (s,
C-N), 692 (s, C-S). MS calculated for C13H12ClNOS [M+H]+ 266.03, found 266.05. Purity >99%,
tR = 6.3 min.

Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
 
Scheme 1. Synthesis of Stereospecific Thioenotriazolodiazepine 5. Conditions: (a) 4-chlorobenzoyl-
o
acetonitrile (1.0 equiv), 2-butanone (1.0 equiv), sulfur (1.0 equiv), piperidine (1.0 equiv), EtOH, 110 C, 10
h; (b) Fmoc-L-Asp(OMe) (1.0 equiv), PyBOP (0.95 equiv), DIPEA (2.75 eq.), DMF, room temperature, 4
o
h; (c) 20% piperidine, room temperature, 1h; (d) silica gel, toluene, 90 C, overnight; (e) i) KOtBu (1.1 eq.),
o o o o
THF, -78 C à -10 C , 30 min; ii) PO(OEt)2Cl (1.2 eq), -78 C à -10 C, 45 min; iii) acetic hydrazide (3.0
o
eq.), BuOH, 90 C, 1h.

(S)-methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4,5-
dimethylthiophen-2-yl)amino)-4-oxobutanoate (2). A 4.0 mL vial was charged with Fmoc-L-
aspartic acid methyl ester (369.4 mg, 1.0 mmol) and 1.0 mL DMF, PyBOP (494.4 mg, 0.95
mmol) and DIPEA (355.4 mg, 2.75 mmol) sequentially. After stirring at room temperature for 10
min, 1 (265.8 mg, 1.0 mmol) was added as solid all at once and was allowed to stir vigorously at
ambient temperature for 4 h. The crude sample was concentrated in vacuo to a minimal
possible volume. Purification by automatic chromatography (4:1 hexane in ethyl acetate, Rf =
0.18) provided the title compound as a bright yellow powder (4.53 g, 97%). 1H NMR (CDCl3) δ
11.61 (s, 1H), 7.57 (d, J = 8.4, 2H), 7.43 (d, J = 8.4, 2H), 5.81 (d, 1H), 4.75 (m, 1H), 3.70 (s,
3H), 3.23 (m, 1H), 2.83 (m, 1H), 2.27 (s, 3H), 1.69 (s, 3H), 1.46 (s, 9H); 13C NMR (CDCl3) δ
193.0, 172.0, 168.8, 155.4, 145.3, 138.5, 138.2, 130.2, 128.6, 127.6, 125.3, 123.1, 113.2, 81.1,
52.8, 52.2, 35.4, 28.3, 15.0, 14.2. FTIR (neat, cm-1) 3105 (s, NH), 2975 (s, N-H), 1731 (s, C=O),
1214 (s, C-O), 669 (s, C-S). MS calculated for C23H27ClN2NaO6S [M+Na]+ 517.12, found 517.13.
Purity >99%, tR = 6.8 min.

(S)-methyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-
oxobutanoate (3). A 20 mL scintillation vial was charged with 2 (810 mg, 1.31 mmol) and 20%
piperidine in DMF (6.0 mL). This mixture was allowed to stir vigorously at room temperature for

  2  
1h. The reaction was quenched by adding saturated NaCl (aq., 40 mL). The aqueous layer was
extracted three times by ethyl acetate (40 mL × 3). The combined organic layer was washed
with brine and dried on anhydrous Na2SO4. The volatiles were removed in vacuo. The title
compound appeared as yellow oil (300 mg, 58%). 1H NMR (CDCl3) δ 12.18 (br s, 1H), 7.60 (d,
J = 8.4, 2H), 7.43 (d, J = 8.4, 2H), 3.91 (t, J = 3.9, 1H), 3.72 (s, 3H), 3.01 (m, 1H), 2.83 (m, 1H),
2.28 (s, 3H), 1.92 (br s, 2H), 1.73 (s, 3H); 13C NMR (CDCl3) δ 193.4, 171.3, 170.8, 165.4, 142.1,
138.9, 137.5, 130.4, 128.7, 128.1, 127.0, 125.0, 52.9, 50.1, 34.8, 14.6, 14.0. FTIR (neat, cm-1)
1290 (s, C-O), 1210 (d, C-O). MS calculated for C18H19ClN2O4S [M+H]+ 395.08, found 395.08.
Purity >99%, tR = 5.8 min.

(S)-methyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-
e][1,4]diazepin-3-yl)acetate (4). A 35 mL sealed tube was charged with compound 3 (300 mg,
0.76 mmol) and toluene (24 mL). The mixture was allowed to heat to 90 °C and refluxed for

Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
overnight. The crude sample was first filtered and then concentrated in vacuo. Purification by
automatic chromatography (3:2 hexane in ethyl acetate, Rf = 0.16) provided the title compound
as a bright yellow crystal (265 mg, 90%). 1H NMR (CDCl3) δ 9.49 (s, 1H), 7.41 (d, J = 8.4, 2H),
7.32 (d, J = 8.4, 2H), 4.25 (m, 1H), 3.73 (s, 3H), 3.43 (m, 1H), 3.17 (m, 1H), 2.27 (s, 3H), 1.58
(s, 3H); 13C NMR (CDCl3) δ 172.4, 169.4, 165.3, 141.5, 136.8, 136.3, 130.2, 129.5, 128.5,
127.5, 126.7, 113.3, 61.2, 51.8, 36.3, 14.4, 12.9. FTIR (neat, cm-1) 1290 (s, C-O), 1210 (d, C-
O). MS calculated for C18H17ClN2O3S [M+H]+ 377.06, found 377.07. Purity >99%, tR = 6.4 min.

Methyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4] triazolo[4,3-

a][1,4]diazepin-6-yl)acetate (5). A 50 mL round bottom flask was charged with compound 4
(125 mg, 0.3 mmol) and THF (1.6 mL). The solution was cooled to -78 °C and potassium tert-
butoxide (37.0 mg, 0.33 mmol, 1.1 eq.). This mixture was warmed up to -10 °C and sustained
for 30 min. The mixture was cooled again to -78 °C and diethyl chlorophosphate (62.1 mg, 0.36
mmol) was added drop wise. The solution was warmed to -10 °C and sustained for 45 min.
After reaction was complete, n-butanol (1.8 mL) and acetic hydrazide (66.7 mg, 900 mmol) were
added sequentially. This mixture was stirred at room temperature for 1 h and heated up to 90 °C
for 1 h. The crude sample was filtered and the residual concentrated in vacuo. Purification by
automatic chromatography (0-100% ethyl acetate in hexane, Rf = 0.3) provided the title
compound. The pure product appeared as a pale yellow crystal (55.0 mg, 40%). 1H NMR
(CDCl3) δ 7.42 (d, J = 7.8, 2H), 7.35 (d, J = 7.8, 2H), 4.62 (m, 1H), 3.79 (s, 3H), 3.65 (m, 2H),
2.69 (s, 3H), 2.43 (s, 3H), 1.70 (s, 3H); 13C NMR (CDCl3) δ 172.1, 163.9, 155.3, 150.0, 136.8,
136.6, 132.3, 130.9, 130.8, 130.4, 129.9, 129.8, 128.7, 116.8, 53.8, 51.9, 36.7, 14.4, 13.1, 11.9.
FTIR (neat, cm-1) 1290 (s, C-O), 1210 (d, C-O). MS calculated for C20H19ClN4O2S [M+H]+
415.09, found 415.09. Purity >99%, tR = 6.6 min. Chiral resolution: t6R = 8.2 min, t6S = 11.5 min
(See Figure S6). Specific rotation activities (αD20) of 5 (also named as MS417) and its
enantiomer (i.e. MS566) were measured to be 19.9 and -20.1, respectively (see Table S4).

Synthesis of FITC-labeled chemical probe. A fluorescein-conjugated thioenotriazolodiazepine

compound (9), also named as MS574, was synthesized by using the synthetic scheme as
described in Scheme 2.

2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin
-6-yl)acetic acid (6). A 4 mL vial was charged with compound 5 (2.6 mg, 6.3 µmol) and THF
(0.4 mL). The solution was stirred vigorously at room temperature for 5 min. To this solution was
added lithium hydroxide monohydrate (2.7 mg, 63 µmol) and deionized water (0.1 mL). The
mixture was stirred at room temperature for 2 h and the reaction was quenched. The organic
content was removed in vacuo and the residual material was dissolved in H2O. The solution was

  3  
adjusted to pH 14 with 1.0 M NaOH (aq.) and the aqueous layer was washed with diethyl ether.
The pH was then carefully adjusted to pH 1 with 1.0 M HCl (aq.), and the solution was extracted
by diethyl ether. The organic phase was dried over MgSO4 and concentrated to provide the title
compound. The pure product appeared as a pale yellow oil (2.6 mg, ~100%). 1H NMR (CDCl3) δ
7.42 (d, J = 7.8, 2H), 7.35 (d, J = 7.8, 2H), 4.62 (m, 1H), 3.65 (m, 2H), 2.69 (s, 3H), 2.43 (s, 3H),
1.70 (s, 3H). MS calculated for C19H17ClN4O2S [M+H]+ 401.08, found 401.08. Purity >99%, tR =
5.2 min.

Downloaded from www.jbc.org at NYU School of Medicine Library, on August 17, 2012
tert-butyl (2-(2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4] triazolo[4,3-
a][1,4]diazepin-6-yl)acetamido)ethyl)carbamate (7). A 4 mL vial was charged with compound
6 (20.0 mg, 49.9 µmol) and DMF (0.5 mL). To this solution was added PyBOP (24.7 mg, 47.4
µmol) and DIPEA (32.2 mg, 250 µmol). The mixture was stirred at room temperature for 10 min
before N-Boc-ethylenediamine (8.0 mg, 50.0 µmol) was added. The reaction was allowed for
overnight. The reaction was quenched with 1:1 (v/v) ratio of ethyl acetate and brine. The
aqueous layer was extracted with ethyl acetate (2×1 mL). The combined organic layer was
washed with brine and dried over sodium sulfate. The organic solvents were removed under
reduced pressure and purification by automatic chromatography (0-10% methanol in
dichloromethane, Rf = 0.1) provided the title compound. The pure product appeared as a
colorless oil (5.2 mg, 20%). 1H NMR (CDCl3) δ 7.41 (d, J = 8.4, 2H), 7.33 (d, J = 8.4, 2H), 6.98
(br s, 1H), 5.19 (br s, 1H), 4.64 (t, J = 6.6, 1H), 3.44-3.34 (m, 2H), 3.33-3.20 (m, 2H), 2.67 (s,
3H), 2.40 (s, 3H), 1.68 (s, 3H), 1.43 (s, 9H). MS calculated for C26H31ClN6O3S [M+H]+ 543.19,
found 543.21. Purity > 95%, tR = 5.2 min.

N-(2-aminoethyl)-2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo
[4,3-a][1,4]diazepin-6-yl)acetamide (8). A 4 mL vial was charged with compound 7 (4.0 mg,
7.4 µmol). To this solution was added 1:1 (v/v) ratio of DCM and TFA. The mixture was stirred
at room temperature for 2 h until completion as indicated by TLC. The organic content was

  4  
removed in vacuo and the residual was put on high vacuum for overnight. The product
appeared as pale yellow oil and was used for the next step without further purification. MS
calculated for C21H23ClN6OS [M+H]+ 443.14, found 443.15. Purity > 99%, tR = 4.8 min.

2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin
-6-yl)acetic amide - a Fluorescein-5-EX conjugate (9) (also named as MS574). A 4 mL vial
was charged with compound 8 (3.3 mg, 7.4 µmol). DMF (300 µL), Fluorescein-5-EX (4.4 mg,
7.4 µmol), and DIPEA (9.5 mg, 73.6 µmol) were added sequentially. The mixture was allowed to
stir at room temperature for overnight. At the time, the reaction was stopped and the solvent
removed by air stream. Purification by HPLC provided the title compound (10-90 % ACN in
water, tR = 12.8 min. The pure product appeared as a bright yellow powder (2.9 mg, 45%). MS
calculated for C46H40ClN7O8S2 [M+H]+ 918.21, found 918.22. Purity > 99%, tR = 5.2 min.

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  5  
 C N C N

R410 R410
K456 K456

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P379 P379

P434
N P434
N

NK-kB NK-kB
C C

Supplementary Figure 1. The NMR solution structure of the BRD4-BD2 bound to an NF-κ
B-K310ac peptide.
Superimposition of the backbone atoms (N, Cα and C’) of the final 20 NMR structures of the
bromodomain in complex with the NF-kB-K310ac peptide (green).
 A ZA B B
BC ZA BC

N140 N140

MS417 MS417

C C

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A A

B
ZA ZA
Y139 Y139
BC BC
Y97 Y97
L95 L95
N140 N140
D144 D144
L92 L92
B B
I146 I146
V87 V87
D145 D145
JQ1 JQ1
MS417 MS417
P82 C P82 C
Q85 Q85
F83 F83
M149 M149

W81 W81
A A

Supplemental Figure 2. (A) Stereoview of the crystal structure highlighting the electron density
of MS417 bound in the BRD4-BD1. (B) Comparison of the 3D crystal structures of the BRD4-BD1
bound to MS417 (light blue) and the BRD2-BD2 bound to JQ1 (grey) (PDB ID 3ONI). Side chains
of the key protein residues located at the ligand binding sites in each BrD are shown, color-coded
by atom type. Carbon atoms are shown in orange in BRD4-BD1/MS417 complex, and green in
the BRD2-BD2/JQ1 complex.
 Affinity of FITC-MS417 to different BrDs
A C BrD Kd (µM)
8 ˜
BRD3-2
BRD2-2 BRD4-BD1 0.085 ± 0.009
˜ BRD4-2 ˜
BRDT-2
˜ BRD3-1 1 BRD4-BD2 0.113 ± 0.010
BRD4-1
7
BRD2-1
KIAA2026 BRDT-1 BRD3-BD1 0.110 ± 0.010
TAF1L-2 TAF1L-1 PCAF ˜
˜ TAF1-2 TAF1-1 ˜
CECR2
GCN5L2 BRD3-BD2 0.147 ± 0.012
ZMYND8 BPTF ˜
CBP 18.0 ± 0.9
6
ZMYND11
PB1-2 PB1-1
PB1-4
PB1-5
PB1-3
ASH1L ˜ BRD7 10.8 ± 1.0
SMARCA2
SMARCA4
TRIM66
TRIM33
BPTF 11.8 ± 0.7
˜ PB1-6 TRIM24
TRIM28
BAZ1B No binding
BRWD1-1
BRWD3-1 BAZ1A
SP140L
BAZ2B SP140 BAZ2B No binding
˜ PHIP-1 BRWD2 BAZ2A ˜
2 ATAD2 No binding

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MLL

5 MLL4
ASH1L No binding
BRD8-2
BRD8-1 SMARCA4 No binding
˜ ATAD2 ˜
ATAD2B
BAZ1B
p300
PCAF No binding
BRD9
BRD1
BRD7
˜
CBP ˜
BRWD3-2
PHIP-1 No binding
4
BRPF1
BRPF3
BRWD1-2 PHIP-2 ˜ 3 TAF1L No binding
TAF1 No binding

B
Nomralized anisotropy

Nomralized anisotropy

1.0 CBP
1.0 BRD4-BD1
BPTF
BRD4-BD2
BRD7

BRD3-BD1 SMARCA4
0.5 0.5
BRD3-BD2 BAZ2B
! PCAF

0.0 0.0
0 2 4 6 8 0 50 100 150 200 250
[Protein] (μM) [Protein] (μM)

Supplemental Figure 3. Binding affinity of FITC-labeled MS417 (i.e. MS574) to different human
BrDs.
(A) Phyologenetic tree depicting a family of human bromodomains. The BrDs that were evaluated in
binding to FITC-labeled MS417 (also named as MS574) are indicated by a dot. These dots are color-
coded for high affinity (red), modest affinity (blue), or no binding (gray) as shown in C. (B) Represen-
tive fluoescence anisotropy binding curves of FITC-MS417 to different BrDs as a function of protein
concentration. (C) Table listing the affinity (Kd) of the FITC-MS417 binding to the BrDs that represent
different subgroups of the human BrD family as illustrated in the phyologenetic tree.
 A
Gene Total NF-!B Enrichment P value
expression genes targets fold
HIV vs. DMSO in GFP Up-regulation 866 30 1.48 0.028
Down-regulation 685 18 1.12 0.52
MS417 vs. DMSO in HIV Down-regulation 892 49 2.48 1.4e-7
Up-regulation 1192 14 0.51 8.3e-3
MS417 vs. DMSO in GFP Down-regulation 1467 69 2.11 1.7e-7
Up-regulation 1999 15 0.32 5.3e-7
!
MS417 MS417
down-regulated genes up-regulated genes
in HIV-infected RTECs in HIV-infected RTECs

1 MS417
13
1 up-regulated
5 0 0 genes in RTECs
1
33

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17
6
5 26 HIV down-regulated
genes in RTECs
14 5 MS417
down-regulated
genes in RTECs
HIV up-regulated
genes in RTECs

B HIV vs. DMSO in GFP MS417 vs. DMSO in HIV

Supplemental Figure 4. Microarray analysis of MS417 effects on gene transcription in human primary
renal tubular epithelial cells (RTECs).
(A) Venn diagram of the NF-κB targets with transcriptional expression affected by both MS417 and HIV infection.
These genes were identified by Significance Analysis of Microarray program using q value of 0.05. Upper, a table
lists enrichment statistics of these NF-κB targets affected by HIV infection and/or MS417. The enrichment fold is
the ratio of the percentage of NF-κB targets in differentially expressed genes to that of NF-κB targets in the whole
gene list. The p value was calculated using Fisher's exact test. (B) Regulation of the NF-κB network derived from
Ingenuity Knowledge database. The genes are color-coded in red or green indicating their up- or down-regulation
under the specified conditions.
A

l)
)

m
TN (μM

g/
(n
1 7
Fα
S4
10.0

M
0

■ ■ ■
8.0 0.1
mRNA level (fold)

1
6.0
0 10

■ ■ ■
4.0 0.1 10
1 10
2.0

0
CCL2 CCL20 CXCL11 CXCL3 LTB CCL3 IL1A IL1B LTA

B C

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FAS TRAF1
4.5 6.0
l)
)
/m
M

4.0
(μ

g
(n

5.0 5.0
17

Fα

3.5
S4

TN
M

mRNA level (fold)

mRNA level (fold)

4.0 0 3.0 4.0
■ ■ ■
mRNA level (fold)

0.1 2.5
3.0 1 3.0
2.0
2.0 0 10 1.5 2.0
*
■ ■ ■

0.1 10 1.0 *
1.0 1 10
* * 1.0
0.5
* * *
*
0 0 0
CXCL5 CXCL6 MS417 (μM) 0 0.1 1 0 0.1 1 0 0.1 1 0 0.1 1
TNFα (ng/ml) 0 0 0 10 10 10 0 0 0 10 10 10

Supplemental Figure 5. Modulation of transcription of NF-κB target and non-target genes by MS417.
(A) MS417inhibits TNFα-induced NF-κB target gene expression in RTEC. The RTEC cells were pre-incubated
with or without MS417 at the indicated doses for 30 minutes in serum-free medium and then stimulated with or
without TNFα for additional 6 hours. The cells were harvested for cytokine and chemokine PCR arrays. Repre-
sentative data from three independent experiments are shown. (B) Some of TNFα-induced NF-κB target genes
were not inhibited by MS417. Renal tubular epithelial cells were pre-incubated with or without MS417 at the
indicated doses for 30 minutes in serum-free medium and then stimulated with or without TNFa for additional 6
hours. The cells were then harvested for cytokine and chemokine PCR arrays. Representative data from three
independent experiments are shown. (C) MS417 inhibits the expression of TNFα-induced apoptosis related
genes FAS and TRAF1. The RTEC cells were pre-incubated with or without MS417 at the indicated doses for 30
minutes in serum-free medium and then stimulated with or without TNFα for additional 6 hours. Cells were then
harvested for real-time PCR analysis.
 Absobance Time (min)

MS417
Absobance

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Time (min)

MS566
Absobance

Time (min)
Absobance

Time (min)

Supplemental Figure 6. The chiral purification chromatograms of MS417.

(A) HPLC chromatogram of MS417 after stereospecific synthesis. The ratio of MS417 over its
enantiomer (MS566) is 85:15; (B) HPLC chromatogram of MS417 after chiral purification with
purity of >99%; (C) HPLC chromatogram of an enantiomer (MS566) of MS417 after chiral purifi-
cation with purity of >99%; (D) HPLC chromatogram of MS417 racemic mixture. The ratio of
MS417 to MS566 is 50:50.
Supplemental Table 1. NMR restraints and statistics of the final 20 structures of the BRD4-
BD2 in complex with a NF-κB-K310ac peptide

NMR distance and dihedral constraints

Distance constraints
Total NOE 2,857
Intra-residue 856
Inter-residue 2,001
Sequential (|i - j| = 1) 609
Medium-range (|i - j| < 4) 725
Long-range (|i – j| > 5) 667
Inter-molecular 20

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Hydrogen bonds 58
Total dihedral angle restraints
Φ angle 79
Ψ angle 79
Ramachandran Map Analysis (%)
Most favored regions 97.8
Additional allowed regions 2.2
Generously allowed regions 0.0
Disallowed regions 0.0
Structure statistics
Violations (mean +/- s.d.)
Distance constraints (A) 0.11 ± 0.0085
Dihedral angle constraints (º) 1.07 ± 0.11
Max. dihedral angle violation (º) 1.33
Max. distance constraint violation (A) 0.12
Deviations from idealized geometry
Bond lengths (A) 0.0011 ± 0.00017
Bond angles (º) 1.1 ± 0.015
Impropers (º) 2.3 ± 0.079
Average pairwise r.m.s. Deviation ** (A)
Heavy 0.58 ± 0.093
Backbone 0.28 ± 0.093
**: The residue number ranges used in full molecule RMSD calculations are 352-455.
Pairwise r.m.s. deviation was calculated among 20 refined structures
%: Procheck residue numbers are 349-369, 400-409, 415-430 and 436-456.
Supplemental Table 2. Crystallography data and refinement statistics.
BRD4-BD1/MS417
Data collection
Space group P212121
Cell dimensions
a, b, c (Å) 36.84, 44.66, 78.24
α, β, γ (°) 90.0, 90.0, 90.0
Resolution (Å) (highest resolution shell) 39.12-1.40
(1.43-1.4)
Measured reflections 152,108
Unique reflections 25,982

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Rmerge 5.5 (33.5)
I/σ 43.7 (4.8)
Completeness (%) 99.3 (98.3)
Redundancy 5.8 (5.5)
Refinement
Resolution (Å) 39.10-1.40
No. reflections 24,656
Rwork/ Rfree (%) 13.6/18.3
No. atoms
Protein 1,076
MS417 28
Water 187
B-factors (Å2)
Protein 16.8
MS417 11.7
Water 28.3
RMSD
Bond lengths (Å) 0.025
Bond angles (º) 2.40
Ramachandran plot % residues
Favored 99.2
Additional allowed 0.8
Generously allowed 0
Disallowed 0

1
 Supplemental Table 3. Summary of body weight, renal function and histology changes
Podocyte Tubular
Body weight BUN (mg/dl) AST & ALT GS index
Hypertrophy casts/cysts

WT + DMSO 22.4 ± 1.3 23.5 ± 3.4 93 ± 11 & 47 ± 4 0 0 0

Tg26 + DMSO 21.8 ± 1.5 39.4 ± 4.6 95 ± 10 & 45 ± 3 16.3 ± 7.1 1.5 ± 0.6 10.4 ± 4.8

WT + MS417 22.8 ± 1.7 24.2 ± 2.2 95 ± 11 & 47 ± 2 0 0 0

Tg26 + MS417 21.2 ± 1.6 26.8 ± 3.5* 96 ± 10 & 47 ± 3 3.1 ± 0.9* 0.5 ± 0.5* 2.4 ± 1.0*

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Note: HIV-1 transgenic (Tg26) mice and the wild-type littermates were treated with vehicle (DMSO) or MS417 at 0.08 mg/kg
from age of 4 weeks and the mice were sacrificed at the age of 8 weeks. Body weight of these mice was recorded and blood
urea nitrogen (BUN) was measured as described. The liver function test (AST & ALT) was measured by the Charles River
Research Animal Diagnosis Service (Wilmington, MA). The kidney sections from these mice were stained for PAS and the
glomerular and tubular injuries were scored as described. N=6, *p<0.05 as compared to Tg26 mice treated with DMSO (0.1%).

 
 Supplemental Table 4. Optical Rotation Measurements
Compound αD
20
Concentration Solvent Path length No. of Measurement
Fmoc-L-Asp(OMe)-OH -25.8 0.01 DMF 10 mm 4
2 18.5 0.01 CHCl3 10 mm 4
3 28.7 0.01 CHCl3 10 mm 4
4 21.1 0.01 CHCl3 10 mm 4
MS417 (5) 19.9 0.01 CHCl3 10 mm 4
MS566 -20.1 0.01 CHCl3 10 mm 4
 
 
 

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