LETTER RESEARCH
Reporter constructs. Gal4–RORa LBD and Gal4–RORc LBD were gifts from
METHODS
Phenex Pharmaceuticals AG. The IL-17 reporter construct was purchased from
SR1001. For the synthesis of SR1001 (N-(5-(N-(4-(1,1,1,3,3,3-hexafluoro-2-
hydroxypropan-2-yl)phenyl)sulphamoyl)-4-methylthiazol-2-yl)acetamide), a solu-
tion of 2-(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (0.88 g, 3.4 mmol),
9
ATCC and has been previously described .
Reporter gene assays. Twenty-four hours before transfection, HEK293 cells were
3
plated in 96-well plates at a density of 153 10 cells per well. Transfections were
2-acetamido-4-methylthiazole-5-sulphonyl chloride (0.79 g, 3.1 mmol) in acetone
performed using Lipofectamine 2000 (Invitrogen). Twenty-four hours after trans-
fection, the cells were treated with vehicle or compound. Twenty-four hours after
treatment, the luciferase activity was measured using the Dual-Glo luciferase assay
system (Promega). Results were analysed using GraphPad Prism software.
Radioligand binding assay. Radioligand binding assays were performed as previ-
(15 ml) and 2,6-lutidine (0.73 ml, 6.2 mmol) was warmed to 60 uC for 18h. The
reaction was judged complete by analytical HPLC (starting materials consumed).
Thesolventwas removedin vacuo, and thecruderesidue was diluted withEtOAc and
aqueous 1 M HCl. The layers were separated, and the organic layer was washed with
1
M HCl, saturated aqueous NaHCO
a solid. Trituration with warm Et O/hexanes afforded N-(5-(N-(4-(1,1,1,3,3,3-hexa-
fluoro-2-hydroxypropan-2-yl)phenyl)sulphamoyl)-4-methylthiazol-2-yl)acetamide
1.2 g, 86% yield) as a light tan solid, .95% pure as judged by analytical HPLC. A
small amount of this was further purified by reverse-phase preparative HPLC to
3 4
, brine, dried MgSO , and concentrated to give
1
1
ously described . For the competition assay, various concentrations of SR1001
2
3
were incubated with receptor in the presence of 3 nM [ H]-25-hydroxycholesterol.
Results were analysed using GraphPad Prism software and the K
i
was determined
(
using the Cheng–Prusoff equation.
8
1
Alpha screen. The Alpha screen assays were performed as previously described .
Assays were performed in triplicate in white opaque 384-well plates (Greiner Bio-
One) under green light conditions (,100lx) at room temperature. The final assay
volume was 20 ml. All dilutions were made in assay buffer (100 mM NaCl, 25 mM
.
1
99%purity to give a colourless solid. HNMR(DMSO-d
0.8 (s, 1H), 8.6(s, 1H), 7.60 (d, 2H), 7.25 (d, 2H), 2.30 (s, 3H), 2.15(s, 3H); CNMR
, 100MHz) d 170.0, 159.6, 153.0, 139.4, 128.4, 126.8, 124.8, 121.9, 121.7,
6
, 400 MHz) d12.5 (s, 1H),
13
(DMSO-d
6
19
120.3, 22.8, 16.3; F NMR (DMSO-d
6
, 376MHz) d 274.1; HRMS (ESI-orbitrap)
HEPES, 0.1% BSA, pH 7.4). The final DMSO concentration was 0.25%. A mix of
calculated for C15
H
14
F
6
N
3
O
4
S
2
, 478.0330; found, 478.0319.
21
1
2 ml of GST–RORc LBD (10nM), beads (12.5 mg ml of each donor and
Mice. All mice were maintained in a specific pathogen-free environment in
accordance with institutional protocol. All procedures were reviewed and
approved by either The Scripps Research Institute or the University of Arkansas
for Medical Sciences Institutional Animal Care and Use Committee. C57BL/6
mice purchased from Jackson laboratories were used for all in vitro experiments
unless otherwise noted. EAE was induced in 8-week-old male wild-type C57BL/6
mice purchased from Harlan. Male DIO mice, 22 weeks of age, were purchased
from Jackson Laboratories and fed a high fat diet (60% kCal% fat) (Research Diets)
for the duration of the study.
acceptor), and 4 ml of increasing concentrations (210 nM to 50 mM) of compound
SR1001 were added to the wells, the plates were sealed and incubated for 1 h. After
this pre-incubation step, 4 ml of biotin-TRAP220-2 peptide (50nM) was added,
the plates were sealed and further incubated for 2 h. The plates were read on
PerkinElmer Envision 2104 and data analysed using GraphPad Prism software.
RNA-mediated interference. EL4 cells were first electroporated with 100nM
total siRNA with the GenePulserXcell Electroporator using siRNA against mouse
RORa and RORc (Dharmacon RNA Technologies) followed by reverse transfec-
tion using 50 nM siRNA according to the instructions for Dharma-FECT 1 trans-
fection reagent and seeded onto a 12-well plate. Twenty-four hours after
transfection, cells were treated with vehicle (DMSO) or SR1001 (10 mM) for
Induction and clinical evaluation of EAE. EAE was induced in C57BL/6 wild-
type mice by subcutaneous injection over four sites in the flank with 200 mg per
mouse MOG35–55 peptide (C S Bio Co.) in an emulsion with IFA supplemented
24 h. Cells were harvested and total RNA was isolated. Quantitative reverse tran-
21
with 2.25 mg ml Mycobacterium tuberculosis, strain H37Ra (Difco). Pertussis
toxin (List Biological Laboratories) dissolved in PBS was injected intraperitoneally
at 200ng per mouse at the time of immunization (day 0) and 48 h later. Mice were
scriptase PCR was performed to analyse mRNA levels of mouse Rora, Rorc, Gapdh
and Il17a using SYBR green technology. Primers sequences to mouse Rora, Rorc,
5,6,19
Il17a and Gapdh and have previously been described . HepG2 cells were treated
15
scored daily on a scale of 0–6, as described previously : 0, no clinical disease; 1,
limp/flaccid tail; 2, moderate hindlimb weakness; 3, severe hindlimb weakness; 4,
complete hindlimb paralysis; 5, quadriplegia or pre-moribund state; 6, death. All
mice were 7–10 weeks of age when experiments were performed. The SR1001 was
similarly to EL4 cells with the following exceptions: HepG2 cells were transfected
with siRNA against human RORA and RORC (Dharmacon RNA Technologies) at
50 nM according to the instructions for Dharma-FECT 1 transfection reagent.
Quantitative reverse transcriptase PCR was performed to analyse mRNA levels of
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dissolved in DMSO at 25 mg ml and the mice were treated (intraperitoneally)
human RORA, RORC, cyclophilin and G6PC using SYBR green technology. The
2
1
21
with 25 mg kg SR1001 (1 ml g body weight of mouse) or vehicle (DMSO,
1
8
primer sequences have previously been described .
21
ml g body weight of mouse) twice per day. The treatment was started 2 days
Quantitative RT–PCR. Splenocyte total RNA was extracted using a RNeasy Plus
Micro Kit (Qiagen) and reverse transcribed using the iScript cDNA biosynthesis
kit(Bio-Rad). Total RNAfrom spinal cord from MOG-immunized mice andlivers
from DIO mice was isolated using TRIzol reagent (Invitrogen) followed by
DNase1 treatment (Invitrogen) and reverse transcribed using the iScript cDNA
biosynthesis kit. Quantitative RT–PCR was performed with a 7900HT Fast Real
Time PCR System (Applied Biosystems) using SYBR green (Roche) as previously
before immunization and continued until the end of experiment. Where indicated
in the figure legends, mice were anaesthetized with halothane and transcardially
perfused with PBS, and spinal cords were removed for RNA and protein isolation.
Cell lines. HEK293 cells and EL4 cells (American Type Culture Collection) were
maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS
and antibiotics (penicillin and streptomycin; Invitrogen). HepG2 cells were main-
tained in minimal essential medium supplemented with 10% FBS and antibiotics.
PBMCs were obtained from Astarte Biologics and maintained in RPMI-1640 with
20
described . Primer sequences for mouse Il17a, Rorc, Rora, Il17f, Il21, Il22, T-bet
5,19,21,22
(also called Tbx21), Gata3 and Foxp3 have been previously described
. The
1
0% FBS and antibiotics.
level of mRNA expression was normalized to that of Gapdh mRNA expression.
T-cell differentiation in vitro. The conditions for the different T-helper cell subsets The following primer sequences were used: Cyp7b1 forward 59-GGGAAG
2
1
21
were: 20 mg ml anti-IL-4 (clone 30340, R&D Systems) and 20 mgml anti-IFN-c AAGCTGAAGACTTACG-39, reverse 59-CCCTATAGGCTTCCTGTCGAT-39;
21
(
clone H2, R&D Systems) for T O (neutral conditions); 20 mg ml anti-IL-4, 20 ng Nr1d1 forward 59-ACCTTTGAGGTGCTGATGGT-39, reverse 59-CTCGCTG
H
2 21
1
ml IL-12 (R&D Systems) and 10 ng ml IFN-c (R&D Systems) for T
H
1 condi- AAGTCAAACATGG-39; Serpine1 forward 59-CTCGCTGAAGTCAAACAT
2 condi- GG-39, reverse 59-TTTTGCAGTGCCTGTGCTAC-39.
tions; 10 mgml anti-IFN-c, 10mg ml anti-IL-4 and 2 ng ml TGF-b (R&D Western blot analysis. HepG2 and EL4 cells were washed once with phosphate-
2
1
21
tions; 20 mg ml anti-IFN-c and 10 ng ml IL-4 (R&D systems) for T
H
2
1
21
21
21
21
Systems) for inducible Treg conditions; 20 mgml anti-IFN-c, 20mg ml anti-IL- buffered saline and then incubated for 10 min at 4 uC in 100ml of TNT lysis buffer
, 1 ng ml TGF-b and 10 ng ml IL-6 (R&D Systems) for T 17 conditions. All (50 mM Tris-Cl, pH 7.5, 150 mM NaCl and 1% Triton X-100) and a complete
cultures were stimulated with 1 mgml anti-CD3 (eBiosciences) and 1 mg ml anti- miniprotease inhibitor mixture (Roche Applied Science). Samples were then har-
CD28 (eBiosciences). For naive T-cell differentiation, CD4 CD25 CD62L CD44
vestedinto1.5-mlmicrocentrifugetubes, vortexedfor30s,andthencentrifuged(10
cells were FACS sorted on a BD FACSAriaII. Naive CD4 T cells were activated with min).ProteinlevelsinthesupernatantsweredeterminedusingaCoomassieprotein
21
21
4
H
21 21
1
2
hi
lo
1
21
21
5
mgml plate-bound anti-CD3 and 1 mg ml anti-CD28 in the presence of 20 mg assay kit (Bio-Rad), and 10mg of protein from each sample was separated by SDS–
21 21 21 21
ml anti-IFN-c, 20mgml anti-IL-4, 1 ng ml TGF-b and 10 ng ml IL-6, PAGE (BioRad, 10%) and then transferred to a polyvinylidene difluoride mem-
similar to splenocyte activation. Four to five days after activation, all cells were re- brane (Millipore) and immunoblotted with primary antibodies: mouse RORa
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stimulated with 5 ng ml phorbol-12-myristate-13-acetate (PMA) (Sigma) and (BioLegend), mouse RORc (BioLegend), human RORa (Perseus Proteomics),
00 ng ml ionomycin (Sigma) for 2h with the addition of GolgiStop (BD human RORc (IMGENEX), or a-tubulin (Sigma) and horseradish peroxidase-
21
5
Bioscience) for an additional 2 h before intracellular staining. Similar re-stimulation conjugated secondary antibodies (Jackson Immunoresearch). Detection of the
withPMAand ionomycin occurred for ELISA. For human PBMCanalysis, cells were bound antibody by enhanced chemiluminescence was performed according to
21
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re-stimulated with 5 ng ml PMA (Sigma) and 500 ng ml ionomycin (Sigma) for the manufacturer’s instructions (Santa Cruz).
h with the addition of GolgiStop (BD Bioscience) for an additional 2 h before Hydrogen/deuterium exchange mass spectrometry. Differential, solution phase
intracellular staining. Cells were cultured in RPMI 1640 medium (Invitrogen) with HDX experiments were performed with a LEAP Technologies Twin HTS PAL
0% FBS and antibiotics. liquid handling robot interfaced with an Orbitrap mass spectrometer (Exactive,
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1
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2011 Macmillan Publishers Limited. All rights reserved