Minor structural change to tertiary sulfonamide RORC ligands led to opposite mechanisms of action

Article abstract of DOI:10.1021/ml500420y

A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity. Compounds behaving as agonists in our biochemical assay also gave rise to an increased production of IL-17 in human PBMCs whereas inverse agonists led to significant suppression of IL-17 under the same assay conditions. The most potent inverse agonist compound showed >180-fold selectivity over the ROR isoforms as well as all other nuclear receptors that were profiled.

Full text of DOI:10.1021/ml500420y

Letter
pubs.acs.org/acsmedchemlett
Minor Structural Change to Tertiary Sulfonamide RORc Ligands Led
to Opposite Mechanisms of Action
Olivier Rene,*,† Benjamin P. Fauber,† Gladys de Leon Boenig,† Brenda Burton,‡ Celine Eidenschenk,†
́
́
Christine Everett,† Alberto Gobbi,† Sarah G. Hymowitz,† Adam R. Johnson,† James R. Kiefer,†
Marya Liimatta,† Peter Lockey,‡ Maxine Norman,‡ Wenjun Ouyang,† Heidi A. Wallweber,†
and Harvey Wong†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡Argenta, Early Discovery, Charles River, 7-9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, U.K.
S
* Supporting Information
ABSTRACT: A minor structural change to tertiary sulfona-
mide RORc ligands led to distinct mechanisms of action. Co-
crystal structures of two compounds revealed mechanistically
consistent protein conformational changes. Optimized phenyl-
sulfonamides were identified as RORc agonists while
benzylsulfonamides exhibited potent inverse agonist activity.
Compounds behaving as agonists in our biochemical assay also
gave rise to an increased production of IL-17 in human
PBMCs whereas inverse agonists led to significant suppression
of IL-17 under the same assay conditions. The most potent
inverse agonist compound showed >180-fold selectivity over
the ROR isoforms as well as all other nuclear receptors that
were profiled.
KEYWORDS: RORc, RORγ, TH17, IL-17, PBMC, agonist, inverse agonist
he retinoic acid receptor-related orphan receptor gamma
of the NR but do not affect the basal transcriptional activity of
the protein. Such molecules may be termed silent ligands or
neutral antagonists.15
1
T(RORγ or RORc) is a key nuclear receptor (NR) of the
ROR family that has been implicated in the production and
regulation of interleukin-17 (IL-17).2 This cytokine is required
In addition to numerous small molecule RORc modulators
previously reported,9−11 compound 1 was recently disclosed as
a potent biaryl sulfonamide inverse agonist that was identified
in a biochemical screen of the Genentech/Roche corporate
collection (Figure 1).16 Co-crystallization of 1 with the human
RORc-LBD [PDB: 4QM0]17 revealed that the benzylic phenyl
ring of 1 interacted with His479, thus preventing His479 from
forming a crucial hydrogen bond with Tyr502 in the protein.
The interaction between His479 and Tyr502 is required to
stabilize the secondary structure of helices 11−12 and induce
coactivator recruitment.18 The disruption of this interaction
may account for the inverse agonist mechanism of action
(MOA)19,20 observed in our biochemical coactivator recruit-
ment assay with compound 1. Further replacement of the biaryl
motif in 1 with an acylpiperazine (2) led to significant
improvement of the physicochemical properties, plasma−
protein binding, and permeability of the series while preserving
RORc inverse agonist potency.17
̈
for the differentiation of naıve pro-inflammatory CD4+ T cells
and for the production of T helper-17 (TH17) cells,2 which
results in the expression of diverse pro-inflammatory cytokines
such as IL-17,2 IL-22,3 and GM-CSF.4 Interleukin-17 is a
critical driver of autoimmune diseases such as psoriasis,5
rheumatoid arthritis (RA),6 multiple sclerosis (MS),7 and
inflammatory bowel disease (IBD).8 Therefore, the modulation
of IL-17 expression through small molecule inhibitors of
RORc9−11 has gained significant momentum as a molecular
target since the first disclosure of a direct link between RORc
signaling and IL-17 production by Littman et al. in 2006.2
Small molecule NRs may modulate transcription through
several mechanisms.12,13 Ligands can induce a conformational
change in the NR that results in the reduction of coactivator
protein recruitment to the NR. Such ligands are termed inverse
agonists.14 Inverse agonists decrease gene transcription, and in
the case of RORc, this results in a reduction of IL-17
production. Conversely, ligands that promote coactivator
protein recruitment and formation of the [NR·coactivator]
complex generally result in increased gene transcription and are
termed agonist ligands.14 A third method of modulation
involves ligands that bind to the ligand-binding domain (LBD)
Received: October 15, 2014
Accepted: December 4, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/ml500420y
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ACS Medicinal Chemistry Letters
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Figure 1. Evolution of Genentech’s tertiary sulfonamide RORc modulators.
Additional optimization of benzylsulfonamide replacements
was explored using parallel synthesis and resulted in the
discovery of the matched pair of phenyl sulfonamide 3 and
benzyl sulfonamide 4. While benzyl analog 4 showed the typical
inverse agonist behavior for the series, phenyl sulfonamide 3
was a high affinity ligand of RORc that showed strong agonist
efficacy in the biochemical coactivator recruitment assay.
Herein, we disclose the discovery of additional phenyl-
sulfonamide and benzylsulfonamide pairs that demonstrate
that a minor change in the structure of the ligand (i.e., one
methylene group) can lead to agonist and inverse agonist
MOAs, respectively.
To better understand how the changes to the sulfonamide
groups affected the binding mode of the ligands and resulted in
divergent MOAs, we cocrystallized inverse agonist ligand 2 and
agonist ligand 3 with the human RORc-LBD and obtained 2.0
and 2.2 Å resolution X-ray structures, respectively.21 In both
cases, the ligands were bound within the ligand binding pocket
of RORc (Figure 2), and approximately one-third of the ligands
(from the acetyl moiety through the piperazine ring)
superimposed nearly identically. The trajectory of the
compounds then diverged slightly, projecting their terminal
phenyl rings from diverging vectors toward helix 11. The
agonist ligand 3 (Figure 2, purple ribbon) packed against
Trp317, His479, and Tyr502, stabilized the agonist con-
formation of helix 12, and recruited the coactivator peptide. On
the other hand, the benzyl moiety of the inverse agonist ligand
2 (Figure 2, yellow ribbon) displaced His479, and prevented
formation of the hydrogen bond with Tyr502 of helix 12. The
disruption of the hydrogen bond between His479 and Tyr502
dislodged helix 12 entirely in the costructure with 2 and
eliminated the binding site for coregulator proteins. This
observation was consistent with the RORc inverse agonist
activity of 2. The C-terminal half of helix 11 bent 16° inward
and filled in the gap created while multiple aromatic side chains
reoriented in a switch-like fashion, and repacked the apex of the
pocket in the absence of helix 12. Similarly, concerted motions
of Trp317 and Phe486 rotated, and filled pockets created by
these changes. Both 2 and 3 formed a single hydrogen bond
from the oxygen of their terminal acetyl groups to an ordered
water molecule at the opposite end of the pocket from helix 12
(Figure S2). The rest of the contacts made to the compounds
were van der Waals interactions with the predominantly
hydrophobic binding site residues.
Figure 2. Binding modes of agonist ligand 3 and inverse agonist ligand
2 within the ligand binding pocket of RORc. The crystal structure of
the agonist ligand 3 (purple ribbon) stabilized helix 12 and revealed a
coactivator peptide (green) bound to the receptor. The crystal
structure of the inverse agonist ligand 2 (yellow ribbon) did not have
resolved density for helices 11−12. Concerted motions of Trp317,
Phe486 and His479 are depicted with arrows.
a
Scheme 1. Synthesis of Sulfonamides 3, 4, and 9−33
a
Reagents and conditions: (a) Pd(OAc)2, RuPhos, NaOt-Bu, 1,4-
dioxane, 100 °C; (b) HCl, H2O, THF, 23 °C, 60% (2 steps); (c)
cyclobutylamine, NaBH(OAc)3, AcOH, 1,2-DCE, 23 °C, quant.; (d)
R-SO2Cl, DIPEA, CH2Cl2, 23 °C, 29−57%.
To further evaluate the effects of different sulfonamide
substituents on the MOA, we synthesized a series of benzyl-
and arylsulfonamides using a late-stage diversification synthetic
approach (Scheme 1). Benzaldehyde intermediate 7 was
obtained using a Buchwald−Hartwig amination reaction22
between acetylpiperazine 5 and acetal-protected bromoarene
6, followed by acetal removal under acidic conditions.
B
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a
Table 1. Structure−Activity Relationships of Phenyl- and Benzylsulfonamide Matched Pairs
b
c
d
e
f
Compd
n
R
RORc IC50 (μM)
RORc SRC1 EC50 (μM) [% efficacy]
cLogP
LLE
MOA
Agonist
3
0
1
2
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
0
H
H
H
0.25
0.069 [+35%]
0.011 [−99%]
0.31 [−64%]
0.063 [+46%]
0.003 [−98%]
0.041 [+46%]
0.010 [−98%]
>10 [−37%]
0.007 [−97%]
0.047 [+33%]
0.004 [−96]
0.050 [+53%]
0.009 [−98%]
0.19 [−63%]
0.033 [−97%]
0.006 [+65%]
0.040 [−92%]
0.014 [+43%]
3.7
3.3
3.7
3.9
3.4
3.9
3.5
3.9
3.5
4.4
3.9
4.4
4.1
4.4
4.1
5.1
4.8
4.1
3.8
3.5
4.7
2.8
3.3
5.1
3.5
4.5
-
4
0.015
0.11
Inverse Agonist
Inverse Agonist
Agonist
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-F
0.076
0.010
0.065
0.025
0.22
2-F
Inverse Agonist
Agonist
3-F
3-F
Inverse Agonist
Inverse Agonist
Inverse Agonist
Agonist
4-F
4-F
0.017
0.055
0.009
0.022
0.019
0.092
0.037
0.006
0.029
0.049
0.036
4.7
2.9
4.5
2.9
4.0
2.3
3.4
3.4
2.6
3.7
3.8
2-Cl
2-Cl
Inverse Agonist
Agonist
3-Cl
3-Cl
Inverse Agonist
Inverse Agonist
Inverse Agonist
Agonist
4-Cl
4-Cl
3,5-diCl
3,5-diCl
3,5-diF
3-OMe
Inverse Agonist
Agonist
0.026 [+54%]
Agonist
a
b
All assay results are reported as the geometric mean of at least two separate runs.24 Biochemical inhibition of the RORc-LBD and [3H2]25-
c
hydroxycholesterol interaction. Activation or inhibition of RORc-LBD recruitment of the SRC1 coactivator; positive “%eff.” denotes agonism and
negative “%eff.” denotes inverse agonism relative to the basal assay signal for apo-RORc LBD in this assay format. Calculated logP (cLogP) value.27
d
e
f
Ligand-lipophilicity efficiency (LLE)25 was calculated using RORc SRC1 EC50 and the cLogP. Mechanism of action (MOA) reported as agonist,
inverse agonist, or silent ligand.
Subsequent reductive amination of aldehyde 7 with cyclobutyl-
amine gave the advanced intermediate amine 8. This late-stage
intermediate allowed for the incorporation of a diverse set of
sulfonyl chlorides and gave sulfonamides 3, 4, and 9−33.23
The analogs were then evaluated in two RORc biochemical
assays.24 The affinity of the analogs for the RORc-LBD was
measured using a radiometric binding assay that monitored the
displacement of [3H2]25-hydroxycholesterol from the RORc-
LBD. Compounds were also evaluated in a time-resolved
fluorescence biochemical assay that monitored the ability of the
human RORc-LBD to bind to a coactivator peptide derived
from steroid receptor coactivator-1 (SRC1). Compounds
disrupting the recruitment of the SCR1 peptide were
determined to be inverse agonists whereas compounds that
enhanced peptide recruitment were agonists. Alternatively,
compounds that did not modulate the level of recruitment but
showed affinity in the radiometric binding assay were identified
as silent ligands. Full inverse agonism (−100% efficacy) was
experimentally measured and corresponded to complete
suppression of basal activity. Full agonism (+100% efficacy)
was calculated as the additive inverse of a complete suppression
of basal activity, since the experimental maximum % efficacy of
a RORc full physiological agonist is currently undefined.
Matched pair analysis of the aryl- and benzylsulfonamides
(Table 1) revealed that phenylsulfonamide 3 was a RORc
agonist (EC50 = 69 nM, +35% efficacy) while the
corresponding benzyl analog 4 was a potent full inverse agonist
(EC50 = 11 nM, −99% efficacy). The phenethylsulfonamide (9)
was also an inverse agonist, albeit with a significantly lower
potency and efficacy (EC50 = 310 nM, −64% efficacy). We then
sought to improve the potency of our ligands by introducing
small nonpolar substituents around the arene to provide
favorable van der Waals interactions with the ligand-binding
pocket. The ligand-lipophilicity efficiency (LLE)25,26 was used
as a metric to ensure the effective use of nonpolar interactions.
Incorporation of a 2-fluorine substituent in compound 10
resulted in an equipotent and equi-efficacious agonist (EC50
=
63 nM, +46% efficacy) when compared to the phenyl analog 3.
However, the same 2-fluoro substituent provided a more
substantial potency improvement with inverse agonist benzyl
compound 11 (EC50 = 3 nM, −98% efficacy). The LLE of
compound 11 was also improved compared to compound 4.
Similar trends were observed with 3-fluorophenylsulfonamide
12 and 3-fluorobenzylsulfonamide 13, with 12 being a slightly
more potent agonist (EC50 = 41 nM, +46% efficacy) than 10.
Interestingly, 4-fluoro substitution (14) led to a loss of effect on
peptide recruitment and weak inverse agonism (EC50 > 10 μM,
−37% efficacy), although this ligand demonstrated significant
affinity for the receptor in the radioligand binding assay (RORc
IC50 = 220 nM). The 4-fluorobenzylsulfonamide 15 was a
potent inverse agonist (EC50 = 7 nM, −97% efficacy).
A similar scan was performed with 2-, 3-, and 4-chloro
substituted phenyl- and benzylsulfonamides 16−21. The 2- and
3-chlorophenylsulfonamides (16 and 18, respectively) were
both similarly potent agonists (EC50 = 47 nM, +33% efficacy
and 50 nM, +53% efficacy, respectively), and their benzylsulfo-
C
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a
Table 2. Structure−Activity Relationship of 4-Substituted Arylsulfonamides
b
c
d
e
f
Compd
R
RORc IC50 (μM)
RORc SRC1 EC50 (μM) [% efficacy]
cLogP
LLE
MOA
Silent
26
27
28
29
30
31
32
33
2,4-diMe
4-CN
0.024
0.28
>10 [+9%]
>10 [−29%]
3.3 [−52%]
0.22 [−37%]
0.15 [−75%]
0.16 [−82%]
0.30 [−39%]
4.7
3.4
3.8
5.2
4.1
4.9
4.6
5.3
Inverse Agonist
Inverse Agonist
Inverse Agonist
Inverse Agonist
Inverse Agonist
Inverse Agonist
Inverse Agonist
4-OMe
2,4-diCI
4-OCF2H
4-OCF3
4-CF3
0.12
1.7
1.5
2.7
1.9
1.9
1.6
0.076
0.031
0.29
0.050
0.018
4-t-Bu
0.12 [−55%]
a
b
All assay results are reported as the geometric mean of at least two separate runs. Biochemical inhibition of the RORc-LBD and [3H2]25-
c
hydroxycholesterol interaction. Activation or inhibition of RORc-LBD recruitment of the SRC1 coactivator; positive “%eff.” denotes agonism, and
negative “%eff.” denotes inverse agonism relative to the basal assay signal for apo-RORc LBD in this assay format. Calculated logP (cLogP) value.27
d
Ligand-lipophilicity efficiency (LLE)25 was calculated using RORc SRC1 EC50 and the cLogP. Mechanism of action (MOA) reported as agonist,
e
f
inverse agonist, or silent ligand.
namide counterparts 17 and 19 displayed full inverse agonist
MOA with RORc SRC1 EC50 values of 4 nM and 9 nM,
respectively. Substitution at the 4-position of arylsulfonamide
20 with the chloride atom led to a reversal of MOA, giving rise
to an inverse agonist (EC50 = 190 nM, −63% efficacy), when
compared to 3. The 4-chlorobenzyl (21) matched pair to 20
also showed inverse agonism (EC50 = 33 nM, −97% efficacy),
although not as potent as other substituted benzylsulfonamides.
Finally, the 3,5-dichloroarylsulfonamide 22 gave rise to our
most potent agonist (EC50 = 6 nM, +65% efficacy), albeit with
a poor LLE of 3.4 resulting from the high lipophilicity of the
dichloroarene. Conversely, the 3,5-dichlorobenzyl analog 23
was a potent inverse agonist (EC50 = 40 nM, −92% efficacy).
Replacing the chlorine atoms of 22 with less lipophilic fluorines
(24) led to a higher LLE agonist (EC50 = 14 nM, +43%
efficacy, LLE = 3.7). A further reduction in lipophilicity was
accomplished by appropriately incorporating a 3-methoxy
substituent (25), which led to further improvement of the
LLE (EC50 = 26 nM, +54% efficacy, LLE = 3.8). This initial
SAR on the right-hand side (RHS) identified single-digit
nanomolar analogs with both agonist and inverse agonist
profiles. Phenylsulfonamides generally behaved as agonists of
RORc, while the benzylsulfonamides acted as inverse agonists.
Intrigued by the reversal of MOA of 4-substituted
arylsulfonamides 14 and 20, we sought to rationalize this
phenomenon by further examining the X-ray costructure of
agonist 3. The unsubstituted phenyl ring of 3 was directed at
Trp317 and formed an edge-to-face π−π interaction (3.6 Å),28
which in turn formed a π stacking interaction with Phe486
(4.2 Å), stabilizing helix 11 and the bound ligand. Substitution
at the 4-position on the phenyl ring in this binding mode would
clash with Trp317. This could cause the pocket to adopt a
different conformation and ultimately disrupt helices 11−12.
We hypothesized that while no substitution at the 4-position
(R = H, 3) led to the observed agonist binding mode in the
RORc SRC1 assay, changes to a small substituent at the 4-
position (R = F, 14) would begin destabilizing the C-terminus
and prevent SRC1 peptide recruitment beyond the basal level,
leading to low inverse agonist efficacy or silent MOA ligands.
Further steric bulk directed toward the C-terminal region (R =
Cl, 20) would fully disrupt the secondary structure of that
region of the protein and prevent RORc interaction with the
coactivator peptide, resulting in a stronger inverse agonist
MOA.29
With this consideration in mind, we synthesized additional
analogs bearing incrementally bulkier functionalities at the para
position of the arylsulfonamide (Table 2) to further explore the
level of efficacy of inverse agonism that could be achieved.
Compound 26, bearing a 4-methyl substituent, showed
significant affinity for RORc in the binding assay (RORc IC50
= 24 nM) but behaved as silent ligand in the RORc SRC1
biochemical assay. Additionally, a more polarized 4-cyano
substituent30 possessed moderate RORc binding affinity, as
exemplified with compound 27 (RORc IC50 = 280 nM).
However, the steric bulk of the nitrile group was not sufficient
to impart full RORc inverse agonism in the RORc SRC1
biochemical assay (EC50 > 10 μM, −29% efficacy). Substitution
with a 4-methoxy group ultimately provided enough steric bulk
and gave rise to stronger RORc inverse agonist 28 (EC50 = 3.3
μM, −52% efficacy). Further increasing the steric demand of
the ligand with dichloro substitution (29) or with
4-difluoromethoxy (30), 4-trifluoromethoxy (31), 4-trifluor-
omethyl (32), or 4-tert-butyl (33) groups afforded similarly
potent RORc inverse agonists with ranging efficacy of −37%,
−75%, −82%, −39%, and −55%, respectively, with 33 being
the most potent (EC50 = 120 nM). In summary, we
demonstrated that arylsulfonamide agonists could be trans-
formed into silent ligands by adding steric bulk to the
4-position of the arene. Additional steric hindrance at the
4-position eventually led to a RORc inverse agonist MOA.
We also explored the effect of replacing the sulfonamide
moiety in 3 and 4 with an amide functional group. Both phenyl-
and benzylamide compounds showed <50% inhibition in the
SRC1 peptide recruitment assay at a 10 μM concentration.
Sulfonamides typically adopt a nonplanar conformation31 that
was also observed in the cocrystal structures of compounds 2
and 3 with the RORc-LBD. Presumably, the planar
conformation of the amide did not allow for the amide analogs
to effectively bind to RORc-LBD and affect the RORc SRC1
peptide recruitment assay.
D
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We progressed two leading agonist/inverse agonist matched
pairs (3−4 and 10−11) into a human peripheral blood
mononuclear cell (PBMC) cytokine production assay to
evaluate their effects on IL-17 production (Table 3).24 RORc
mechanisms of action with RORc in both biochemical and
primary human blood cell assays. We utilized X-ray cocrystal
structural data to identify and characterize two distinct binding
modes consistent with these biochemical and cellular MOAs.
Optimized phenylsulfonamides were identified as RORc
agonists while benzylsulfonamides exhibited potent inverse
agonist activity. Our most potent inverse agonist showed >180-
fold selectivity over all other RORc isoforms and other nuclear
receptors that were profiled. Finally, compounds behaving as
agonists in our biochemical assay also gave rise to an increased
production of IL-17 in human PBMCs, while inverse agonists
led to significant suppression of IL-17 under the same assay
conditions. Thus, we were able to observe translation of
biochemical MOA into the same MOA in human primary
blood cells.
Table 3. Potency and Mechanism of Action of Phenyl- and
a
Benzylsulfonamides in hPBMC IL-17 Production Assay
IL-17 PBMC
EC50 (μM)
IL-17 PBMC
efficacy (%)
IFNγ EC50 CTG EC50
Compd
(μM)
(μM)
3
4
0.78
0.35
0.80
0.32
+77
−79
+71
−72
>10
>10
>10
>10
>20
>20
>20
>20
10
11
a
All assay results are reported as the geometric mean of at least two
separate runs. All assays were conducted using peripheral blood
mononuclear cells (PBMCs) isolated from human whole blood.24
Positive %efficacy denotes a potentiation of IL-17 production.
Negative %efficacy denotes an inhibition of IL-17 producton.
Interferon-γ (INF-γ) and CellTiter-Glo (CTG) cell measurement
assays were used as positive controls to monitor for non-TH17 cell
cytokine activity and adverse off-target effects on cell health,
respectively.24
ASSOCIATED CONTENT
■
S
* Supporting Information
Cocrystal structure data and compound characterization. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
agonists 3 and 10 gave rise to an increased production of IL-17
(EC50 = 0.78 μM, +77% efficacy and 0.80 μM, +71% efficacy,
respectively), which was in agreement with their MOA
determined in the RORc SRC1 biochemical assay. Conversely,
compounds 4 and 11, which were shown to be RORc inverse
agonists in the SRC1 peptide recruitment biochemical assay,
both led to a suppression of IL-17 production in the human
PBMC assay (EC50 = 0.35 μM, −79% efficacy and 0.32 μM,
−72% efficacy, respectively). In addition, none of the tested
compounds affected interferon-γ (INF-γ) biosynthesis or cell
health as assessed by CellTiter-Glo (CTG) cellular ATP
measurements, confirming that the compounds did not exhibit
off-target cytokine activity or gross toxicity. These results
demonstrated that closely related compounds with only minor
structural differences could be ligands that increased (phenyl-
sulfonamides) or decreased (benzylsulfonamides) IL-17
production in human PBMCs.
Corresponding Author
*Phone: +1-650-467-0236. E-mail: rene.olivier@gene.com.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
We thank Drs. Krista Bowman and Jiansheng Wu, and their
respective Genentech research groups, for performing protein
expression and purification activities. We also thank Crystallo-
graphic Consulting, LLC for diffraction data collection.
Diffraction data were collected at beamline 08ID-1 at the
Canadian Light Source, which was supported by the NSERC,
the NRC, the Canadian Institutes of Health Research, the
Province of Saskatchewan, Western Economic Diversification
Canada, and the University of Saskatchewan at beamline 5.0.2
of the Advanced Light Source. The Berkeley Center for
Structural Biology was supported in part by the NIH, the
NIGMS, and the Howard Hughes Medical Institute. The
Advanced Light Source was supported by the Director, Office
of Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under Contract No. DE-AC02-
05CH11231.
The potent RORc inverse agonist 11 (RORc SRC 1 EC50
=
3.1 nM, IL-17 PBMC EC50 = 320 nM) was profiled for its
selectivity in a panel of HEK293 cell Gal4-ROR construct
human NR-dependent transcriptional reporter assays. The
panel included the three isoforms of ROR (RORa, RORb, and
RORc) and monitored for suppression of basal transcription
activity in the absence of any exogenous agonist.24 In addition,
the following NRs were evaluated in both agonist mode (no
exogenous agonist ligand added) and antagonist mode
(exogenous agonist ligand added): farnesoid X receptor
(FXR), liver X receptors (LXR)-α and LXRβ, and pregnane
X receptor (PXR).24 Compound 11 was a potent inverse
agonist of RORc in the Gal4 reporter assay (EC50 = 15 nM)
and showed no detectable activity against the RORa isoform
while it was 180-fold selective against RORb (EC50 = 2.7 μM).
The compound also displayed favorable selectivity against other
NRs in the antagonist mode. For example, it was >400-fold
selective over FXR (EC50 = 6.1 μM) and >600-fold selective
over LXRα (EC50 = 9.2 μM). No activity was detected against
LXRβ and PXR. Additionally, 11 did not exhibit any agonist
activity in all the NRs profiled.
Notes
The authors declare no competing financial interest.
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change to our tertiary sulfonamide series led to opposite
E
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