Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with “short” and “long” inverse agonists

Article abstract of DOI:10.1038/s41401-020-00552-w

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the “short” inverse agonist 6. With the increase in the size of the 6-position substituents, the “short” inverse agonist 6 first reversed its function to agonists and then to “long” inverse agonists. The cocrystal structures of RORγt complexed with the representative “short” inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the “long” inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of “short” inverse agonist 6 and “long” inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that “short” or “long” inverse agonists led to different behaviors of helixes H11, H11’, and H12 of RORγt. The “short” inverse agonist 6 destabilizes H11’ and dislocates H12, while the “long” inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

Full text of DOI:10.1038/s41401-020-00552-w

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ARTICLE
Crystallography-guided discovery of carbazole-based retinoic
acid-related orphan receptor gamma-t (RORγt) modulators:
insights into different protein behaviors with “short” and
“long” inverse agonists
Ming-cheng Yu1, Feng Yang1,3, Xiao-yu Ding2,3, Nan-nan Sun1,4, Zheng-yuan Jiang1, Ya-fei Huang1, Yu-rong Yan1, Chen Zhu1,
Qiong Xie1,5, Zhi-feng Chen2,3, Si-qi Guo3, Hua-liang Jiang2,3,6, Kai-xian Chen2,3, Cheng Luo2,3,6, Xiao-min Luo2,3, Shi-jie Chen2,3 and
Yong-hui Wang1
A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered
through 6-position modification guided by insights from the crystallographic profiles of the “short” inverse agonist 6. With the
increase in the size of the 6-position substituents, the “short” inverse agonist 6 first reversed its function to agonists and then to
“long” inverse agonists. The cocrystal structures of RORγt complexed with the representative “short” inverse agonist 6 (PDB: 6LOB),
the agonist 7d (PDB: 6LOA) and the “long” inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor
differences were found in the binding modes of “short” inverse agonist 6 and “long” inverse agonist 7h. To further reveal the
molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that “short” or
“long” inverse agonists led to different behaviors of helixes H11, H11’, and H12 of RORγt. The “short” inverse agonist 6 destabilizes
H11’ and dislocates H12, while the “long” inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two
types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with
different regulatory mechanisms.
Keywords: RORγt; cocrystal structures; MD simulation; “short” inverse agonists; agonists; “long” inverse agonists
Acta Pharmacologica Sinica (2020) 0:1–11; https://doi.org/10.1038/s41401-020-00552-w
INTRODUCTION
indicates that RORγt modulators are promising in the treatment of
various autoimmune diseases and cancer [5–7].
Retinoic acid receptor-related orphan receptor gamma-t (RORγt)
belongs to the nuclear receptor (NR) transcription factor super-
family. It plays a key role in driving the differentiation of naive
CD4+ T cells into T helper 17 (Th17) cells [1]. RORγt contains a
variable N-terminal domain (A/B or AF1), a conserved DNA-
binding domain (DBD), a flexible hinge region, and a C-terminal
ligand-binding domain (LBD) with a ligand-dependent activation
function 2 (AF2) region that recruits coactivators by interacting
with helix 12 (H12). Small molecules binding to the LBD can
stabilize or destabilize the activation state of AF2 to modulate
the function of RORγt, thus acting as agonists or inverse agonists
[2–4]. For years, RORγt has attracted significant attention as a drug
target for its special role in the Th17/IL-17 pathway. RORγt inverse
agonists such as JTE-451, ESR-114, AUR-101, and VTP-43742 for
autoimmune diseases and RORγt agonists such as LYC-55716 for
cancer immunotherapy are now in Phase II clinical trials, which
Some research groups accidentally discovered RORγt agonists
through the study of inverse agonists with minor structural
modifications [8–10]. Cocrystal structures of each pair of inverse
agonists and agonists revealed a stable hydrophobic network (the
so-called “HYF lock”) among His479 (H11), Tyr502 (H12), and
Phe506 (H12) in the active state of the RORγt LBD. Recently,
Takeda designed RORγt agonist 2 through a structure-based
functional switching approach from inverse agonist 1 (Fig. 1a) [11].
By molecular dynamics (MD) simulation, the researchers found
that the conformational change of Trp317 and the hydrogen bond
interaction of His479 and Tyr502 were important in the functional
switch between inverse agonists and agonists.
In our recent study, the functional switch phenomenon was
observed between agonists and two types of inverse agonists
(Fig. 1b), namely, “short” and “long” inverse agonists, which were
1Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China; 2University of Chinese Academy of Sciences, Beijing 100049, China; 3Drug
Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 4Key
Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University,
Shanghai 200032, China; 5Fudan Zhangjiang Institute, Shanghai 201203, China and 6School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study,
UCAS, Hangzhou 310024, China
Correspondence: Xiao-min Luo (xmluo@simm.ac.cn) or Shi-jie Chen (shijiechen@simm.ac.cn) or Yong-hui Wang (yonghuiwang@fudan.edu.cn)
These authors contributed equally: Ming-cheng Yu, Feng Yang, Xiao-yu Ding
Received: 21 July 2020 Accepted: 2 October 2020
© CPS and SIMM 2020

Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
2
Fig. 1 Recently reported RORγt modulators. Functionally switchable modulators found by Takeda (a) and our recent study (b).
Fig. 2 Crystallography-guided design of novel carbazole-based RORγt agonists and inverse agonists.
named based on the size of the inverse agonists relative to the
corresponding agonist [12]. Through removal from or addition to a
minor structural moiety, RORγt agonist 4 could be converted into
“short” inverse agonist 3 and “long” inverse agonist 5. Notably, the
two types of RORγt inverse agonists behaved differently in cofactor
peptide recruitment. The “short” inverse agonist 3 recruited
corepressor peptides and dispelled coactivator peptides, while
the “long” inverse agonist 5 dispelled both. However, the detailed
mode of action (MOA) at the molecular level has not been studied
in depth, and the underlying mechanism of “short” and “long”
inverse agonists remains unclear. We believe that the difference
between the two types of RORγt inverse agonists deserves a
systematic study, as it not only demonstrates the regulatory
mechanism of RORγt but also may guide the discovery of novel
modulators with differentiated drug action and safety profiles.
conducted with a Biotage Initiator™ microwave synthesizer
(Biotage, Uppsala, Sweden). NMR data were recorded with a
Bruker 400 MHz NMR system. Chemical shifts (δ) are expressed in
parts per million (ppm) relative to tetramethylsilane (TMS) as an
internal standard. Mass spectra were measured on an Agilent
1100 series LC/MSD 1947d spectrometer (Agilent Technologies,
Inc., Santa Clara, CA, USA). High-resolution mass spectra (HRMS)
were obtained on an AB SCIEX TripleTOF 5600+ mass spectro-
meter (AB SCIEX, LLC., Redwood City, CA, USA). The contents of
compounds for biological evaluation were examined with an
Agilent 1260 Infinity LC system (Agilent Technologies, Inc.) with
methanol/water (40:60 to 95:5) as the eluent. Unless specified, the
purity of the target compounds was >95%, which was considered
to be pure enough for biological assays. The synthetic procedures,
1H-NMR, 13C-NMR, and HRMS are detailed in the Supporting
Information section.
Previously, we reported
a series of carbazole-containing
compounds, such as compound 6 (Fig. 2), as RORγt inverse
agonists [13]. Herein, based on insights from the freshly resolved
cocrystal structure, we classified compound 6 as a “short” inverse
agonist and identified the 6-position of the carbazole moiety as
the site for structural modification to achieve the functional switch
from the “short” inverse agonist 6 to agonists and “long” inverse
agonists. In this paper, we describe our insights into stabilizing or
destabilizing the AF2 domain from cocrystal structures resolved
and report the design, synthesis, and biological evaluation of 6-
substituted carbazole-based RORγt modulators with substituents
(R groups) gradually increasing in size to obtain RORγt “short”
inverse agonists, agonists or “long” inverse agonists (7a-7j, Fig. 2).
The molecular mechanisms of the RORγt agonists and two types
of inverse agonists were then revealed by MD simulation studies.
Different behaviors of helixes H11, H11’ and H12 of the protein
with “short” or “long” inverse agonists were further elucidated.
Compounds 6 and 7a were prepared according to the
procedures described in our previous work [13]. The synthesis of
the carbazole-based compounds 7b-7j is depicted in Scheme 1.
Starting from commercially available 4-halogeno-nitroarenes
(8 and 17) or 4-nitrophenol (15), the desired ether intermediates
(9, 16, and 18) were generated through nucleophilic substitution
reactions. Then, the nitro-containing intermediates were reduced
to yield the corresponding anilines (10 and 19). Carbazole-based
intermediates (12 and 20) were obtained via Buchwald-Hartwig
cross coupling reactions, followed by palladium-catalyzed cycliza-
tion reactions. The intermediates were then alkylated, and ester
hydrolysis followed by amide formation with (4-(ethylsulfonyl)
phenyl)methanamine (23) resulted in the target compounds 7b-
7j. The synthesized compounds were then evaluated in the RORγt
dual FRET assay and Gal4 cell-based reporter gene assay.
Protein expression and purification
Human nuclear receptor RORγt (residues 260–507)-GGG-SRC1
(SRC1 sequence: EKHKILHRLLQDS) was amplified by polymerase
chain reaction (PCR) and then analyzed by electrophoresis on a
1% agarose gel. RORγt LBD was cloned into the His-Flag-SUMO
tag pRSF DUET1 vector. The proteins were expressed in
MATERIALS AND METHODS
Chemical synthesis
All reagents used were commercially available and were used as
received unless otherwise indicated. The microwave reaction was
Acta Pharmacologica Sinica (2020) 0:1 – 11

Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
3
H
N
NO2
Cl
NH2
Cl
NO2
Cl
c
a
b
R1
O
R1
R1
O
Cl
O
O
F
O
O
11
8
9
10
H
N
N
N
d
e
f
( I )
R1
O
O
R1
O
R1
OH
O
O
O
O
12
13
14
7c
7h
: R1 = Et
: R1 = Cyclohexyl
N
O
g
7i
: R1
: R1
=
=
N
O
S
O
R1
NH
O
O
O
7j
N
t-Bu
NO2
NO2
NO2
h
R2
H
N
HO
O
15
17
16
NH2
c, k
j
R2
R2
O
O
O
NO2
O
i
19
20
F
O
( II )
18
7b
7d
7e
7f
: R2 = Me
: R2 = i-Pr
: R2 = t-Bu
: R2 = Cyclobutyl
: R2 = Cyclopentyl
N
e, f, g
O
S
O
R2
NH
O
7g
O
Scheme 1 Synthesis of carbazole-based RORγt modulators a. a Reagents and conditions: (a) NaH, DMF and 1-(4-hydroxypiperidin-1-yl)ethan-
1-one (21)/1-(4-hydroxypiperidin-1-yl)-2,2-dimethylpropan-1-one (22)/alkyl alcohols at 0 °C, followed by 8 at 60 °C for 3 h, yield: 49.8%–65.2%.
b SnCl2·2H2O, MeOH at rt for 12 h, yield: 78.9%–92.1%. c rac-BINAP, Pd(OAc)2, K2CO3, methyl 4-bromobenzoate, and toluene under reflux for
2 h, yield: 27.3%–63.0%. d Tributylphosphonium tetrafluoroborate, Pd(OAc)2, K2CO3, and DMA under reflux for 5 h, yield: 46.8%–52.7%. e NaH
and DMF at 0 °C followed by bromoethane at rt for 20 min, yield: 62.6%–90.1%. f KOH, H2O, and MeOH at 80 °C for 1 h, yield: 55.6%–100.0%.
g DIPEA, HATU, (4-(ethylsulfonyl)phenyl)methanamine (23) and DCM at rt for 2 h, yield: 45.8%–88.9%. h Bromoalkanes, K2CO3 and DMF at 70 °C
for 5 h, yield: 83.4%–96.2%. i Potassium tert-butoxide and THF at 60 °C for 6 h, yield: 38.6%. j Zn, HCOONH4, MeOH, and H2O at 80 °C, 2 h, yield:
61.6%–66.9%. k Pd(OAc)2 and HOAc at 130 °C, 1 h, yield: 29.8%–44.7%.
Escherichia coli strain BL21(DE3). The cells were grown in LB
(Luria − Bertani broth) at 37 °C. When the OD600 nm reached 0.6,
the temperature was shifted to 16 °C, and the cells were induced
with 0.4 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) for
16 h. The cells were collected by supercentrifugation at 4000
rpm for 15 min. RORγt protein was first purified by nickel affinity
chromatography (His Trap HP, GE Healthcare, General Electric
Company, NJ, USA) and then further purified by gel-filtration
RORγt dual FRET assay
The assay was performed in an assay buffer consisting of 50 mM
NaF, 50 mM 3-(N-morpholino)propanesulfonic acid (pH = 7.4),
0.05 mM 3-[(3-cholamidopropyl) dimethylammonio]propanesulfo-
nate, 0.1 mg/mL bovine serum albumin, and 10 mM dithiothreitol
in 384-well plates. The total volume was 25 μL/well. The europium-
labeled SRC1 solution was prepared by adding an appropriate
amount of biotinylated SRC and europium-labeled streptavidin
into assay buffer, with final concentrations of 20 and 10 nM,
respectively. The allophycocyanin (APC)-labeled-LBD solution was
prepared by adding an appropriate amount of biotinylated RORγt-
LBD and APC-labeled streptavidin at final concentrations of 20 and
10 nM, respectively. After 15 min of incubation at room tempera-
ture, a 20-fold excess of biotin was added and incubated for
10 min at room temperature to block the remaining free
streptavidin. Equal volumes of europium-labeled SRC and APC-
labeled RORγt-LBD were dispensed into 384-well assay plates at
chromatography on
a Superdex 75 10/300 column (GE
Healthcare). Finally, the purified RORγt protein was concentrated
in a 10 k enrichment tube (Millipore, Merck KGaA, Darmstadt,
Germany) and flash frozen at −80 °C. The RORγt LBD used in the
binding assay was stored at −80 °C in buffer containing 25 mM
HEPES (pH = 7.4), 200 mM NaCl, and 5% glycerol. The RORγt-
GGG-SRC1 protein used for crystallization was stored at −80 °C
in buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl, and
2 mM dithiothreitol.
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Insights to “short” and “long” RORγt inverse agonists
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4
25 μL volume/well. The 384-well assay plates had 100 nL of test
compound in DMSO predispensed into each well. The plates were
incubated for 1 h at room temperature and then read on Envision
in LANCE mode configured for europeum-APC labels. Detailed
data are presented in Supplementary Table S1.
concentrations. The fluorescence signals were gathered with a
slope of 2.5 °C·min−1 from 25 °C to 95 °C using a real-time PCR
machine (QuantStudio™ 6 Flex, Applied Biosystems). The output
data were analyzed, melting temperatures were calculated by
Protein Thermal Shift™ v1.0 software (Life Technologies), and
curves were plotted in GraphPad Prism.
RORγt GAL4 cell-based reporter gene assay
The hRORγt LBD coding sequence was inserted into a pBIND
expression vector (Promega, Fitchburg, WI, USA) to express ROR-
GAL4 binding domain chimeric receptors. This expression vector
and a reporter vector (pGL4.35, which carries a stably integrated
GAL4 promoter-driven luciferase reporter gene [luc2P/9XGAL4
UAS/Hygro]) were cotransfected into HEK293T host cells. Upon
agonist binding to the corresponding ROR-GAL4 chimeric
receptor, the chimeric receptor binds to the GAL4 binding sites
and stimulates the reporter gene. In the presence of an inverse
agonist, the agonist will bind competitively to the nuclear receptor
and activate reporter gene transcription. HEK293T cells were
cultured in a culture medium composed of DMEM containing 5%
charcoal-treated FBS at 37 °C under a 5% CO2 atmosphere, as
recommended by the ATCC. Before the assay, the cells were
washed with PBS to remove phenol red and suspended in phenol
red-free medium (phenol red-free DMEM containing 5% charcoal-
treated FBS and penicillin-streptomycin (10,000 U/mL)) to a proper
concentration. HEK293T cells (6 × 106) were seeded into a 100 mm
dish and incubated for 16 h. To a reagent mixture of TransIT-293
transfection reagent (Mirus Bio, WI, USA) and Opti-MEM (Invitro-
gen, CA, USA), plasmid DNA (used as 0.5 mg/mL stocks) containing
5 μg RORγt plasmid and 5 μg pGL4.35 luciferase plasmid was
added. The mixture was added to the cells in a 100 mm dish and
incubated for 5–6 h. Test compounds were serially diluted in
DMSO to 300 nM. Compounds (25 nL) were transferred to a 384-
well plate (white opaque) using Echo 550. Then, the cells were
seeded at 15,000 cells/well into a 384-well plate using phenol red-
free DMEM containing 5% charcoal-treated FBS and 0.25 μM
ursolic acid. Cells were incubated for 16–20 h at 37 °C (white
opaque) using Echo 550. Then, 25 μL of SteadyGlo™ Luciferase
Assay Reagent (Promega) was added to each well of the 384-well
plate. The plate (avoiding light) was shaken for 5 min on a plate
shaker (4625-1CECN/THZ Q, Thermo Fisher Scientific, MA, USA).
The luminescence value was recorded on an Envision 2105 plate
reader (PerkinElmer, MA, USA). Activation/inhibition values of the
compounds at 300 nM were determined by the average value of
duplicate tests.
Crystallization, data collection, and structure determination
First, his-thrombin RORγt (residues 260–507)-GGG-SRC1 proteins
were prepared at 10 mg/mL, and initial crystal trials were
conducted utilizing the hanging drop vapor diffusion method in
24-well plates at 25 °C. The apo crystals were grown by mixing
1 μL of the protein (10 mg/mL) with 1 μL of reservoir solution
containing 0.4 M NaCl, 8% PEG4000, and 0.1 M PIPES, pH 6.5. The
inerratic crystals appeared in 1 day and grew to their full size in
~1 week. The cocrystals of inverse agonist compound 6 were
obtained by soaking the compound into apo RORγt-LBD-SRC1
crystal. Nevertheless, crystals of RORγt-LBD-SRC1 in complex with
agonist compound 7d and inverse agonist compound 7h were
prepared by incubation of 10 mg/mL protein with 0.5 mM
compounds. After 5 h of incubation, the cocrystals were obtained
fortunately by mixing 1 μL of the protein with 1 μL of reservoir
solution containing 0.4 M NaCl, 8% PEG4000, and 0.1 M PIPES, pH
6.5. The diffraction data of compounds 6, 7d, and 7h were
collected on beamlines BL17U1 and BL19U1 at Shanghai
Synchrotron Radiation Facilities at 100 K [17, 18]. Data sets were
processed (indexing and integration) using the program HKL3000
or HKL2000 [19]. The structures were determined by molecular
replacement using the programs PHENIX [20]. The models were
refined using the program PHENIX and rebuilt with COOT [21]. The
stereo images and electron density maps were generated
using PyMol.
Model construction and preparation
Initial structures were obtained from our crystal coordinates (PDB
code: 6LOA, 6LOB, and 6LO9), including compound 6-RORγt,
compound 7d-RORγt and compound 7h-RORγt. For the models of
MD simulation, the triglycine linker and cofactor SRC1 were
removed from the crystal structures. Each protein structure was
prepared by assigning the bond order, adding missing hydrogen,
and deleting water molecules, followed by Impref minimization
with Maestro (Schrödinger, LLC, NY, USA). For ligand optimization,
the Gaussian 09 (Gaussian 09, Revision D. 01, Gaussian, Inc., LA,
USA) program was used to calculate the partial atomic charges of
ligands (compounds 6, 7d, and 7h) at the HF/6-31 G* level. The
restrained electrostatic potential charges of the ligands were
derived with an antechamber. The simulation systems were
subsequently constructed by means of tleap in AmberTools 16
(AmberTools 16, University of California, CA, USA). Each complex
was solvated in a cubic water box of TIP3P water molecules,
maintaining a distance of 8 Å from any protein atom. A suitable
number of counterions was added to neutralize the charge of
each system.
Microscale thermophoresis (MST) assay
The microscale thermophoresis (MST) method has been described
in detail previously [14–16]. For an optimal protein to dye (RED-
tris-NTA, Nanotemper, Munich, Germany) ratio for subsequent His-
tag labeling, MST experiments were conducted in buffer contain-
ing 25 mM HEPES (pH 7.4), 150 mM NaCl and 0.05% pluronicTM F-
127. In addition, 150 nM RORγt (residues 265–509) protein and
5 nM dye were used for the assay. The compound was serially
diluted from ~10 μM to 5 nM. The medium MST power and 11%
excitation power were applied to the signal detection of protein in
the 25.0 °C thermostat set point. The instrument used in the
experiment is monolith NT Automated (Nanotemper, Munich,
Germany). Data analyses were performed using Nanotemper
Analysis software (MO Affinity Analysis), and curves were plotted
in GraphPad Prism.
MD simulations
All simulations were performed by Amber 16 with GPU accelera-
tion (AmberTools 16). The ff14SB force field was used for each
protein [22], and the general Amber force field was used for each
molecule [23]. Each system was minimized with the steepest
descent minimization of 5000 steps and a conjugate gradient
minimization of 5000 steps by freezing heavy atoms of complex
with a harmonic restraint of force constant of 1 kcal/(mol·Å2).
Then, each system was heated to 300 K gradually within 0.2 ns.
Then, this step was followed by another 0.1 ns equilibrium at 300 K
in the constant-pressure, constant-temperature ensemble (NPT).
Finally, 1000 ns conventional MD production without any restric-
tion was performed. Particle mesh Ewald was applied to calculate
the electrostatic interactions [24]. A cutoff distance of 12 Å was
Protein thermal shift assay (PTS)
In the assay, 7.5 μM purified RORγt-LBD proteins and 5× SYPRO®
Orange (Molecular Probes, Life Technologies, Inc., Eugene, OR)
were mixed into a fast 96-well optical plate (Applied Biosystems,
Foster City, CA, USA) in buffer consisting of 25 mM HEPES (pH 7.4)
and 150 mM NaCl. Then, the compounds were diluted to different
concentrations by DMSO and added to the mixture at different
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Insights to “short” and “long” RORγt inverse agonists
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5
Fig. 3 Crystal structure of RORγt(265-509)-LBD with compound 6 (PDB code: 6LOB, resolution of 2.40 Å). Left: Surface representation that
shows compound 6 (pink) binding to RORγt (blue-white), along with a 2mFo-DFc OMIT electron density map at a contour level of 2.0δ shown
as a blue mesh around the compound. Right: Compound 6 forms extensive interactions with the RORγt-LBD. Hydrogen bond interactions are
indicated by dashed lines in orange. Distances from C6 of the carbazole ring are marked in green. For clarity, only important residues (blue-
white) at the binding site are shown.
used for nonbonding van der Waals and electrostatic interaction
calculations. Data analysis was performed on the trajectories of
the MD production stage. The MD trajectory postprocessing and
data analysis were implemented inside the cpptraj module in
Amber16. The representative structure for each system was
derived from the last 400 ns stage, in addition to the initial
conformation. All the structure diagrams were processed with
PyMOL software (Version 1.2r3pre, Schrödinger).
Table 1 and Supplementary Table S1). Based on this result, we
kept increasing the size of the substituent group on the carbazole
ring. As we expected, the first carbazole-based agonist was
discovered. A methoxyl group at the 6-position reversed the
function and led to agonist 7b with an EC50 of 8.0 nM and a
moderate % maximum activation (52.9%, Table 1). Enlarging the
substituent by an ethoxyl group led to agonist 7c with a higher
efficacy (% max activation = 61.9%, Table 1), and agonist 7d with
an isopropoxyl group exhibited the best agonist activity with a
potent maximum activation of 83.7%. Interestingly, 7e, 7f and 7g
with larger substituents, such as tert-butoxyl, cyclobutoxy, and
cyclopentoxy groups, started to show a decrease in efficacy, giving
RESULTS
Identification of the “Short” RORγt inverse agonist
In a FRET assay where a surrogate agonist was added, 6 exhibited
RORγt inhibitory activity with an IC50 of 167 nM and a maximum
percentage inhibition (% max inhibition) of 153% [13]. In a dual
FRET assay without a surrogate agonist added, 6 showed RORγt
inhibitory activity with an IC50 of 45.3 nM and a % max inhibition
of 71.7%, indicating that it is an inverse agonist. To confirm a
direct interaction of RORγt-LBD with compound 6, we first
conducted a MST assay and PTS assay. As shown in Supplemen-
tary Fig. S1a, compound 6 displayed a Kd value of 637.5 109.6
nM. In addition, compound 6 changed the Tm value (ΔTm) in a
concentration-dependent manner, with the ΔTm values of 8.7 °C
and 8.1 °C at 25 μM and 7 μM, respectively. Then, inverse agonist 6
was cocrystallized with RORγt LBD (PDB code: 6LOB, resolution of
2.40 Å) to help elucidate the binding mode. Data collection and
refinement statistics are included in Supplementary Table S2. With
the hydrogen bond between the carboxamide linker and Phe377
and with the sulfonyl group interacting with Leu287 and Arg367
through hydrogen bonds, compound 6 made strong polar
interactions at the hydrophilic site. However, the interaction at
the AF2 domain was not well established. Although the 6-position
of the carbazole-ring directly pointed to AF2, it was ~6.5 Å, 6.7 Å,
and 7.1 Å from His479 (center of imidazole ring), Tyr502 (O) and
Trp317 (center of benzene ring), respectively, which left a cavity of
~2–3 atoms in size between the 6-position of the carbazole-ring
and these key residues (Fig. 3 and Supplementary Fig. S2a).
These observations indicate that compound 6 is a “short”
inverse agonist, which leads to the hypothesis that introducing R
group substituents to the 6-position will stabilize or destabilize the
AF2 domain to potentially turn the “short” inverse agonist 6 into
agonists and then “long” inverse agonists. Therefore, a series of
carbazole-based analogs with substituents at the 6-position
gradually increasing was designed and synthesized.
a mitigated % max activation (59.1%, 48.8%, and 42.6%,
respectively, Table 1 and Supplementary Table S1) in the dual
FRET assay. The functional switch phenomenon observed in the
dual FRET assay was confirmed by the cell-based reporter gene
assay. In the Gal4 cell-based reporter gene assay, the compounds’
activation percentages at 300 nM ranged from −48.12% (inverse
agonist 6) to 48.60% (agonist 7d) and then to a reduced activation
level of 13.30% (agonist 7g), which was consistent with our
hypothesis that introducing R substituents to approach the AF2
domain will functionally reverse “short” inverse agonists to
agonists (Table 1).
Compound 7d was then measured in the MST assay and PTS
assay (Supplementary Fig. S1b). The results revealed a higher
affinity of 53.3 17.4 nM and a similar ΔTm changing mode
compared with those of compound 6. Fortunately, we succeeded
in acquiring a cocrystal structure of RORγt-LBD with 7d (PDB code:
6LOA, resolution of 2.51 Å). This structure showed that 7d
occupied the RORγt-LBD and fit well in the electron density
map. In addition, the cocrystal structures of compounds 6 and 7d
overlapped well in both the carbazole moiety and the sulfonyl
moiety toward Phe377, Phe378, Phe388, Leu287, and Arg367
(Supplementary Fig. S3). The terminal isopropoxyl group of
agonist 7d was observed pointing to the AF2 domain, 4.8 Å and
3.4 Å apart from His479 (center of imidazole ring) and Tyr502(O),
packing against the hydrogen bond between His479(NE2) and
Tyr502(OH) (Fig. 4a and Supplementary Fig. S2b). In addition, the
formation of an alkyl-π interaction of 4.7 Å toward the phenyl ring
of Trp317 was found. These results could explain the stabilized
AF2 domain and the observed inverse agonist-agonist functional
switch.
From agonists to “Long” inverse agonists
However, when the substituents increased to cyclohexyloxy
group, the compound started to show a functional switch in the
opposite direction. With an IC50 of 88.3 nM and a % max inhibition
of 40.7% in the dual FRET assay (Supplementary Table S1),
compound 7h functionally changed to a so-called “long” inverse
agonist. The inhibitory activity was also confirmed by a moderate-
From “Short” inverse agonists to agonists
With the substituents growing, inverse agonist 7a first showed a
decrease in % max inhibition and a rise in potency (RORγt dual
FRET IC50 = 20.6 nM, % max inhibition = 45.5%) compared with 6
(RORγt dual FRET IC50 = 45.3 nM, % max inhibition = 71.7%,
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Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
6
Table 1. Structure-activity relationship of 6-position substituted carbazole compounds.
N
O
S
O
H
N
R
O
b
RORγ dual FRET XC50
nM (% max acti.)a
Compound
R
Gal-4,%acti.@300nMa
Compound Type
6
H
45.3 10.2 (-71.7)
20.6 7.5 (-45.5)
8.0 0.1 (52.9)
-48.12
-53.91
34.72
40.15
“Short” inverse agonist
“Short” inverse agonist
Agonist
7a
7b
7c
CH3
OCH3
OC2H5
15.0 4.6 (61.9)
Agonist
7d
7e
7f
10.1 0.2 (83.7)
11.9 3.0 (59.1)
6.1 0.2 (48.8)
48.60
30.88
16.10
13.30
-67.40
-16.30
-28.50
Agonist
Agonist
O
O
O
O
Agonist
7g
7h
7i
17.4 0.5 (42.6)
88.3 26.2 (-40.7)
147.0 24.3 (-84.3)
43.2 8.9 (-94.4)
Agonist
“Long” inverse agonist
“Long” inverse agonist
“Long” inverse agonist
O
O
N
O
O
N
7j
O
aEC50 for agonists and IC50 for inverse agonists.
bDual FRET XC50 (% act.) value and Gal-4 % act. value are the average values of at least two determinations.
to-potent % max inhibition of 67.4% in the cell-based reporter
gene assay. With the substituents growing continuously, the
inhibition efficacy of compound 7i increased to 84.3%, and
compound 7j functioned as the best “long” inverse agonist with a
similar % max inhibition of 94.4% in the dual FRET assay (Table 1).
However, in the GAL4 cell-based reporter gene assay, compounds
7i and 7j only displayed inhibitory activities of 16.3% and 28.5%,
respectively. In contrast to the results of the “short” ones, the
efficacies in the dual FRET assay and cell-based reporter gene
assay have some gap. This finding might be explained by the
reduced cell permeability according to the QikProp predictions
(Supplementary Table S3). Similarly, MST and PTS assays were also
conducted with compound 7h to determine its binding ability. A
decreased Kd value of 159.4 65.4 nM and ΔTm values of ~8.6 °C
and 7.8 °C were obtained (Supplementary Fig. S1c). Notably, we
obtained a cocrystal structure of RORγt-LBD with 7h (PDB code:
6LO9, resolution of 1.85 Å) for further molecular mechanistic
exploration. The distances between the mass center of the
cyclohexyl ring of 7h and the key residues were similar to those in
the agonist 7d-RORγt complex. However, the cyclohexyl group of
7h bound in a boat conformation instead of a chair conformation,
indicating an existing collision between the 4-position carbon
atom (C4) of the cyclohexyl group and the AF2 domain (Fig. 4b
and Supplementary Fig. S2c).
With the above cocrystal structures in hand, we superimposed
the three structures of 6, 7d, and 7h to compare the overall
binding modes. As shown in Fig. 5, His479 and Tyr502 rotate
slightly, as indicated by the red arrow in crystal structures. Since
the aryl sulfone moiety of ligands 6, 7d, and 7h superimposed
well, we surmised that the stabilization of AF2 was affected by the
terminal substituents on the carbazole moiety, which made
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7
Fig. 4 Crystal structures of RORγt(265-509)-LBD with modulators. a With compound 7d, PDB code: 6LOA, resolution of 2.51 Å. b With
compound 7h, PDB code: 6LO9, resolution of 1.85 Å. Left: Surface representation that shows compound binding to RORγt (blue-white), along
with a 2mFo-DFc OMIT electron density map at a contour level of 2.0δ shown as a blue mesh around the compound. Right: The compound
forms extensive interactions with the RORγt-LBD. Hydrogen bond interactions are indicated by dashed lines in orange. The green lines
represent distances.
obvious fluctuation was observed during 430–500 and 820–1000
ns, whereas the C6-His479 distance curve remained flat (4.77
0.45 Å) during the entire simulation time. In the “long” inverse
agonist 7h-RORγt system, the distance curve of C6-His479
decreased slightly at ~620 ns, followed by a significant fluctuation
in the C6-Tyr502 distance during 801–808 ns. This result indicated
that the behaviors of His479 and Tyr502 in the three systems were
significantly different.
In addition to the time-dependent distance, the side-chain
dihedral angle was analyzed to reveal the specific conformational
changes of His479 and Tyr502. The distribution of Chi1 and Chi2
Fig. 5 The superimposed crystal structures of ligands 6, 7d, and
during all simulation times is visualized in Fig. 6c, d. It was obvious
that dihedral angles appeared with different frequencies, espe-
cially the Chi1 angles. Since significant conformational changes in
His479 and Tyr502 were detected in the late MD simulation, the
following dihedral angle analysis and representative structure
presentation would be focused on the last 400 ns stage. For ease
of comparison among the three systems, the Chi2 angle was
restricted as −110° for His479 and −90° for Tyr502, where the
fixed Chi2 angles were determined by the starting systems. The
conformation in the lowest energy minima for each system is
marked in the left panels, indicating that the His479 Chi1s for 6-
RORγt and 7d-RORγt were preferred in the trans position (Chi1s
near 175° and 155°, respectively), whereas the His479 Chi1 for 7h-
RORγt was preferred in the g- position (Chi1 near −75°). As shown
in the right panel of Fig. 6c, the His479 side chain might be
crashed by a chair-form cyclohexyl group of 7h and had a
deflection away from the ligand. In particular, the g- position for
Tyr502 Chi1 was preferred in 6-RORγt, while the g+ position was
most frequent in 7h-RORγt (Fig. 6d), which all differed from the
trans position represented in 7d-RORγt.
The graphical representation of conformation states of His479
and Tyr502 in H11-H12’-H12 during MD simulations is presented
(Fig. 6e-g). In the “short” inverse agonist 6-RORγt system, because
of the unoccupied cavity around the 6-position of the carbazole
ring, His479 flipped toward the ligand, and the Tyr502 side chain
then shifted outwards from the inside pocket (Fig. 6e), which led
to the disruption of the His479-Tyr502 hydrogen bond. Not
surprisingly, the cyclohexyl group in the “long” inverse agonist 7h
7h within the LBD of RORγt. Left: An overall picture of a
superimposed crystal structure. Right: Interaction picture of ligands
in the pocket. Each arrow (red) respectively represented the
direction.
apparent agonist/inverse agonist differences in biological
function.
MD simulation study
To understand the role of terminal substituents of ligands in the
switching function of RORγt, we conducted a 1 μs MD simulation.
As reported previously, the hydrogen-bonding interaction
between His479 and Tyr502 played a key role in stabilizing the
position of H12 in the active form [25]. Therefore, we first
monitored the distance between His479(NE2) and Tyr502(OH)
during the MD trajectory (Fig. 6a). In our MD simulation study, no
dynamic change in distance was observed (4.17 0.89 Å) in 7d-
RORγt, whereas the distances gradually increased after 600 ns in 6-
RORγt and 7h-RORγt, which was consistent with the response of
the agonist lock between His479 and Tyr502 in RORγt. To
determine the dominant reason for the His479-Tyr502 hydrogen
bond disruption, we monitored the time-dependent distances
between the 6-position carbon atom (C6) of the carbazole scaffold
and His479 (center of imidazole ring)/Tyr502(O) (Fig. 6b). In the
agonist 7d-RORγt system, the distances between C6 and the
residues were quite stable. In the “short” inverse agonist 6-RORγt
system, the time-dependent distance curve of C6-Tyr502 exhibited
apparent fluctuations during 608–615 ns and 810–814 ns. Less
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Fig. 6 Conformational changes of His479 and Tyr502 in three systems. a Time-dependent distances between His479(NE2) and Tyr502(OH),
and the cutoff of the hydrogen bond distance is 3.5 Å. b Time-dependent distances between the C6 atom of each ligand and two residues,
His479 (center of imidazole ring) and Tyr502(OH). Chi1 and Chi2 angle distribution for His479 (c) and Tyr502 (d) (left three panels). Contours
are drawn at <0 (black), 0 (red), 0.5…, 19.5, 20 (blue), and >20 (white) kcal/mol. Representative snapshots of the local energy minima in marked
regions (right panel) during simulations. e–g Graphical representation of the conformational states of His479 and Tyr502 observed during MD
simulation. e “Short” inverse agonist 6 complexes are shown in the pink series (at 0 ns, 612 ns, 810 ns, and 1000 ns); (f) “Long” inverse agonist
7h complexes are shown in the orange series (at 0 ns, 810 ns, 820 ns, and 1000 ns); (g) Agonist 7d complexes are shown in the green series (at
0 ns and 1000 ns). Conformation snapshots over simulation time are colored in various shades that start with white and fade down to the
darker. Each arrow represents the change of the His479 or Tyr502 side chain in direction.
clashed with His479, followed by the Tyr502 side-chain flipping
away from the pocket (Fig. 6f). Agonist 7d with an isopropoxy
group, however, helped to form the His479-Tyr502 hydrogen
bond and relatively stabilized the conformation of H12 (Fig. 6g).
Intrigued by the different behaviors of Tyr502 and His479 in
“short” and “long” inverse agonist-RORγt systems, we suspected
that the conformation of the whole RORγt in the two systems may
also show different changes. Therefore, we calculated the root
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Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
9
Fig. 7 Positional RMSD (in Å) for atoms of the H11, H11’ and H12 along the simulation time (ns). RMSDs of heavy atoms (a: in the
compound 6-RORγt system; b: in the compound 7h-RORγt system) and backbone atoms (c: in the compound 6-RORγt system; d: in the
compound 7h-RORγt system) of H11 (Lys469 to Leu489), H11’ (Pro491 to Ala497), and H12 (Pro500 to Phe506) as a function of simulation time.
mean square fluctuation for each residue and found that the H11-
H11’-H12 portion was highly flexible in both systems (Supple-
mentary Fig. S4). To obtain the individual changing modes of H11,
H11’, and H12, we separately calculated the root mean square
deviation (RMSD). With “short” inverse agonist 6, H11 heavy atoms
remained relatively stable. However, the RMSD of H12 heavy
atoms coordinated with the general changing trends of the C6-
Tyr502 curve during 0–810 ns and decreased during 810–815 ns. It
increased again after 815 ns, together with the H11’ heavy atoms
(Fig. 7a). Interestingly, although the behavior of the H11 and H11’
backbones remained consistent with their heavy atoms, the RMSD
value of the H12 backbone remained flat during the whole
simulation time (Fig. 7c), indicating that the introduction of
compound 6 could affect the side chain of H12 during 0–815 ns
and the backbone of H11’ after 815 ns. However, in the 7h-RORγt
system, the three helixes appeared to be more flexible during
0–650 ns (Figs. 7b, d). H11’ became stable, and the RMSD of H11
heavy atoms increased slowly after 650 ns, 80 ns ahead of the rise
in the RMSD of the H11 backbone. After 750 ns, the RMSD of H12
heavy atoms started to increase, followed by an increase in the
RMSD of the H12 backbone after 800 ns. It seemed that the “long”
inverse agonist 7h could influence the helix structures of H11 and
H12 by affecting the side chains.
Since α-helixes were generally stabilized by internal hydrogen
bonds between every four amino acids, we then evaluated the
stability of helixes H11, H11’, and H12 in the compound 6-RORγt
system and compound 7h-RORγt system by tracking the distance
between residue X (backbone atom O) and residue X + 4
(backbone atom N). Representative time-dependent distance
curves in H11, H11’, and H12 are presented in Fig. 8a. In the 6-
RORγt system, although hydrogen bonds in H11 and H12
remained stable during the whole simulation time, the connection
between Val493(O) and Ala497(N) in H11’ loosened slowly after
800 ns, followed by the fluctuation of C6-Tyr502 distance during
810–814 ns and a gradual increase of the Pro491(O)-Gln495(N)
distance after 815 ns. The results suggested that the increased
instability of H11’ in the “short” inverse agonist 6-RORγt system
could result in H12 movement as a whole, and the swinging H12
further loosened the secondary structure of H11’ as feedback. In
another case of the “long” inverse agonist 7h, the hydrogen bond
distances of Arg482(O)-Phe486(N) and Leu483(O)-Gln487(N) in
H11 started to increase from 799 ns and 797 ns, respectively, after
His479 shifted its location ~620 ns, immediately before
a
significant increase in the C6-Tyr502 distance (801–808 ns) and
an addition in the Pro500(O)-Glu504(N) distance (803–810 ns).
Therefore, it was reasonable to conclude that in the “long” inverse
agonist 7h-RORγt system, the shift of His479 separated the
secondary structure of H11, leading to the fluctuation of Tyr502,
and then unwound H12.
On the basis of the evidence in this study, we proposed the
following models to describe the different behaviors of RORγt
induced by our carbazole-containing “short” and “long” inverse
agonists. For the “short” inverse agonist 6, lacking a substituent
on the 6-position of the carbazole ring to reach the hydrophobic
AF2 domain, His479 first flipped toward the pocket side quickly
and then destroyed the His479-Tyr502 hydrogen bond, leading
to a loosened H11’ and dislocation of H12. In this process, the
main protein scaffolds of H11 and H12 were stable, but the helix
structure of H11’ and the side-chain of H12 were affected
(Fig. 8b). The “long” inverse agonist 7h, with its cyclohexyl group
approaching the AF2 domain, clashed sterically with the
His479 side chain. His479 then influenced the subsequent
residues Arg482 and Leu483, resulting in the movement of
Tyr502 toward the exterior part of the binding pocket, which led
to the Tyr502-Pro500 collision and the disconnection between
Pro500(O) and Glu504(N). As a result, the helix structures of H11
and H12 were partly destroyed (Fig. 8c). Furthermore, a DSSP
analysis was conducted to confirm the models proposed from
our observations (Supplementary Fig. S5). We determined that
destroying whether the location of H12 by the “short” inverse
agonist 6 or the helix structure of H12 by the “long” inverse
agonist 7h always affected the action of H12 to recruit
coactivators, which would thus interrupt or repress the
transcriptional function of RORγt.
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Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
10
Fig. 8 Results of structural monitoring of the H11, H11’ and H12 from the MD trajectory. a The tracked time-dependent distances for
important hydrogen bonds of H11, H11’ and H12 in the compound 6-RORγt system and compound 7h-RORγt system. Arg482-Phe486 stands
for the distance from the oxygen atom at the Arg482 backbone to the backbone nitrogen atom of Phe486. Leu483-Gln487, Pro491-Gln495,
Val493-Ala497 and Pro500-Glu504 stand for their own distance in the same way. Graphic representations of H11, H11’ and H12 (b: with “short”
inverse agonist 6, c: with “long” inverse agonist 7h, white: crystal structure, pink and orange: schematic structures at the end of MD
simulations).
DISCUSSION
“short” or “long” inverse agonists. Further analyses showed that
the “long” inverse agonists could change the skeletal structures of
H11 and H12, the helixes that were important to recruit
coactivators, while the “short” inverse agonists were more likely
to break the helix structure of H11’ and to influence side chains of
H11 and H12, resulting in the dislocation of H12. To the best of our
knowledge, this is the first study to discuss the difference between
“short” and “long” inverse agonists by proposing detailed
molecular mechanisms.
According to previous reports, inverse agonists with a common
scaffold may still significantly differ in gene expression, such as
CXCR4 and CTLA4, and may affect RORγt-DNA interactions with
different mechanisms [27]. Therefore, more experiments, including
ChIP-seq and RNA-seq, are needed to understand their unique
regulatory functions. We believe that the crystallography-guided
design of functionally switchable RORγt modulators and the
models we proposed to analyze the behaviors of agonists, “short”
and “long” inverse agonists will be helpful to better understand
the RORγt modulators at the molecular level and to make the drug
discovery process more efficient.
RORγt inverse agonists and agonists have attracted extensive
attention as novel therapeutic molecules for autoimmune diseases
and cancer immunotherapy in clinical trials. In previous work, the
functional characteristics of agonists and inverse agonists have
been elucidated [2, 11]. In our study, we first discovered a series of
6-substituted RORγt modulators, including agonists, “short” and
“long” inverse agonists, through insights into the cocrystal
structure of RORγt with inverse agonist 6, indicating a potent
approach to identify agonists and certain types of inverse
agonists. By using MD simulation data, we revealed that agonists
stabilize and inverse agonists destroy the hydrogen bond
between His479 and Tyr502, which confirmed previous work.
Although some reports suggested that the conformation of
Trp317 might affect the inverse agonist-agonist functional switch
[26], there was no obvious conformational change in Trp317 in our
study (Supplementary Fig. S6).
Furthermore, as reported recently [12], “short” inverse agonists
and “long” inverse agonists differed in the cofactor recruitment
assay. However, the molecular mechanisms between “short”
inverse agonists and “long” inverse agonists remain unclear.
Herein, starting from the minor difference observed in cocrystal
structures, we analyzed the side-chain dihedral angles of His479
and Tyr502 and found that the two residues flipped toward
opposite directions when introducing “short” inverse agonist 6 or
“long” inverse agonist 7h, indicating a different MOA between
In conclusion, we designed a series of carbazole-based RORγt
modulators using crystallography-guided design and succeeded
in the identification of agonists, “short” and “long” inverse
agonists in one scaffold. The “short” and “long” inverse agonists
were found to behave differently through MD simulation analysis.
The different molecular mechanisms of “short” and “long” inverse
Acta Pharmacologica Sinica (2020) 0:1 – 11

Insights to “short” and “long” RORγt inverse agonists
MC Yu et al.
11
agonists can be important to the drug discovery process and
therefore deserve more experiments to define their regulatory
functions in the future.
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ACKNOWLEDGEMENTS
We are extremely grateful to the National Centre for Protein Science Shanghai
(Protein Expression and Purification system) for their instrument support and
technical assistance. We thank the staff from the BL17U1 and BL19U1 beamlines at
the Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data
collection. We gratefully acknowledge the financial support from the National Natural
Science Foundation of China (Grant Numbers: 81703415; 81573276; 81874287;
81973163), the K. C. Wong Education to CL, the Fudan-SIMM Joint Research Fund
(Grant Number: FUSIMM20174007), the CAS Strategic Priority Research Program
(Grant Number: XDA12020372), the Shanghai Biopharmaceutical Science and
Technology Supporting Plan (Grant Number: 17431902100; 19431900100), the
National Science and Technology Major Project (Grant Number: 2018ZX09711002-
003-014), the Natural Science Foundation of Shanghai (Grant Number: 19ZR1436700),
and the China Postdoctoral Science Foundation (Grant Number: 2019M651383).
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AUTHOR CONTRIBUTIONS
MCY, FY, XYD, NNS, QX, HLJ, KXC, CL, XML, SJC, and YHW developed the hypothesis,
designed the experiments, and revised the manuscript. MCY, ZYJ, YFH, YRY, and CZ
designed and synthesized the compounds. FY, ZFC, and SQG performed the
crystallographic studies. MCY, XYD, and NNS conducted the MD simulation and data
analysis. All authors read and approved the final manuscript.
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-020-00552-w)
contains supplementary material, which is available to authorized users.
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