Covalent Occlusion of the RORγt Ligand Binding Pocket Allows Unambiguous Targeting of an Allosteric Site

Article abstract of DOI:10.1021/acsmedchemlett.1c00029

The nuclear receptor RORγt is a key positive regulator in the differentiation and proliferation of T helper 17 (Th17) cells and the production of proinflammatory cytokines like IL-17a. Dysregulation of this pathway can result in the development of various

Full text of DOI:10.1021/acsmedchemlett.1c00029

pubs.acs.org/acsmedchemlett
Letter
Covalent Occlusion of the RORγt Ligand Binding Pocket Allows
Unambiguous Targeting of an Allosteric Site
Femke A. Meijer,§ Maxime C. M. van den Oetelaar,§ Richard G. Doveston, Ella N. R. Sampers,
and Luc Brunsveld*
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ABSTRACT: The nuclear receptor RORγt is a key positive
regulator in the differentiation and proliferation of T helper 17
(Th17) cells and the production of proinflammatory cytokines like
IL-17a. Dysregulation of this pathway can result in the develop-
ment of various autoimmune diseases, and inhibition of RORγt
with small molecules thus holds great potential as a therapeutic
strategy. RORγt has a unique allosteric ligand binding site in the
ligand binding domain, which is distinct from the canonical,
orthosteric binding site. Allosteric modulation of RORγt shows
high potential, but the targeted discovery of novel allosteric ligands
is highly challenging via currently available methods. Here, we
introduce covalent, orthosteric chemical probes for RORγt that occlude the binding of canonical, orthosteric ligands but still allow
allosteric ligand binding. Ultimately, these probes could be used to underpin screening approaches for the unambiguous and rapid
identification of novel allosteric RORγt ligands.
KEYWORDS: Nuclear receptors, RORγt, covalent probes, allosteric modulators
examples of highly potent RORγt inverse agonists that bind to
he retinoic acid receptor-related orphan receptor gamma
Tt (RORγt) is a nuclear receptor (NR) that plays an this allosteric site.15−19 These ligands exert their effect via a
reorientation of H12 into a conformation that prevents
coactivator binding.15,18,19
important regulatory role in the immune system via the Th17/
IL-17a pathway.1−3 Inhibition of RORγt by inverse agonists
has been shown to be a promising strategy for the treatment of
autoimmune diseases and thus has been the focus of several
drug discovery programs.4,5 The majority of RORγt ligands
bind to a highly conserved canonical binding pocket, termed
the orthosteric binding site, within the ligand binding domain
(LBD) of RORγt (Figure 1C).6−10 RORγt appears to be
transcriptionally active, even in the absence of an agonist
ligand.11 However, agonists (e.g., cholesterol and its deriva-
tives) that bind to the orthosteric site enhance RORγt
transcriptional activity further by stabilizing the active
conformation of helix 12 (H12) in a way that promotes the
recruitment of transcriptional coactivators.12 RORγt inverse
agonists binding this site (e.g., digoxin) destabilize the active
conformation of H12, inhibiting coactivator recruitment.13
The conserved nature of the RORγt orthosteric binding
pocket presents some challenges for drug discovery, for
example, because of competition with endogenous ligands.14
Most interestingly, and unique among the NR family, RORγt
has a second binding site, termed allosteric binding site
(located at a topographically distinct place in the LBD)
(Figure 1C).15 This allosteric site offers ample opportunities
for innovative NR drug discovery. The indazole MRL-871,
thiazole compound 13 (Glenmark), and isoxazole FM26 are
Despite the potential of NR allosteric inverse agonists, the
number of examples and their chemical diversity have
remained rather limited.15−17,19−22 Furthermore, understand-
ing of the structure−activity relationships (SARs) and scaffold
diversity of allosteric RORγt inverse agonists is of importance
to tune the potency, selectivity, and pharmacokinetic profiles of
potential pharmaceutical lead molecules. However, unambig-
uous screening for allosteric ligands is challenging, since both
orthosteric and allosteric ligands can show an inhibitory
response on RORγt (Figure 1A, left). Currently, discrimination
between orthosteric and allosteric ligands is not trivial and the
orthosteric pocket can host a plethora of chemically diverse
compounds. Illustrative of the above, the mode of action for
MRL-871 was only described in retrospect.15 Even in a
targeted program, the methods currently available require a
series of biophysical and structural experiments to discriminate
Received: January 17, 2021
Accepted: March 3, 2021
Published: March 8, 2021
© 2021 The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acsmedchemlett.1c00029
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Figure 1. (A) Schematic representation of RORγt orthosteric site occlusion. When both binding sites are available, orthosteric and allosteric ligands
are identified in coactivator binding assays. When the orthosteric binding site is occluded, allosteric ligands can be unambiguously identified. (B)
Crystal structure of PPARγ ligated to GW9662 (blue) at the Cys285 residue (green) in the orthosteric binding site (PDB: 3B0R). (C) Crystal
structure of RORγt (PDB: 6T4I) showing the orthosteric (green) and allosteric (orange) ligand binding site. The Cys320 residue in the orthosteric
site is shown in red. (D) Chemical structures of PPARγ covalent ligands GW9662 (1) and SB1404 (2). (E) Enlarged view of GW9662 ligated to
PPARγ. (F) The thiol of Cys320 in RORγt is postulated to ligate to the electron-deficient aryl ring of GW9662 or derivatives.
between orthosteric and allosteric inverse agonism.15 There-
fore, the development of a molecular approach for the specific
identification of allosteric ligands is highly desirable.
established coactivator recruitment assay, would provide a
more robust approach (Figure 1A, right). An important feature
of the prospective orthosteric probe is that it should have
minimal effect on the characteristics of the allosteric pocket
and on RORγt coactivator binding.
Our approach took inspiration from studies of the
structurally related NR peroxisome proliferator-activated
receptor γ (PPARγ). PPARγ contains a cysteine residue within
the orthosteric ligand binding pocket (Cys285) (Figure 1B),
which has previously been targeted by covalent ligands (Figure
1E).23−25 In particular, covalent modification of Cys285 by the
electron deficient aryl chlorides GW9662 (1) and SB1404 (2)
(Figure 1D) occlude the binding, and thus also the activity, of
certain PPARγ agonists.24−28 RORγt contains an analogous
A potential strategy to achieve the goal defined above would
be via a competitive binding assay, whereby the displacement
of a well-characterized allosteric probe ligand can be
monitored, such as the one previously reported.15,19 However,
competition assays are notoriously problematic for identi-
fication of the weaker binding initial chemical entities.
Additionally, orthosteric inverse agonists could potentially
lower the allosteric probe affinity, which would lead to false
positive results that diminish the reliability of the assay. We
therefore postulated that using a molecular probe to occlude
the RORγt orthosteric binding site, in combination with an
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Figure 2. (A−C) Dose−response curves of a TR-FRET coactivator recruitment assay with RORγt by titration of MRL-871 and different
orthosteric covalent probes. Data recorded in triplicate from two independent experiments (one representative data set shown). Error bars
represent the SD of the mean. (D) Schematic representation of the TR-FRET coactivator recruitment assay. When RORγt is in its apo state, the
coactivator binds to the LBD, resulting in FRET pairing from an anti-His terbium cryptate donor to the d2-labeled coactivator. When the covalent
orthosteric probe ligates to the protein, coactivator recruitment is blocked by its (partial) inverse agonistic character.
cysteine residue within the orthosteric ligand binding pocket
(Cys320, RORγ numbering; see Figures 1C and S1).
Therefore, we were interested to determine if this residue
could also be targeted with covalent molecular probes that
would block ligand binding to the orthosteric site without
significantly affecting coactivator binding. Examples of RORγt
covalent inverse agonists have been reported, but they, thus far,
target other cysteine residues that are not in proximity to the
orthosteric binding pocket.22
GW9662 and SB1404 were synthesized via an amide
coupling reaction between the commercially available 2-
chloro-5-nitrobenzoyl chloride and the appropriate amine
(Scheme S1).24,27 A single GW9662 molecule fully ligated to
RORγt, following optimization of the ligation conditions24
(Table S1), as shown by quadrupole time-of-flight mass
spectrometry (Q-TOF MS) (Figure S2A). In order to prove
binding to Cys320, a RORγt Cys320Ala mutant was generated.
No ligation of GW9662 to this RORγt mutant was observed
which verified the expected Cys320 ligation site (Figure S2B).
Surprisingly, SB1404, which has a smaller methyl group instead
of the phenyl substituent, did not show any ligation to RORγt,
while it did fully ligate to PPARγ. This might be because
SB1404 has a lower affinity for the RORγt LBP compared to
GW9662. This observation highlights the difference between
the binding pockets of the two NRs.
orthosteric inverse agonist T090131731,32 shows that the nitro
moiety of GW9662 and the CF3 groups of T0901317 have the
same orientation and distance toward H11, which could
explain the inverse agonistic behavior of GW9662 (Figure S4).
A ligand binding TR-FRET coactivator recruitment assay
was used to determine if GW9662 ligation effectively occluded
orthosteric ligand binding to RORγt, without affecting
allosteric ligand binding (see Figure 3F/G). To probe this,
three orthosteric agonists (cholesterol (CHL), 20α-hydrox-
ycholesterol (20-OH), and desmosterol (DSM)), an orthos-
teric inverse agonist (digoxin), and two allosteric inverse
agonists (MRL-871 and FM26) were tested (see chemical
structures in Figure 3H).12,13,15,19 For the apo RORγt protein,
the cholesterol derivatives show agonistic character, increasing
coactivator recruitment to the LBD (Figure 3A), consistent
with literature reports.2,12 The orthosteric inverse agonist
digoxin and the allosteric inverse agonists reduced coactivator
recruitment in a dose-dependent manner as expected (Figure
3A).13,19 These titration experiments were then repeated with
the GW9662-ligated RORγt (Figure 3B). In this experiment,
the orthosteric agonists did not cause any increase in
coactivator recruitment to RORγt, indicating occlusion of the
orthosteric binding site. However, the allosteric ligands MRL-
871 and FM26 also did not demonstrate a change in
coactivator recruitment, due to the full inverse agonistic
behavior of GW9662. Because of its full inverse agonistic
behavior, GW9662 is thus not suitable for the screening of
allosteric inverse agonists. We therefore set out to identify a
GW9662 analogue that would act either as true covalent
antagonist (/agonist) or as a partial covalent inverse agonist,
that would maintain some RORγt sensitivity to allosteric ligand
binding.
A TR-FRET coactivator recruitment assay29 was used to
investigate the effect of GW9662 on the activity of RORγt, i.e.,
the ability of RORγt to recruit coactivators (Figure 2D). The
dose−response curve in Figure 2A shows inhibition of
coactivator recruitment upon titration of GW9662 to the
protein (similar behavior to the full allosteric inverse agonist
MRL-871), demonstrating that the probe acts as a full inverse
agonist for RORγt with an IC50 value of 86 5 nM. A possible
reason for this inverse agonistic character could be derived
from the docking pose, which shows a conformation of
GW9662 where the warhead (nitro-phenyl moiety) points
toward H11 (Figure S3A), which could result in destabilization
of the active conformation of H12, inhibiting coactivator
recruitment.30 Alignment of the docking pose of GW9662 to
the crystal structure of RORγt bound to the known RORγt
An initial library of 12 GW9662-derivatives was synthesized
(compounds 3−14, Table 1), containing the same warhead,
but with modifications in place of the phenyl moiety, to
investigate its influence on the ligation efficiency and activity of
RORγt. These modifications were made with the aim to
identify a probe that ligates to the RORγt Cys320 but does not
behave as a full inverse agonist. The modifications were varied
in terms of bulkiness, π−π stacking capacity, aromaticity, and
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Figure 3. TR-FRET coactivator recruitment ligand binding assay with RORγt (unligated and ligated) by titration of various orthosteric and
allosteric ligands. (A) Apo (unligated) RORγt protein. (B) GW9662-ligated RORγt. (C) Compound 9-ligated RORγt. (D) Compound 19-ligated
RORγt. (E). Compound 20-ligated RORγt. Data recorded in triplicate from two independent experiments (one representative data set shown).
Error bars represent the SD of the mean. Abbreviations: n.a., not active. (F/G) Schematic representation of the TR-FRET coactivator recruitment
assay, using the ligated protein. (F) Orthosteric ligand binding will be occluded, showing no effect on the initial coactivator recruitment capacity.
(G) Allosteric ligand binding will result in reduced coactivator binding and therefore a lower FRET pairing. (H) Chemical structures of orthosteric
agonists (cholesterol (CHL), desmosterol (DSM), and 20α-hydroxycholesterol (20-OH)), orthosteric inverse agonist digoxin, and allosteric
inverse agonists MRL-871 and FM26.
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Table 1. Chemical Structures of GW9662 Derivatives and
Their RORγt Ligation
Table 2. Chemical Structures of GW9662 Derivatives, Their
RORγt Ligation, IC50 SD (nM) (Data Recorded in
Triplicate in Two Independent Experiments) And Full/
a
Partial Inverse Agonistic Character
substitution pattern. The compounds were synthesized in a
similar manner to GW9662 (Scheme S1B).24,33−35 First, the
ligation efficiency was explored for all derivatives via Q-TOF
analysis of the reacted protein. Although GW9662 fully ligated
to the protein (Figure S6), its derivatives ligated with varying
efficiency (Table 1, Figures S7 and S8) (ligation conditions
shown in Table S1). From the ligation data, it can be
concluded that a ring system (preferably aromatic) is necessary
at the phenyl position of GW9662 in order to obtain full
ligation to the protein. A methyl substitution on the phenyl
ring is only fully tolerated at the ortho position (compound 9),
indicating that bulk at meta/para positions lowers ligation
(compound 7 and 8). Furthermore, an electron-withdrawing
substituent on the phenyl ring improves the ligation efficiency
compared to an electron-donating group (compound 13 vs
10), most probably caused by the higher electrophilicity of the
warhead. Interestingly, all probes (except for compounds 12
and 14) also showed full covalent attachment to PPARγ,
indicating that PPARγ shows less differentiation in the ligation
of the compounds than RORγt, which is probably due to the
larger size of the PPARγ binding pocket.
a
Abbreviations: inv. ago., inverse agonist; rem. act., remaining activity;
n.d., not determined.
The compounds that fully ligated to RORγt (9 and 11) were
taken forward for evaluation of the binding behavior in a TR-
FRET dose−response assay. Compound 11 showed the same
full inverse agonistic behavior as GW9662 but was slightly less
potent (IC50 value of 122 6 nM) (Figure 2A, Table 2). More
interestingly, in contrast to the other two probes, compound 9
showed partial inverse agonistic behavior, thus not completely
blocking coactivator recruitment to the LBD, with 55%
remaining activity (Figure 2A, Table 2). The difference in
activity between GW9662 and compound 9 could be explained
by an inverse binding conformation of 9 (with the warhead
pointing toward the orthosteric pocket instead of toward H11)
which was supported by an in silico docking experiment
(Figure S3B).
A focused SAR study was performed around compound 9 to
obtain more insight into the desirable partial behavior of this
probe. A library of nine compound 9 derivatives (compounds
15−23, Table 2) was designed, by varying the size, polarity,
and electron density of the ortho-substituent. The probes were
synthesized as described above (Scheme S1B). All the probes
from this set of derivatives fully ligated with RORγt (Table 2,
Figures S9−S16), except for compound 16 that has a tert-butyl
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Figure 4. Thermal shift assay performed for Apo RORγt (A), compound 9-ligated RORγt (B), compound 19-ligated RORγt (C), and compound
20-ligated RORγt (D), for ligands cholesterol (CHL), 20α-hydroxycholesterol (20-OH), desmosterol (DSM), digoxin, MRL-871 and FM26 (2
equiv compound relative to protein). The melting temperatures (ΔTm in °C) are shown. Data recorded in triplicate from two independent
experiments (one representative data set shown). Error bars represent the SD of the mean.
substituent. Although an ethyl substituent and a naphthalene
moiety appeared to be tolerated (15 and 21), the tert-butyl
moiety of 16 is likely too bulky for optimal binding.
Furthermore, the polarity of the substituent (22 and 23) did
not affect the ligation behavior and even a double-ortho methyl
substitution was tolerated (19).
All probes (except 16) were tested in the TR-FRET dose−
response assay and showed varying inverse agonistic behavior
(Figure 2B/C). A partial inverse agonistic behavior, similar to
compound 9, was observed for probes 15 and 18 (increased
size of the substituent), 19 (bis-ortho substitution), and 20
(electron-deficient substituent) (Figure 2B). Compound 17
with a fluoro substituent also demonstrated partial behavior,
however inducing a greater decrease in coactivator recruitment
than the previous four probes (Figure 2C), which might be due
to its size being more comparable to GW9662. Compound 23
(hydroxyl modification) resulted in full inverse agonism,
similar to GW9662, and 22 (amine modification) also
approached the bottom plateau, but with a lower potency
than 23. Compound 21, with the large naphthalene
substituent, showed a significantly lower potency than the
detect allosteric inverse agonist binding. Clear occlusion of the
orthosteric site for agonist binding was observed for RORγt
ligated to 9, 19, and 20 (Figure 3C−E), in comparison to the
apo protein (Figure 3A). In addition, the orthosteric inverse
agonist digoxin was also ineffective. In contrast, and most
importantly, the allosteric inverse agonists MRL-871 and
FM26 still induced a clear inverse agonistic response in RORγt.
Also, the potency of MRL-871 and FM26 was not negatively
affected by the occlusion of the orthosteric site, revealing that
the partial inverse agonism of the covalent probes translates to
responsiveness of the allosteric site for ligand binding. The
other partial inverse agonistic probes 15 and 18 show similar
occlusion of the orthosteric site while still allowing allosteric
binding, albeit with a smaller assay window (Figure S5).
Interestingly, in all cases, the IC50 values for the allosteric
ligands MRL-871 and FM26 were actually decreased to
different extents (3-fold and 13-fold, respectively) in
comparison to the data for the apo protein (Figure 3A/C−
E). This increased potency of the allosteric ligands in the
presence of an orthosteric probe is likely caused by a
cooperative effect between both binding sites, as observed in
previous studies.18,19,36
other probes (IC50 = 1080
153 nM), not reaching the
bottom plateau, which makes it hard to confidently character-
ize it as a partial or full inverse agonist.
A thermal shift assay (TSA) was used as an orthogonal
method to confirm occlusion of orthosteric ligand binding by
the most interesting probes (9, 19, and 20) without affecting
allosteric modulation. With the apo RORγt protein, all
orthosteric ligands showed a significant thermal stabilization
(ΔTm between 2.5 and 5.8 °C), except for cholesterol (Figure
4A). In contrast, for RORγt ligated to 9, 19, and 20, a thermal
stabilization effect was not observed anymore for these
orthosteric ligands and they even caused a small destabilization
effect (ΔTm between −0.1 and −2.7 °C) (Figure 4B−D).
These results again demonstrate orthosteric site occlusion by
the covalent probes. The allosteric ligands MRL-871 and
FM26 show a moderate to high thermal stabilization effect for
the apo protein (respectively ΔTm of 7.5 and 2.1 °C) (Figure
4A). When RORγt was ligated to a covalent probe, this thermal
stabilization effect by the allosteric ligands was preserved and
even higher ΔTm values were observed in all cases (Figure 4B−
D). These results again prove that the allosteric binding is still
The modifications with a similar size to the methyl group in
compound 9 (e.g., ethyl, methoxy, bismethyl, trifluoromethyl)
appear to result in a partial inverse agonistic behavior, while
smaller or polar substituents (e.g., fluoro, hydroxyl, amine)
show a full inverse agonistic character. A structural explanation
could be derived from the docking studies, where the partial
inverse agonists generally show a docking pose similar to
compound 9 (warhead pointing toward the orthosteric site),
while for the full inverse agonists a docking pose similar to
GW9662 was observed. Combined, compound 9 and four
derivatives were found to be covalent partial inverse agonists
and suitable candidates as covalent orthosteric probes.
The TR-FRET ligand binding assay was used to investigate
the combination of occlusion of the orthosteric site and
potential for allosteric binding. With the partial inverse agonist
probes, a screening window is expected to be preserved to
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functional with the orthosteric site blocked and that affinity is
even slightly enhanced. This increased stabilization by the
allosteric ligands in the presence of the orthosteric covalent
probes is consistent with the cooperative behavior observed in
the TR-FRET ligand binding assays. Although the observed
cooperative behavior might question the usefulness of the
probes at first sight, it will not be an issue from a screening
perspective, since it will not lead to false hits but will just
enhance an allosteric IC50 value by a limited degree.
AUTHOR INFORMATION
■
Corresponding Author
Luc Brunsveld − Laboratory of Chemical Biology, Department
of Biomedical Engineering and Institute for Complex
Molecular Systems, Technische Universiteit Eindhoven, 5612
AZ Eindhoven, The Netherlands; orcid.org/0000-0001-
5675-511X; Email: l.brunsveld@tue.nl
Authors
Although allosteric RORγt inverse agonists have high
potential for NR drug discovery, the number of examples
and chemical diversity have remained limited. An enlargement
of the allosteric RORγt ligand library is therefore essential in
order to enhance understanding of the SAR and to tune
potency and selectivity. Unambiguous screening for allosteric
ligands is challenging, since both orthosteric and allosteric
ligands will result in an inhibitory response on the protein and
discrimination between them is not trivial.
Femke A. Meijer − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems, Technische Universiteit
Eindhoven, 5612 AZ Eindhoven, The Netherlands;
orcid.org/0000-0003-4412-9968
Maxime C. M. van den Oetelaar − Laboratory of Chemical
Biology, Department of Biomedical Engineering and Institute
for Complex Molecular Systems, Technische Universiteit
Eindhoven, 5612 AZ Eindhoven, The Netherlands
Richard G. Doveston − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems, Technische Universiteit
Eindhoven, 5612 AZ Eindhoven, The Netherlands; Leicester
Institute of Structural and Chemical Biology and School of
Chemistry, University of Leicester, Leicester LE1 7RH, United
Kingdom
Here, we introduced a method for occlusion of the RORγt
orthosteric binding site via the ligation of covalent chemical
probes to a native cysteine residue. This allows for the
unambiguous targeting of the allosteric binding site, which has
the potential to facilitate the rapid identification of allosteric
inverse agonists. The reference compound GW9662 showed
full ligation to Cys320 of RORγt but acts as a full inverse
agonist and completely inhibits coactivator binding, preventing
the detection of allosteric inverse agonists. From a small library
of GW9662 derivatives, the methyl-substituted compound 9
was identified as a covalent partial inverse agonist. Further SAR
studies around compound 9 resulted in the discovery of four
additional covalent probes with a partial inverse agonistic
character, for which 19 and 20 appeared to be the most
promising probes (containing a bis-ortho-methyl and trifluor-
omethyl modification). The partial character of these probes
can most probably be explained by an inverse binding
conformation compared to GW9662 as was supported by
docking experiments. Co-crystallization attempts of the probes
with RORγt were unsuccessful but could provide more
structural evidence. TR-FRET and thermal shift assays
revealed complete occlusion of the orthosteric binding site
with the covalent probes, while allosteric ligand binding was
not inhibited and even occurred with enhanced affinity. This
cooperative behavior of the orthosteric inverse agonistic
covalent probes with the allosteric ligands is an interesting
observation, since these cooperative effects had previously only
been observed with orthosteric agonists. The covalent probes
are excellent tools that could underpin an assay format that
unambiguously screens for allosteric RORγt modulators.
Additionally, these covalent orthosteric ligands could be used
as inspiration for the development of covalent orthosteric
inverse agonists for RORγt, for which future studies could
focus on the efficacy and toxicity of covalent RORγt targeting.
Ella N. R. Sampers − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems, Technische Universiteit
Eindhoven, 5612 AZ Eindhoven, The Netherlands
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00029
Author Contributions
§F.A.M. and M.C.M.O. contributed equally. M.C.M.O. and
E.N.R.S. performed synthesis; F.A.M., M.C.M.O., and E.N.R.S.
performed biochemical studies; F.A.M., M.C.M.O., R.G.D.,
and L.B. designed the studies. The manuscript was written
through contributions of all authors.
Funding
This work was supported by The Netherlands Organization for
Scientific Research through Gravity program 024.001.035 and
VICI Grant 016.150.366 and the European Union through a
MSCA Individual Fellowship (R.G.D., H2020-MSCA-IEF-
2016, Grant Number 705188). This work was carried out on
the Dutch national e-infrastructure with the support of SURF
Cooperative.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Rens M.J.M. de Vries for his efforts in
crystallography experiments and Joost L.J. van Dongen for
performing HRMS measurements.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00029.
ABBREVIATIONS
■
20-OH, 20α-hydroxycholesterol; CHL, cholesterol; DSM,
desmosterol; H12, helix 12; IPTG, isopropyl-b-D-thiogalacto-
side; mAb, monoclonal antibody; LBD, ligand binding domain;
NR, nuclear receptor; PPARγ, peroxisome proliferator-
activated receptor γ; Q-TOF, quadrupole time-of-flight;
RORγt, retinoic acid receptor-related orphan receptor γ t;
Additional figures, synthetic procedures, compound
characterization, details description of methods con-
cerning protein expression, in silico measurements,
ligation, and biochemical assays (PDF)
Compound characterization checklist (XLS)
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(19) Meijer, F. A.; Doveston, R. G.; De Vries, R. M. J. M.; Vos, G.
M.; Vos, A. A. A.; Leysen, S.; Scheepstra, M.; Ottmann, C.; Milroy, L.-
G.; Brunsveld, L. Ligand-Based Design of Allosteric Retinoic Acid
Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists. J.
Med. Chem. 2020, 63, 241−259.
SAR, structure−activity relationship; Th17, T helper 17; Tm,
melting temperature; TR-FRET, time-resolved FRET
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