Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

Article abstract of DOI:10.1021/acs.jmedchem.0c01137

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine recept

Full text of DOI:10.1021/acs.jmedchem.0c01137

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Article
Design and Characterization of an Intracellular Covalent Ligand for
CC Chemokine Receptor 2
̌
́
Natalia V. Ortiz Zacarías, Kirti K. Chahal, Tereza Simkova, Cas van der Horst, Yi Zheng, Asuka Inoue,
Emy Theunissen, Lloyd Mallee, Daan van der Es, Julien Louvel, Adriaan P. IJzerman, Tracy M. Handel,*
Irina Kufareva,* and Laura H. Heitman*
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ABSTRACT: Covalently acting inhibitors constitute a large and
growing fraction of approved small-molecule therapeutics as well as
useful tools for a variety of in vitro and in vivo applications. Here, we
aimed to develop a covalent antagonist of CC chemokine receptor 2
(CCR2), a class A GPCR that has been pursued as a therapeutic target
in inflammation and immuno-oncology. Based on a known intra-
cellularly binding CCR2 antagonist, several covalent derivatives were
synthesized and characterized by radioligand binding and functional
assays. These studies revealed compound 14 as an intracellular
covalent ligand for CCR2. In silico modeling followed by site-directed
mutagenesis confirmed that 14 forms a covalent bond with one of
three proximal cysteine residues, which can be engaged interchange-
ably. To our knowledge, compound 14 represents the first covalent
ligand reported for CCR2. Due to its unique properties, it may
represent a promising tool for ongoing and future studies of CCR2 pharmacology.
covalent drugs, including insurmountability and prolonged
duration of action.13−15 The later advantage may be
particularly useful in disease contexts in which high receptor
occupancy and prolonged inhibition are required for an
effective response.16 As tool compounds, covalent ligands have
been increasingly used for structure elucidation as they can
stabilize inherently flexible receptor−ligand complexes.9
Examples include the recent crystal structures of the
cannabinoid CB1 receptor17 and the adenosine A1 receptor.18
Furthermore, covalent probes represent valuable starting
points for a wide variety of chemical biology and proteomic
profiling applications.9,12
Recently, the X-ray structures of CCR2 isoform A
(CCR2a)19 and isoform B (CCR2b)20 have been solved in
complex with small-molecule antagonists. Besides providing
structural insight into the binding poses of the antagonists, the
crystal structure of CCR2b revealed an intracellular binding
site, which accommodates small molecules that inhibit CCR2
without directly competing with the binding of chemokines.20
INTRODUCTION
■
CC chemokine receptor 2 (CCR2) is a G protein-coupled
receptor (GPCR) expressed on the surface of various immune
cells, including monocytes, basophiles, and natural killer cells.1
Activation of CCR2 by its endogenous chemokine ligands,
such as CCL2, results in leukocyte trafficking toward sites of
inflammation as part of the immune response.2 However,
dysregulation of CCR2 signaling can lead to leukocyte
accumulationa hallmark of the inflammatory response
and ultimately to a variety of inflammatory and immune
diseases.3 Preclinical studies have suggested a critical role of
CCR2/CCL2 signaling in atherosclerosis,4 diabetes,5 neuro-
pathic pain,6 and cancer,7 among others. However, despite
extensive efforts to develop CCR2 antagonist therapeutics,
most clinical candidates have failed due to lack of efficacy.8
Thus, a better understanding of the structure and biological
function of CCR2, both in vitro and in vivo, is necessary for the
development of clinically successful CCR2 antagonists.
Covalent ligands have recently re-emerged as valuable tool
compounds and therapeutic agents for various targets and
diseases.9−11 Covalent ligands contain a reactive chemical
group, or “warhead”, which allows them to bind to their target
in an irreversible manner.12 In the field of GPCRs, covalent
ligands have been primarily used as tool compounds due to
major safety concerns; however, recent studies have high-
lighted several potential advantages for the development of
Received: July 2, 2020
Published: February 18, 2021
© 2021 The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acs.jmedchem.0c01137
J. Med. Chem. 2021, 64, 2608−2621
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Figure 1. Chemical structures and binding mode of sulfonamide derivatives. (a) Chemical structures of the parent compound SD-24, non-covalent
control 7, and the sulfonamide scaffold used for the design of putative covalent ligands. (b) Binding mode of compound 7 in the intracellular
binding pocket of CCR2. (c) Docking of compound 7 into the crystal structure of CCR2 (PDB 5T1A); compound 7 makes a hydrogen bond with
Cys75ICL1
.
a
Scheme 1. Synthesis of Compounds 7 and 11−14
a
Final compounds were synthesized using the following reagents and conditions: (a) K2CO3, DMF, 70 °C, 3.5 h or overnight; (b) SnCl2·2H2O,
EtOAc, rt, overnight; (c) 3, indium, MeCN, rt, overnight, or 4, DMAP, pyridine, microwave 95 °C, 3 h; (d) NaOH, dioxane, rt, 2 h, or NaOH,
dioxane, 60 °C, 2.5 h; (e) i. tert-butyl-N-(2-aminoethyl)carbamate, EDC, HOBt, dioxane, rt; ii. TFA, DCM, rt, for 9; or 2-bromoethan-1-amine,
EDC, HOBt, dioxane, rt for 10; (f) 9, TEA, CS2, TsCl, THF for 11; (g) 9, acryloyl chloride, TEA, acetonitrile, −78 °C to rt for 12; (h) 9, 4-
(fluorosulfonyl) benzoic acid, EDC, HOBt, TEA, dioxane, rt for 13; (i) 10, KSCN, EtOH, reflux, for 14.
Due to their noncompetitive manner of inhibition, these
intracellular allosteric modulators might be more efficacious in
the treatment of inflammatory diseases characterized by high
levels of endogenous chemokines.21 An intracellular covalent
ligand could be valuable for further investigation of CCR2
pharmacology and role in diseases. Thus, based on previously
described high-affinity intracellular ligands for CCR2,22 we
aimed to design and synthesize a novel covalent intracellular
ligand for this receptor. Biochemical characterization, in silico
modeling, and a mutational study resulted in the identification
of compound 14 as a covalent, negative allosteric modulator
(NAM) that binds intracellularly and forms a covalent bond
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with one of three proximal cysteine residues of CCR2.
Compound 14 represents a promising starting point for a
variety of applications to further study CCR2.
tion-dependent manner, displaying high to moderate binding
affinities for hCCR2b (Ki < 100 nM, Table 1). Of note, for
Table 1. Binding Affinities (pKi) of Synthesized
Sulfonamide Derivatives Determined in [3H]-CCR2-RA
RESULTS
■
b
Displacement Assays
Covalent Derivatization of a Sulfonamide Antagonist
Yields Novel CCR2 Inhibitors. The design of covalent
ligands for CCR2 was based on a previously described
sulfonamide series, whose representative SD-2422 was
identified as a high-affinity, intracellular ligand for CCR2
(Figure 1a).23 Previous structure−activity studies also showed
that it is possible to extend these derivatives on the “left hand”
phenyl moiety, particularly at the para position, as CCR2
activity is not affected after introduction of cyclic amines linked
by an ethylacetamide moiety.24 Thus, we decided to introduce
a reactive warhead in the para position, using the described
ethylacetamide linker. A close analogue of SD-24, with the
carboxylic group in the para position (sulfonamide 34 by
Peace et al.,22 corresponding to compound 7 in our study
(Figure 1a)) was used as a non-covalent control. Because the
predicted binding mode of compound 7 featured a hydrogen-
bond between its carboxyl group and Cys75 in the first
intracellular loop (ICL1) of CCR2 (Cys75ICL1, Figure 1b), we
envisioned four warheads with varying selectivity toward
cysteine: isothiocyanate, which targets both cysteine and lysine
residues;25,26 acryloyl, a Michael acceptor that preferentially
targets cysteine over lysine;27 a non-selective fluorosulfonyl,
with reactivity toward serine, threonine, tyrosine, lysine,
cysteine, and histidine;28 and thiocyanate, which selectively
reacts with cysteine.12,29 In addition, a trifluoromethyl group
was incorporated in the design as this moiety has been shown
to improve the activity of sulfonamide derivatives toward
CCR224 (Figure 1a).
The synthesis of final compounds 7 and 11−14 is shown in
Scheme 1. Briefly, commercially available starting materials
(methyl 4-hydroxybenzoate and 5-chloro-2-fluoronitroben-
zene) were combined under basic conditions to generate
compound 1 in high yield. Reduction of the aromatic nitro
group using tin(II) chloride as a reducing agent30 followed by
sulfonylation of the release amine using indium in acetoni-
trile31 or DMAP in pyridine as a catalyst yielded 5 or 6,
respectively. Compounds 5 and 6 were subjected to
saponification with sodium hydroxide in dioxane to yield the
final compound 7 and the intermediate compound 8. The
ethylacetamide linkers were attached to 8 by a peptide
coupling reaction32 followed by the introduction of the
different electrophilic warheads. Transformation of the amine
in 9 to an isothiocyanate in 11 was performed using carbon
disulfide and tosyl chloride (TsCl) as previously described.33
Reaction of the terminal amine in 9 with acryloyl chloride in
the presence of triethylamine yielded the final compound 12,
while the fluorosulfonyl warhead on 13 was attached by
peptide coupling of 9 with 4-(fluorosulfonyl)benzoic acid.32
Finally, the thiocyanate warhead was incorporated by SN2
substitution on the bromide intermediate 10, yielding the final
compound 14 (Scheme 1).
compound
pKi SEM (Ki, nM)
8.2 0.03 (6)
7.5 0.04 (31)
7
a
11
12
13
14
a
7.7 0.14 (22)
a
7.2 0.16 (89)
a
8.4 0.06 (4)
a
As these compounds might bind covalently, we only refer to the
affinities as apparent affinities. Data are presented as mean SEM of
at least three individual experiments performed in duplicate.
b
compounds 11−14, binding affinities are reported as “apparent
(p)Ki” values due to the putative covalent interaction between
these compounds with CCR2, which precludes the determi-
nation of equilibrium binding parameters. The non-covalent
control, compound 7, displaced [3H]-CCR2-RA with a Ki
value of 6 nM (Table 1). Compound 13 with a non-selective
fluorosulfonyl warhead exhibited the highest (15-fold) loss of
affinity compared to compound 7 (apparent Ki, 89 nM).
Compound 13 was followed by compound 11 with the
isothiocyanate warhead, which showed a 5-fold reduction in
affinity compared to compound 7 (apparent Ki, 31 nM).
Compounds containing an acryloyl (12) or thiocyanate (14)
warhead displayed the highest apparent affinities toward
CCR2. Compound 12 displaced [3H]-CCR2-RA with an
apparent Ki of 22 nM, while 14 displaced it with an apparent Ki
of 4 nM, the highest affinity in this series of compounds (Table
1). Based on the apparent affinities, we decided to continue
with compound 14 for further characterization.
Because putative covalent drugs can be intrinsically unstable,
the stability of 7 and 14 was assessed under the conditions
used in radioligand binding assays (data not shown). Both
compounds (25 μM) remained intact for over 6 h in assay
buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 0.1% CHAPS)
at room temperature. Furthermore, to demonstrate that
compound 14’s reactivity with CCR2 is significantly higher
than the intrinsic reactivity of the warhead, compound 14 (25
μM) was coincubated with 80 μM reduced glutathione (GSH)
at room temperature for over 6 h. This treatment did not result
in the formation of a GSH-adduct, suggesting a low intrinsic
reactivity of the thiocyanate warhead under the assay
conditions. Finally, increasing the concentration of compound
to 1 mM and the incubation temperature to 37 °C, resulted in
gradual degradation of compound 14 over 4 h, indicating lower
stability at higher temperatures. Compound 7 remained intact
under these conditions.
Radioligand Binding Assays with Preincubation and
Washing Suggest Covalent Binding of Compound 14 to
CCR2. To determine whether compound 14 binds irreversibly
to CCR2, the time dependency of its affinity, in comparison
with the non-covalent control, was determined using the (R)-
pure isomer [3H]-CCR2-RA-[R]. Specifically, the affinity of
compounds 7 and 14 to U2OS-CCR2 membranes was
measured in a [3H]-CCR2-RA-[R] displacement assay with a
short incubation time of 20 min, which was preceded or not by
a longer preincubation of the membranes with compounds 7
or 14 (Figure 2a,b). Compared to the coincubation experi-
The binding affinities of compounds 7 and 11−14 were
determined in membranes from U2OS cells stably expressing
hCCR2b (U2OS-CCR2). [3H]-CCR2-RA, a racemic version
of the CCR2-RA-[R] antagonist previously shown to target the
intracellular allosteric site in CCR2,20,23,34,35 was used as a
radiolabeled probe. In competition binding assays, all
compounds fully displaced [3H]-CCR2-RA in a concentra-
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Table 2. Time-Dependent Characterization of the Affinity
(pKi) of Compounds 7 and 14 Obtained from [3H]-CCR2-
RA-[R] Displacement Assays with U2OS-CCR2
d
Membranes
pKi,0h SEM (Ki,0h
,
pKi,4h SEM (Ki,4h
,
a
b
c
compound
nM)
nM)
Ki shift
7
14
8.4 0.08 (4.4)
8.7 0.10 (2.4)
8.2 0.02 (6.1)
9.2 0.15 (0.7)**
0.7 0.1
3.8 0.5
a
Affinity after 20 min coincubation of unlabeled ligands with [3H]-
b
CCR2-RA-[R] and no preincubation. Affinity after 4 h preincubation
with unlabeled ligands followed by 20 min coincubation with
c
radioligand. Ki shift represents the ratio of Ki,4h/Ki,0h calculated per
experiment. Data shown are the mean SEM of the individual Ki
d
ratios. Data are presented as mean SEM of at least three individual
experiments performed in duplicate. Differences in pKi,0h versus pKi,4h
values were analyzed using a paired, two-tailed, Student’s t-test, with
significance indicated as **p < 0.01.
which is apparent as a shift to the left in the concentration-
displacement curve (Figure 2b). This indicates that, over time,
more of the compound is covalently bound to the receptor,
and thus, less of the compound is needed to achieve similar
levels of displacement.
To assess the irreversibility of the interaction with the
receptor, a [3H]-CCR2-RA-[R] washout experiment was
employed. In this assay, U2OS-CCR2 cell membranes were
preincubated with 10×IC50 concentration of compounds 7 or
14 for 2 h followed by four extensive washing and
centrifugation cycles in order to remove the non-covalently
bound ligands. After the washing steps, [3H]-CCR2-RA-[R]
was added and the sample further incubated for 2 h before
measuring radioligand binding. Radioligand binding was
compared to vehicle control in the absence of compounds 7
or 14 (set as 100% binding). [3H]-CCR2-RA-[R] binding was
fully recovered in membranes preincubated with 7 (117 6%
binding), indicating that this compound was completely
washed away during the washing and centrifugation cycles
(Figure 2c). In contrast, less than 30% binding of [3H]-CCR2-
RA-[R] was recovered in membranes preincubated with
compound 14 (27
0.4%), indicating that, even after
Figure 2. Radioligand binding assays with preincubation and washing
suggest a covalent interaction. (a, b) Time-dependent characterization
of affinity of compounds 7 (a) and 14 (b). Concentration-dependent
inhibition of [3H]-CCR2-RA-[R] binding to U2OS-CCR2 cell
membrane preparations following a 20 min coincubation which was
preceded (4 h) or not (0 h) by preincubation with unlabeled ligands.
Preincubation shifts the apparent affinity of compound 14. pKi values
obtained from non-linear regression fits of these data are described in
Table 2. (c) [3H]-CCR2-RA-[R] binding to U2OS-CCR2 membranes
pretreated or not with iodoacetamide (IA) followed by preincubation
with 10×IC50 concentration of compounds 7 and 14 and repeated
washouts. Following the washouts, the inhibition of radioligand
binding by compound 14 persists in non-IA-treated membranes but is
recovered in membranes pretreated with IA, indicating the
extensive washing, a significant amount of this compound
remained bound to the receptor (Figure 2c). As the
thiocyanate warhead is expected to interact with cysteine
residues, we performed the same washout assay following
membrane pretreatment with iodoacetamide (IA), a highly
reactive alkylating agent that modifies cysteine sulfhydryl
groups and makes them unavailable for covalent interactions.
When U2OS-CCR2 membranes were pretreated with IA for
30 min in the dark before preincubation with compounds 7 or
14, [3H]-CCR2-RA-[R] binding was fully recovered for both
compounds: 93
5% for compound 14 and 104
5% for
compound 7 (Figure 2c). A comparable effect was observed by
pretreating the membranes with 1 mM N-ethylmaleimide,
another common cysteine modifier36 (Figure S1). This loss of
wash-resistant capacity of compound 14 in membranes
pretreated with IA confirmed that cysteine residues were
indeed responsible for the irreversible binding of 14 with
CCR2.
involvement of cysteine residues. Data represent the mean
SEM
of at least three independent experiments performed in duplicate.
Significant differences between indicated groups were determined
using an unpaired t-test with Welch’s correction: **p < 0.01, ***p <
0.001 or ns, not significant.
ment, the affinity of compound 7 was similar following a 4 h
preincubation, in agreement with its reversible mode of
interaction (Figure 2a and Table 2). In contrast, the apparent
affinity of compound 14 increased almost 4-fold after
preincubation, from a Ki of 2.4 nM to 0.7 nM (Table 2),
Compound 14 Is More Potent than Its Non-covalent
Analogue and Wash-Resistant in Functional Assays.
After demonstrating that the interaction of compound 14 with
CCR2 was wash-resistant, we investigated whether this
compound was able to inhibit the receptor in a functional
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Figure 3. Functional assays suggest a covalent interaction. (a) Inhibition of CCL2-stimulated [35S]GTPyS binding by increasing concentrations of
compounds 7 and 14. (b) After washout, compound 7 is not able to inhibit CCL2-induced [35S]GTPyS binding, while compound 14 remains
capable of completely inhibiting [35S]GTPyS binding. *p < 0.05 or ns, not significant, according to unpaired t- test with Welch’s correction. (c, d)
Inhibition of CCL2-stimulated β-arrestin recruitment by increasing concentrations of compounds 7 (c) and 14 (d) with or without two washing
steps before addition of CCL2. Data represent the mean SEM of at least three independent experiments performed in duplicate. pIC50 values
obtained from these graphs are described in Table 3.
d
Table 3. Functional Characterization of Compounds 7 and 14 from [35S]GTPγS and β-Arrestin Recruitment Assays
pIC50 SEM (IC50, nM)
a
b
c
compound
[35S]GTPγS binding
β-arrestin
β-arrestin_washed
7
14
7.1 0.05 (87)
7.5 0.07 (33)
7.8 0.15 (16)
8.4 0.12 (4)
7.0 0.09 (103)**
8.0 0.08 (10)*
a
b
Inhibition of [35S]GTPyS binding to U2OS-CCR2 cell membranes at 25 °C, after stimulation with 20 nM CCL2. Inhibition of β-arrestin
recruitment determined with a NanoBiT CCR2 assay, after stimulation with 200 nM CCL2. Inhibition of β-arrestin recruitment determined with a
NanoBiT CCR2 assay, after two washing steps followed by stimulation with 200 nM CCL2. Differences in pIC50 values between unwashed and
washed samples were analyzed using a paired, two-tailed, Student’s t-test, with significance shown as *p < 0.05 and **p < 0.01. Data are presented
as mean SEM of at least three individual experiments performed in duplicate.
c
d
assay. First compounds 7 and 14 were characterized in a
[35S]GTPγS binding assay on U2OS-CCR2 cell membranes. In
this assay, both compounds behaved as negative allosteric
modulators (NAMs) as they were able to inhibit, in a
concentration-dependent manner, [35S]GTPγS binding to the
membranes induced by a submaximal concentration of CCL2
(20 nM). The non-covalent control 7 inhibited [35S]GTPγS
recovered to 98 14%, which is comparable to levels in the
presence of CCL2 alone and significantly different from the
“unwashed” situation, (p < 0.05, Figure 3b). In contrast, the
inhibition of [35S]GTPγS binding to membranes preincubated
with compound 14 persisted and remained at the level of −24
30%, comparable to the “unwashed” situation (Figure 3b).
This indicates that, as for the competition binding experiments,
compound 7 was washed away and lost its effectiveness in this
functional assay, whereas the binding of compound 14 was
wash-resistant and led to persistent inhibition of the receptor
activity.
binding with a pIC50 of 7.1
0.05, while compound 14
showed a higher pIC50 of 7.5 0.07 (Figure 3a and Table 3).
Of note, both compounds decreased the basal activity of CCR2
at the highest concentrations (Figure 3a).
At a single saturating compound concentration (600 and
250 nM for compounds 7 and 14, respectively), [35S]GTPγS
binding to CCR2 was reduced to 34 2% by compound 7 and
Next, we explored the functional effects of irreversible
compound binding in live HEK293t cells transiently trans-
fected with hCCR2b. We used a variation of a previously
described NanoBiT luminescence complementation assay
measuring chemokine-induced β-arrestin recruitment to
CCR2,37 to which we added a washout step to distinguish
rapidly dissociating compounds from long residence time
fully inhibited to −5
7% by compound 14 (Figure 3b).
However, when the same measurement was performed after
four cycles of membrane washing and centrifugation,
[35S]GTPγS binding to membranes preincubated with 7 was
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Figure 4. Cysteine 75 seems to be involved in a covalent bond with compound 14. (a) Docking of compound 14 into the crystal structure of CCR2
(PDB 5T1A), showing the cysteine residues with potential to interact with this ligand: Cys75ICL1 within 4 Å; Cys70ICL1 at 6.4 Å; and Cys232ICL3 at
14 Å. (b) Proposed binding mode of compound 14, displaying the covalent interaction between the thiocyanate warhead and Cys75ICL1. (c)
Washout-radioligand experiments performed after preincubation of compound 14 (60 or 160 nM for triple mutant) or compound 7 (200 nM) in
membranes from CHO cells transiently transfected with CCR2 mutants. Data represent the mean SEM of at least three independent experiments
performed in duplicate. ****p < 0.0001, one-way ANOVA with Dunnett’s post hoc test.
binders. Increasing concentrations of compounds 7 and 14
were preincubated with the cells prior to two washing steps
and a 90 min incubation with compound-free assay buffer,
which was followed by cell stimulation with a fixed
concentration of CCL2 (200 nM) for another 10 min. In
parallel, a control experiment was performed where cells were
stimulated with CCL2 in the presence of compounds 7 or 14.
In the unwashed control situation, compounds 7 and 14
inhibited CCL2-induced β-arrestin recruitment with 5-fold and
8-fold higher potencies, respectively, than those measured in
the [35S]GTPγS binding assay (Table 3). After two washing
Structural Basis of the Covalent Binding of Com-
pound 14 to CCR2 Elucidated by Molecular Docking. As
suggested by the results of the washout experiment in the
presence of IA (Figure 2c), compound 14 is likely to form an
irreversible interaction with a cysteine residue in the
intracellular binding pocket of CCR2. In the crystal structure
of CCR2-RA-[R] bound to hCCR2b-T4L (PDB 5T1A20),
there are two cysteine residues in proximity of the ligand:
Cys75ICL1 within 4 Å and Cys70ICL1 at 6.4 Å. Additionally,
following the removal of T4L and re-building of the native
ICL3 of CCR2, we discovered that it contains Cys232ICL3 at 14
Å from the intracellular binding pocket. Although this distance
(14 Å) is too large to be spanned by the ethylacetamide linker,
the flexible nature of ICL3 may enable closer approach of
Cys232ICL3 to the pocket (Figure 4a). The binding pose of
compound 14 was predicted via covalent docking, assuming
that the closest of the three cysteines, Cys75ICL1, is the
covalent attachment point. The predicted pose was indeed (i)
consistent with that of non-covalent analogs from the same
series and (ii) compatible with the linker attachment to
Cys75ICL1 (Figure 4b).
steps, compound 7 displayed a pIC50 of 7.0
corresponding to a 6-fold reduction in potency compared to
the unwashed control (pIC50 of 7.8 0.15) (Table 3 and
0.09,
Figure 3c). For compound 14, we observed a smaller shift in
potency after the washing steps: from a pIC50 of 8.4 0.12 in
unwashed cells to a pIC50 of 8.0
0.08 after washing,
corresponding to only a 2.5-fold reduction in potency (Table 3
and Figure 3d). In other words, even in the context of a live-
cell assay where covalent compound binding to CCR2 is
affected by the reducing intracellular environment, compound
14 was more resistant to washing than compound 7, consistent
with its covalent mechanism of action.
Covalent Binding of 14 to CCR2 Is Mediated by a
Unique Switching Mechanism between Cysteine Resi-
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Table 4. Binding Affinities (pIC50) of 7 and 14 to Membranes of CHO Cells Expressing the Different CCR2 Constructs,
a
Obtained from [3H]CCR2-RA-[R] Displacement Assays
pIC50 SEM (IC50, nM)
construct
7
14
CCR2-RA-[R]
WT
C70S
C75S
C232S
C70S/C75S/C232S
7.9 0.02 (12)
7.7 0.09 (22)*
8.2 0.05 (6)**
7.9 0.005 (12)
8.3 0.02 (5)**
8.8 0.10 (2)
7.9 0.06 (12)
7.9 0.02 (13)
8.0 0.05 (11)
8.0 0.09 (9)
8.1 0.02 (8)
8.4 0.04 (4)*
8.4 0.10 (5)*
8.8 0.06 (1)
7.8 0.02 (16)****
a
Data are presented as mean SEM of at least three individual experiments performed in duplicate. Differences in pIC50 values compared to WT
were analyzed using a one-way ANOVA with Dunnett’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
dues. To assess which of the three cysteines suggested by
docking was responsible for the covalent binding of compound
14 (Figure 4a,b), we designed binding experiments using
CCR2 variants in which single-point mutations of cysteine to
serine were made: C70ICL1S, C75ICL1S, and C232ICL3S. These
CCR2 mutants, together with CCR2 wild-type (WT) as a
control, were transiently transfected into CHO cells, and cell
membranes were prepared. We first determined if the
mutations affected the binding of the radiolabeled probe,
[3H]-CCR2-RA-[R], to CCR2 using homologous displace-
ment assays. As shown in Table 4, the affinity of CCR2-RA-[R]
for the single CCR2 mutants was similar to WT CCR2 (pIC50
results suggest that other residues might also be involved in the
formation of a covalent bond with compound 14 or become
involved when Cys75ICL1 is not available. Hence, we mutated
all three cysteine residues to serine, yielding the triple C70S/
C75S/C232S mutant. The [3H]-CCR2-RA-[R] binding assay
showed that the affinity of compound 14 was significantly
decreased in this triple mutant (Table 4) from a pIC50 of 8.8
for the WT receptor to a pIC50 of 7.8 for the mutant. In
contrast, the affinity of compound 7 was increased to a similar
level as for the C75S mutant (Table 4). Lastly, washout
experiments with compound 14 revealed that binding of [3H]-
CCR2-RA-[R] was recovered to levels comparable to
compound 7 for this mutant (Figure 4c). This recovery
indicates that the irreversible interaction between compound
14 and CCR2 was lost when the three cysteine residues were
mutated to serine. Overall, these data suggest that compound
14 interacts primarily with Cys75ICL1 but can switch to
Cys70ICL1 or Cys232ICL3 as secondary residues when Cys75ICL1
is not available.
Compound 14 Is Selective toward CCR2 vs Closely
Homologous Chemokine Receptors CCR1 and CCR5.
Some CCR2 intracellular antagonists also bind to the highly
homologous CCR148 and CCR5.23,52 Therefore, we inves-
tigated whether compounds 7 and 14 are also able to inhibit
CCL3-induced [35S]GTPγS binding in U2OS-CCR1 and
U2OS-CCR5 cell membranes. Compared to CCR2, both
compounds displayed a much lower inhibitory potency on
CCR1 and CCR5, with pIC50 values of 5.3 and 5.4 (Figure S3
and Table S1). This is remarkable since CCR5 has two out of
three candidate cysteines in the intracellular binding site
(Figure S3A): only Cys75ICL1 of CCR2 is substituted by a
serine in CCR5. This again underlines the role of CCR2
Cys75ICL1 as a covalent attachment point for compound 14.
On the contrary, in CCR1, none of the three intracellular
pocket cysteines are conserved, which may also partially
explain the lack of activity of compound 14 toward this
receptor.
of 7.9
0.06), confirming the integrity of the intracellular
pocket in all mutants. Next, we used [3H]-CCR2-RA-[R]
displacement to characterize mutant receptor binding to
compounds 7 and 14. The affinity of compound 14 for both
the C70S and C75S CCR2 mutants was significantly decreased
compared to WT CCR2 (pIC50 of 8.8 0.10, Table 4). In the
case of compound 7, its affinity was significantly decreased for
the C70S mutant and increased for the C75S mutant in
comparison to the WT CCR2 (pIC50 of 7.9
0.02). The
affinities of both compounds 7 and 14 were not affected by the
C232S mutation, indicating that this cysteine might not be
involved in the binding of these compounds to CCR2 (Table
4).
Next, a [3H]-CCR2-RA-[R] washout assay was performed
using membrane preparations from WT and mutant CCR2
expressing CHO cells (Figure 4c). Similar to the washout
assays in U2OS-CCR2 membranes, compound 7 was
completely washed away from the WT receptor, leading to
full recovery of [3H]-CCR2-RA-[R] binding (∼100%), while
compound 14 only led to 9% binding of [3H]-CCR2-RA-[R],
in agreement with its irreversible nature (Figure 4c). For
compound 7, full recovery of radioligand binding was observed
in the three mutants. In the case of compound 14, mutants
C70S and C232S showed similar [3H]-CCR2-RA-[R] binding
levels as for the WT receptor, indicating that compound 14
still binds covalently despite these mutations. However, [3H]-
CCR2-RA-[R] binding was significantly increased to 30% in
the C75S mutant (p < 0.0001), indicating a partial loss of
covalent binding in this mutant. These data suggest that
Cys75ICL1 is partially responsible for the irreversible nature of
compound 14. However, the recovery of [3H]-CCR2-RA-[R]
binding was not complete, i.e., to similar levels as compound 7.
To rule out the possibility that the serine group introduced at
position 75 covalently interacts with the thiocyanate warhead
through its nucleophilic hydroxyl group, we mutated Cys75ICL1
to alanine; however, alanine mutation of this residue (C75A)
did not improve the recovery of [3H]-CCR2-RA-[R] binding
in comparison with the C75S mutant (Figure S2). These
DISCUSSION AND CONCLUSIONS
■
Many studies have suggested a critical role of CCR2 in the
pathology of inflammatory and immune diseases;3 yet, no
CCR2 antagonists have been approved for clinical use so far.
Thus, novel approaches are needed to better study and target
this receptor. Covalent ligands represent useful tools to study
the structure and function of GPCRs;9,12 furthermore, due to
their “infinite residence time”, covalent inhibitors may lead to
enhanced in vivo efficacy as a result of their extended duration
of action and insurmountability.13−15 Several orthosteric and
allosteric antagonists have been previously described for
CCR2,38 but no covalent binders have been described so far.
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Some of the existing CCR2 antagonists bind to an allosteric
binding site located in the intracellular region of the receptor,
from where they inhibit the receptor in a noncompetitive and
insurmountable manner.20,23,35 In an attempt to combine the
advantages provided by covalent inhibition and those of
intracellular ligands, we designed, synthesized, and charac-
terized a novel covalent intracellular ligand for CCR2 based on
a previously described sulfonamide scaffold.
While searching for the target amino acid of the thiocyanate
warhead, we unexpectedly discovered that compound 14 is
able to interact with more than one cysteine residue in the
intracellular pocket of CCR2. First, the radioligand washout
assays in the presence of IA narrowed the potential list of
candidates to cysteine residues (as opposed to, e.g., serines or
lysines). Docking of compound 14 into an in silico model of
CCR2 suggested three proximal candidate cysteines: Cy-
s70ICL1, Cys75ICL1 (the closest and most likely to interact), and
Cys232ICL3. The irreversibility of compound 14 was indeed
affected when Cys75ICL1 was mutated to serine; however, a
complete loss of covalent binding required elimination of all
three cysteines. This suggests that the flexibility of the
compound linker and the receptor loops allows compound
14 to adopt multiple binding poses and interact with
alternative nucleophilic residues when Cys75ICL1 is removed.
A similar phenomenon has been described for other GPCRs,
such as adenosine A2A receptor,44 cannabinoid CB2 receptor,49
and muscarinic receptors,50 in which more than one residue
was found to mediate the covalent binding of the chemical
probes. Furthermore, a recent study suggested that CCR2
antagonists can induce several inactive conformations of the
receptor, further supporting receptor flexibility.51
With intracellularly acting antagonists for chemokine
receptors, receptor selectivity can be a challenge due to the
high degree of residue conservation at their binding sites.21
Many existing intracellularly acting antagonists of CCR2 also
inhibit related chemokine receptors such as CCR148 and
CCR5.23,52 Compound 14 performed very favorably in this
respect: we demonstrated that it is highly selective toward
CCR2 compared to CCR1 and CCR5 in a [35S]GTPγS
binding assay. This is likely because the action of compound
14 is mediated by Cys75ICL1 that is unique to CCR2 among all
chemokine receptors: most of them possess a serine residue in
this position. Despite the ability of compound 14 to interact
with secondary sites when Cys75ICL1 is not present, the
compound was specific for CCR2, further supporting
Cys75ICL1 as the main site for covalent interaction. Thus,
targeting of Cys75ICL1 may lead to improved selectivity of
intracellularly acting CCR2 antagonists.
Previous research in our group identified sulfonamide
derivatives, similar to compound 7 in the present study, as
intracellular antagonists for CCR2.22,23 Based on the predicted
proximity of these compounds to cysteine residues in the
intracellular region of CCR2, we synthesized sulfonamide
derivatives containing four electrophilic warheads with differ-
ent reactivity profiles: isothiocyanate, acryloyl, fluorosulfonyl,
and thiocyanate. Compound 14 with a thiocyanate war-
head12,29 was prioritized for further characterization based on
the results of the equilibrium binding assays. However,
equilibrium parameters (Ki or IC50) are poor indicators of
the “true” binding affinity or potency of covalent ligands due to
their two-step mechanism of inhibition, which results in a time-
dependent shift in affinity.39 Therefore, we further charac-
terized compound 14 in binding assays with a preincubation
step. Preincubation led to an increase in the apparent affinity of
compound 14 to CCR2, consistent with an increase in
receptor occupancy, and thus covalent binding, over time.
However, a shift in affinity over time can also be obtained with
slowly dissociating, long residence time ligands, as observed
with long residence time antagonists for CCR5 and
cannabinoid CB1 receptors.40,41 Thus, we next confirmed the
irreversible nature of the interaction of compound 14 with
CCR2 using “washout” radioligand binding assays, which rely
on extensive washing to ensure the removal of unbound, non-
covalent ligands. Such assays have been previously used to
validate the irreversible nature of covalent ligands for other
GPCRs.42−46 The binding of compound 14 was resistant to
washing and in fact was not completely abolished even with
repeated extensive washes, consistent with a covalent
mechanism.
Two functional assays, one involving GTPγS binding to
receptor-expressing membranes and another involving β-
arrestin recruitment to the receptor in live cells, provided
further evidence of the covalent nature of compound 14. In the
former assay, repeated membrane washes completely abro-
gated the ability of the non-covalent analogue compound 7 to
inhibit CCL2-induced GTPγS binding to CCR2-expressing
membranes; however, the inhibition by compound 14 was
unaffected. In the latter assay, the inhibitory potency of
compound 14 toward CCL2-induced β-arrestin recruitment to
CCR2 was reduced by washing to a significantly lesser extent
than that of compound 7. However, and in contrast to the
GTPγS binding assay, compound 14 was not fully resistant to
washing in the β-arrestin assay. This may be because the
shorter compound preincubation in the β-arrestin recruitment
assay (20 min versus 2 h in the [35S]GTPγS assays) was not
sufficient to allow compound 14 to fully bind CCR2 in an
irreversible manner. Another reason could be the highly
reducing nature of the intracellular environment in live cells,
which might limit the formation of the disulfide bond between
the cysteine residue and the compound warhead.47 Interest-
ingly, in the [35S]GTPγS binding assay, both compounds 7 and
14 displayed inverse agonism: a behavior also observed with
other CCR2 intracellular ligands.48
To conclude, we report the design, synthesis, and
pharmacological characterization of compound 14, the first
intracellular covalent NAM for CCR2. The results of binding
and functional assays support the idea of an irreversible
interaction between compound 14 and CCR2, which leads to a
long-lasting inhibitory effect. In silico structure-based docking
and receptor mutagenesis studies suggest Cys75ICL1 in the
intracellular binding site of CCR2 as the primary site of
covalent binding for compound 14, although secondary
interaction sites were also identified. Overall, compound 14
may represent a useful tool for further studies of CCR2
structure and function in a variety of in vitro and in vivo
systems, which might ultimately assist the translation of
preclinical findings into the clinic.
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All solvents and reagents were
purchased from commercial sources and were used without further
1
purification. H spectra were recorded on a Bruker AV 400 MHz
liquid spectrometer at room temperature (rt) using CDCl3, MeOD, or
DMSO as a solvent. Chemical shifts are reported in ppm relative to
internal standard tetramethylsilane (TMS) or solvent resonance.
Purity of the compounds was determined by HPLC with a C18
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column (50 × 4.6 mm, 3 μm), flow rate = 1.3 mL/min, using a
gradient of 10−90% MeCN/H2O (0.1% TFA) and measuring UV
absorbance at 254 nm. Purity of all final compounds used in biological
assays was at least 95%. HPLC traces of all final compounds 7 and
11−14 are shown in Figures S4 and S5. Reactions were monitored by
TLC using Merck TLC Silica gel 60 F254 aluminum sheets.
Compounds were visualized by UV irradiation or by staining with a
KMnO4 solution in H2O. A Biotage Initiator microwave synthesizer
was used for the reactions performed in a microwave reactor. For the
flash chromatography, Davisil silica gel (40−63 μm) was used. The
automatic flash chromatography was performed on an Isolera One
Automatic Flash Chromatography System by Biotage with pre-packed
flash cartridges (ISCO RediSep or Biotage ZIP Sphere). Mass spectra
were measured using a Shimadzu Prominence LCMS-2020 system
and a Gemini C18 Phenomenex column (50 × 3 mm, 3 μm).
Experimental Procedures. Methyl 4-(4-Chloro-2-nitrophenoxy)-
benzoate (1). Commercially available methyl 4-hydroxybenzoate (1
eq) and 5-chloro-2-fluoronitrobenzene (1 eq) were dissolved in
dimethylformamide (DMF, 1 mL/mmol) together with potassium
carbonate (2 eq) in a round-bottom flask. The mixture was stirred at
70 °C under nitrogen atmosphere for 3.5 h. After the solvent was
evaporated, the residue was dissolved in EtOAc and washed with
water. The organic layer was dried over MgSO4 and concentrated in
evaporated, after which precipitation with DCM and PE gave 6. Yield:
1.15 g, 63%. 1H NMR (400 MHz, CDCl3) δ 8.03 ( d, J = 2.1 Hz, 1H),
7.93 (d, J = 8.8 Hz, 2H), 7.79 (dd. J = 8.4, 2.1 Hz, 1H), 7.73 (d, J =
2.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H),
7.00 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 6.65−6.60 (m, 2H), 3.92 (s,
3H).
4-(4-Chloro-2-((4-chloro-3-(trifluoromethyl)phenyl)-
sulfonamido)phenoxy)benzoic Acid (8). A mixture of 2 M NaOH (6
mL) and dioxane (30 mL) was added to intermediate 6, and the
reaction mixture was stirred for 2.5 h at 60 °C. Upon completion, the
reaction was diluted with H2O and acidified with aqueous HCl. The
product was then extracted with EtOAc (2 × 150 mL), dried over
MgSO4, and evaporated to yield 8. Yield: 1.64 g, 85%. 1H NMR (400
MHz, CDCl3) δ 8.03 (d, J = 1.8 Hz, 1H), 7.99 (d, J = 8.6 Hz, 2H),
7.80 (dd, J = 8.3, 2.0 Hz, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.52 (d, J =
8.4 Hz, 1H), 7.13 (dd, J = 8.8, 2.5 Hz, 1H), 6.98 (s, 1H), 6.80 (d, J =
8.7 Hz, 1H), 6.68−6.63 (m, 2H).
N-(2-Aminoethyl)-4-(4-chloro-2-((4-chloro-3-(trifluoromethyl)-
phenyl)sulfonamido)phenoxy)benzamide Hydrochloride (9). A
mixture of intermediate carboxylic acid 8 (1 eq), N-Boc-ethylenedi-
amine (1 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC,
2 eq), and hydroxybenzotriazole (HOBt, 0.1 eq) was dissolved in
dioxane (0.015 M) and stirred at rt for at least 20 h until completion.
The reaction mixture was diluted with EtOAc, and the organic phase
was washed with water and brine, dried over MgSO4, and
concentrated. The crude product was purified on a silica gel column
chromatography with DCM/MeOH to give the Boc-protected
intermediate compound tert-butyl(2-(4-(4-chloro-2-((4-chloro-3-
(trifluoromethyl)phenyl)sulfonamido)phenoxy)benzamido)ethyl)-
carbamate (581 mg, 61%). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J
= 1.5 Hz, 1H), 7.83 (dd, J = 8.4, 1.8 Hz, 1H), 7.75−7.68 (m, 3H),
7.54 (d, J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.33 (s, 1H), 7.08 (dd, J = 8.8,
2.5 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 6.64 (d, J = 8.7 Hz, 2H), 5.07
(s, 1H), 3.56−3.53 (m, 2H), 3.40 (d, J = 4.6 Hz, 2H), 1.43 (s, 9H).
The intermediate was dissolved in DCM (0.1 M), and trifluoro-
acetic acid (TFA, 4 mL/mmol) was added dropwise. The mixture was
stirred at rt for 3 h. After completion, the solvents were evaporated
and the residue was dissolved in H2O and alkalized with 2 M NaOH.
The aqueous phase was then extracted with EtOAc and washed
successively with water and brine. After drying over MgSO4 and
concentrating, the residue was taken up in EtOAc and 1 M HCl (1
1
vacuum to afford 1. Yield: 13.9 g, 45.1 mmol, 90%. H NMR (400
MHz, DMSO) δ 8.29 (d, J = 2.6 Hz, 1H), 8.03−7.96 (m, 2H), 7.86
(dd, J = 8.9, 2.6 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.20−7.13 (m,
2H), 3.84 (s, 3H).
Methyl 4-(2-Amino-4-chlorophenoxy)benzoate (2). Intermediate
1 was dissolved in EtOAc (0.05 M) before addition of SnCl2·2H2O (5
eq). The reaction was stirred overnight at rt under a nitrogen
atmosphere. Upon completion, the reaction was quenched with 1 M
NaOH and extracted with EtOAc. The organic layer was then washed
with H2O and dried over MgSO4 to yield 2. Yield: 4.13 g, 14.9 mmol,
75%. 1H NMR (400 MHz, DMSO) δ 7.98−7.88 (m, 2H), 7.01−6.93
(m, 2H), 6.89 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.57 (dd,
J = 8.5, 2.6 Hz, 1H), 5.36 (s, 2H).
Methyl 4-(4-Chloro-2-((3,4-dichlorophenyl)sulfonamido)-
phenoxy)benzoate (5). Intermediate 2 was dissolved in CH3CN
before addition of indium (0.1 eq) and commercially available 3,4-
dichloro-benzenesulfonylchloride 3 (1 eq). The mixture was stirred
overnight at rt after purging the system with nitrogen. The solvents
were then evaporated, and the residue extracted with EtOAc and
washed successively with H2O and brine. The organic layer was dried
over MgSO4, filtered, and evaporated, after which the product was
purified by flash column chromatography (CHCl3/petroleum ether
1
mL/mmol) was added. Evaporation gave 9. Yield: 358 mg, 92%. H
NMR (400 MHz, MeOD) δ 8.66 (t, 1H), 8.03 (d, J = 2.1 Hz, 1H),
7.84 (dd, J = 8.3, 2.1 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.63 (d, J =
2.5 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.8, 2.6 Hz, 1H),
6.82 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 3.64 (q, J = 5.9 Hz,
2H), 3.14 (t, J = 5.9 Hz, 2H).
1
3:1) in order to obtain pure 5. Yield: 3.61 g, 7.41 mmol, 50%. H
NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H),
7.76 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 11.1,
2.2 Hz, 2H), 7.33 (dd, J = 8.8, 2.5 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H),
6.64 (d, J = 8.8 Hz, 2H), 3.83 (s, 3H).
4-(4-Chloro-2-((4-chloro-3-(trifluoromethyl)phenyl)-
sulfonamido)phenoxy)-N-(2-isothiocyanatoethyl)benzamide (11).
Intermediate 9 (1 eq) was dissolved in THF (0.02 M), and
triethylamine (6 eq) was added. The mixture was cooled to 0 °C
before adding carbon disulfide (4 eq), and the reaction mixture was
stirred for approximately 2 h at 0 °C until complete conversion to an
intermediate. Tosyl chloride (3.5 eq) was added, and the mixture was
stirred for an additional 19 h. The reaction was quenched with a
phosphate buffer, and the products were extracted into DCM. The
organic phase was washed with brine dried over MgSO4 and
concentrated. The crude compound was purified with flash column
chromatography using DCM/MeOH as eluents to yield final
4-(4-Chloro-2-((3,4-dichlorophenyl)sulfonamido)phenoxy)-
benzoic Acid (7). A mixture of 2 M NaOH (10 mL) and dioxane (10
mL) was added to intermediate 5, and the reaction mixture was
stirred at rt for 2 h. Solvents were evaporated, and the residue was
extracted with DCM. The organic layer was extracted with H2O. The
aqueous layers were pooled and acidified with 6 M HCl. Filtration of
1
the precipitate gave final compound 7. Yield: 37 mg, 8%. H NMR
(400 MHz, DMSO) δ 12.81 (s, 1H), 10.60 (s, 1H), 7.81 (s, 1H),
7.81−7.75 (m, 2H), 7.63 (d, J = 8.4 Hz, 1H), 7.57 (dd, J = 8.4, 2.0
Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.33 (dd, J = 8.4, 2.4 Hz, 1H), 7.04
(d, J = 9.6 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H); MS: ESI [M − H]−:
469.8; HPLC: 10.2 min.
Methyl 4-(4-Chloro-2-((4-chloro-3-(trifluoromethyl)phenyl)-
sulfonamido)phenoxy)benzoate (6). Intermediate 2 (1 eq) and
commercially available 4-chloro-3-(trifluoromethyl)benzenesulfonyl
chloride 4 (1.1 eq) were dissolved in pyridine (0.4 M) and N,N-
dimethylaminopyridine (0.1 eq) was added. The mixture was stirred
at 95 °C for 3 h under microwave irradiation. The mixture was
extracted with DCM, and the organic layer was washed with aqueous
HCl, water, and brine. The organic phase was dried over MgSO4 and
1
compound 11. Yield: 6 mg, 6%. H NMR (400 MHz, CDCl3) δ
8.07 (d, J = 2.1 Hz, 1H), 7.83 (dd, J = 8.4, 2.2 Hz, 1H), 7.72 (d, J =
2.5 Hz, 1H), 7.71−7.67 (m, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.32 (s,
1H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 6.69−
6.62 (m, 2H), 6.54 (t, J = 5.9 Hz, 1H), 3.83−3.76 (m, 2H), 3.76−
3.69 (m, 2H); MS: ESI [M − H]−: 587.9; HPLC: 13.0 min.
N-(2-Acrylamidoethyl)-4-(4-chloro-2-((4-chloro-3-
(trifluoromethyl)phenyl)sulfonamido)phenoxy)benzamide (12). In-
termediate 9 (1 eq) was dissolved in acetonitrile (5 mL) and cooled
to −78 °C in an acetone/dry ice bath. Triethylamine (3.5 eq) and
acryloyl chloride (1.1 eq) were added. The reaction mixture was then
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allowed to warm to room temperature and stirred for 2 h. The
mixture was poured into water and acidified with aqueous HCl, and
the aqueous layer was extracted with ethyl acetate twice. The organic
layers were washed with water, dried over MgSO4, and concentrated.
Purification by preparative TLC with EtOAc/acetone 100:1 as an
eluent gave the final compound 12. Yield: 10 mg, 20%. 1H NMR (400
MHz, CDCl3) δ 8.09 (d, J = 2.1 Hz, 1H), 7.82 (dd, J = 8.4, 2.2 Hz,
1H), 7.71 (d, J = 2.1 Hz, 1H), 7.70−7.68 (m, 2H), 7.53 (d, J = 8.4
Hz, 1H), 7.44 (s, 1H), 7.43 (s, 1H), 7.09 (dd, J = 8.8, 2.5 Hz, 1H),
6.73 (d, J = 8.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 2H), 6.51 (s, 1H), 6.30
(dd, J = 17.0, 1.3 Hz, 1H), 6.14 (dd, J = 17.0, 10.2 Hz, 1H), 5.69 (dd,
J = 10.2, 1.3 Hz, 1H), 3.60 (s, 4H); MS: ESI [M − H]−: 600.0;
HPLC: 11.7 min.
4-((2-(4-(4-Chloro-2-((4-chloro-3-(trifluoromethyl)phenyl)-
sulfonamido)phenoxy)benzamido)ethyl) carbamoyl)-
benzenesulfonyl Fluoride (13). A mixture of intermediate 9 (1 eq),
commercially available 4-fluorosulfonylbenzoic acid (1 eq), EDC (2
eq), and HOBt (0.1 eq) was dissolved in dioxane (0.015 M) and
stirred at rt for at least 20 h until completion. The reaction mixture
was diluted with EtOAc, and the organic phase was washed with water
and brine, dried over MgSO4, and concentrated. The crude product
was purified on a silica gel column chromatography with DCM/
MeOH to give 13. Yield: 8 mg, 31%. 1H NMR (400 MHz, CDCl3) δ
8.07 (s, 4H), 8.06 (d, J = 2.1 Hz, 1H), 7.89 (t, J = 4.4 Hz, 1H), 7.82
(dd, J = 8.4, 2.1 Hz, 1H), 7.71 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 8.8 Hz,
2H), 7.52 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.10 (dd, J = 8.8, 2.5 Hz,
1H), 6.93 (t, J = 5.2 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.65 (d, J = 8.8
Hz, 2H), 3.81−3.69 (m, 4H); MS: ESI [M − H]−: 732.0; HPLC:
12.8 min.
Hans den Dulk (Leiden University, the Netherlands) and originally
obtained from ATCC. All other chemicals were from standard
commercial sources.
Plasmids Used in the Study. pCDNA3.1+ plasmids contacting the
FLAG-tagged wild-type human CCR2, the human CCR2 mutants
C70SICL1, C75SICL1, and C232SICL3, and the triple mutant C70S/
C75S/C232S were cloned in-house.
The NanoBiT luminescence complementation system has been
previously described.55,56 Here, we used the pCAGGS LgBiT-β-
arrestin1-EE construct containing the NanoBiT LgBiT fused to the N-
terminus of clathrin-binding-deficient variant57 of human β-arrestin1,
first described by Shihoya et al..58 We also used the pCAGGS
hCCR2b-SmBiT construct in which the SmBiT was fused to the C-
terminus of human CCR2b37 with a flexible 15 AA linker
(GGSGGGGSGGSSSGG). The plasmids were propagated in E. coli
using ampicillin (100 μg/mL) as a bacterial selection marker.
Cell Culture. U2OS-CCR2 and CHO cells were grown as a
monolayer in 10 cm ø or 15 cm ø culture plates at 37 °C and 5% CO2.
U2OS-CCR2 cells were cultured in McCoy’s 5A medium containing
10% fetal calf serum (FCS), 2 mM glutamine, 0.1 mM non-essential
amino acids, 25 mM HEPES, 1 mM sodium pyruvate, 200 IU/mL
penicillin, 200 μg/mL streptomycin, 100 μg/mL G418, 40−50 μg/
mL hygromycin, and 125 μg/mL zeocin. Cells were subcultured twice
a week after trypsinization at a ratio of 1:3 to 1:8. Dialyzed fetal calf
serum was used before membrane preparation of U2OS-CCR2 cells.
Empty CHO cells were cultured in DMEM/F12 medium
supplemented with 10% (v/v) newborn calf serum, 200 IU/mL
penicillin, 200 μg/mL streptomycin, and 2 mM glutamine and
subcultured twice a week after trypsinization at a ratio of 1:30 to 1:50.
HEK293t cells for NanoBiT β-arrestin recruitment experiments were
cultured in 75 mL tissue culture treated flasks in DMEM
supplemented with 10% FCS and subcultured three times a week at
a ratio of 1:10 after trypsinization.
N-(2-Bromoethyl)-4-(4-chloro-2-((4-chloro-3-(trifluoromethyl)-
phenyl)sulfonamido)phenoxy)benzamide (10). A mixture of
carboxylic acid 8 (1 eq), 2-bromoethylamine (1 eq), EDC (2 eq),
and HOBt (0.1 eq) was dissolved in dioxane (0.015 M) and stirred at
rt for at least 20 h until completion. The reaction mixture was diluted
with EtOAc, and the organic phase was washed with water and brine,
dried over MgSO4, and concentrated. The crude product was purified
on a silica gel column chromatography with EtOAc/petroleum ether
Transfections. Transfections of CHO cells with FLAG-tagged WT
or mutant CCR2 were performed using a previously described PEI
method.23 Briefly, empty CHO cells were grown to ∼50% confluence
in 15 cm ø culture plates and transfected with a DNA/PEI mixture in
150 mM NaCl solution containing 10 μg of plasmid mixed with PEI
(1 mg/mL) at a mass ratio of 1:6. Before transfection, the DNA/PEI
mixture was incubated for 20 min and the culture medium of the cells
was refreshed. After 24 h, sodium butyrate (final concentration of 3
mM) was added to the plates to increase receptor expression.59
Finally, cells were incubated for another 24 h at 37 °C and 5% CO2.
For NanoBiT β-arrestin recruitment experiments, HEK293t cells were
transiently transfected with CCR2b-SmBiT and LgBiT-β-arrestin1-EE
plasmids using TransIT-LT1 transfection reagent (Mirus Bio,
Madison, WI, USA). Empty cells were grown in 6 cm ø culture
plates to 60−80% confluence. Prior to transfection, the culture
medium on the cells was replaced with fresh DMEM +10% FBS.
DNA/reagent mixture was prepared with 3 μg of CCR2b-SmBiT
DNA and 3 μg of LgBiT-β-arrestin1-EE DNA mixed with 18 μL of
TransIT-LT1 reagent in 600 μL of serum-free OptiMEM medium,
according to the manufacturer’s instructions. The DNA/reagent
mixture was incubated for 30 min at room temperature and added
dropwise to the cells. The transfected cells were incubated for another
24 h at 37 °C and 5% CO2 before performing the NanoBiT assays.
Membrane Preparation. Membranes from U2OS-CCR2 or CHO
cells transiently transfected with wild-type (WT) human CCR2 or
human CCR2 mutants were prepared as previously described, using
several centrifugation and homogenization steps.35 Final membrane
pellets were resuspended in ice-cold Tris buffer (50 mM Tris-HCl pH
7.4, 5 mM MgCl2), homogenized with an Ultra Turrax homogenizer
(IKA-Werke GmbH & Co. KG, Staufen, Germany), and stored in
aliquots of 100 or 250 μL at −80 °C. A standard BCA protein
determination assay was used to measure the membrane protein
concentrations (Pierce BCA protein assay kit).60
1
to yield 10. Yield: 62 mg, 51%. H NMR (400 MHz, CDCl3) δ 8.06
(s, 1H), 7.85−7.77 (m, 3H), 7.73−7.69 (m, 1H), 7.51 (d, J = 8.4 Hz,
1H), 7.09 (dd, J = 8.7, 2.5 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 6.60 (d,
J = 8.8 Hz, 2H), 4.44 (t, J = 9.5 Hz, 2H), 4.05 (t, J = 9.5 Hz, 2H).
4-(4-Chloro-2-((4-chloro-3-(trifluoromethyl)phenyl)-
sulfonamido)phenoxy)-N-(2-thiocyanatoethyl)benzamide (14). A
mixture of KSCN (6 eq) and the intermediate (1 eq) was dissolved in
EtOH (0.025 M), and the mixture was refluxed for 3 days. After
completion of the reaction, the solvent was evaporated and the
residue was dissolved in EtOAc, washed with water and brine, dried
over MgSO4, and concentrated. Preparative TLC with DCM/MeOH
1
25:1 as an eluent gave the final compound 14. Yield: 4 mg, 21%. H
NMR (400 MHz, CDCl3) δ 8.05 (d, J = 2.1 Hz, 1H), 7.82 (dd, J =
8.4, 2.1 Hz, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.73−7.68 (m, 2H), 7.54
(d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 7.11 (dd, J = 8.7, 2.5 Hz, 2H), 6.75
(d, J = 8.8 Hz, 1H), 6.68−6.63 (m, 2H), 6.62 (t, J = 5.8 Hz, 1H), 3.88
(q, J = 6.0 Hz, 2H), 3.26 (t, J = 6.0 Hz, 2H); MS: ESI [M − H]−:
587.9; HPLC: 12.5 min.
Biology. Chemicals and Reagents. [3H]-CCR2-RA-[R] (specific
activity 59.6 Ci mmol−1) was custom-labeled by Vitrax (Placentia,
CA), and [35S]GTPγS (specific activity 1250 Ci mmol−1) was
purchased from PerkinElmer (Groningen, The Netherlands). The
CCR2 ligands CCR2-RA-[R] and JNJ-27141491 were synthesized as
previously described.53,54 Human CCL2 was purchased from
PeproTech (Rocky Hill, NJ). Bovine serum albumin (BSA, fraction
V), guanosine 5′-diphosphate (GDP), and iodoacetamide (IA) were
from Sigma Aldrich (St. Louis, MO, USA). Pierce bicinchoninic acid
(BCA) protein assay kit and coelenterazine (CTZ-n) were obtained
from Thermo Fisher Scientific (Rockford, IL). Polyethyleneimine
(PEI) was purchased from Polysciences Inc. (Warrington, Pennsylva-
nia). Tango CCR2-bla osteosarcoma cells stably expressing human
CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad,
CA). Chinese hamster ovary (CHO) cells were kindly provided by
Radioligand Binding Assays. [3H]-CCR2-RA and [3H]-CCR2-RA-
[R] Binding Assays. For all radioligand binding assays, membranes
from U2OS-CCR2 cells or CHO cells transiently transfected with WT
or mutant CCR2 were first thawed and homogenized using an Ultra
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Turrax homogenizer (IKA-Werke GmbH & Co.KG, Staufen,
Germany). Membranes were then diluted in assay buffer (50 mM
Tris-HCl pH 7.4, 5 mM MgCl2, 0.1% CHAPS) to a final
concentration of 5−30 μg membrane protein in a total volume of
100 μL. For displacement assays, membranes were coincubated for 2
h at 25 °C with multiple concentrations of competing ligand, ranging
from 0.01 nM to 10 μM, and a fixed concentration of radioligand (∼6
nM [3H]-CCR2-RA or [3H]-CCR2-RA-[R]). At this concentration,
we ensured that the total radioligand binding did not exceed 10% of
the total radioactivity added in order to prevent radioligand depletion.
In all cases, nonspecific binding was determined using 10 μM JNJ-
27141491. In the case of preincubation experiments, membranes were
preincubated for 4 h with increasing concentrations of competing
ligand before addition of ∼6 nM [3H]-CCR2-RA-[R] and further
coincubation of 20 min. In all assays, incubations were terminated by
rapid vacuum filtration through pre-wetted GF/B filter plates using a
PerkinElmer FilterMate harvester (PerkinElmer, Groningen, The
Netherlands). Filters were subsequently washed 10 times with ice-
cold wash buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 0.01%
CHAPS) and dried at 55 °C for at least 30 min. Finally, filter-bound
radioactivity was measured in a P-E 2450 Microbeta2 counter
(PerkinElmer) after addition of 25 μL of Microscint scintillation
cocktail (PerkinElmer).
minutes at 25 °C before stopping the reaction with ice-cold wash
buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2). Filtration and
radioactivity measurement were performed as described under the
Binding Assays section.
Washout Assays with [35S]GTPγS. Washout assays with
[35S]GTPγS were performed as described under the Washout Assays
section. Briefly, U2OS-CCR2 membrane homogenates (40 μg) were
preincubated in the absence or presence of a single concentration of 7
or 14 (600 or 250 nM, respectively) in a final volume of 400 μL of
buffer containing 50 mM Tris-HCl (pH 7.4) and 5 mM MgCl2. After
four cycles of centrifugation and washing, the remaining pellets were
transferred to test tubes in a final volume of 320 μL containing
[35S]GTPγS assay buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2,
100 mM NaCl, 1 mM EDTA, and 0.05% BSA), saponin (0.5 mg/
mL), GDP (10 μM), and an EC80 concentration of CCL2 (20 nM).
For unwashed samples, only the last centrifugation step was
performed in order to resuspend the sample in the same volume of
320 μL. All samples were then preincubated for 30 min at 25 °C
before addition of 80 μL of [35S]GTPγS (0.3 nM). Reactions were
stopped after 90 min at 25 °C by rapid filtration as described under
the Washout Assays section, but using [35S]GTPγS wash buffer (50
mM Tris-HCl pH 7.4, 5 mM MgCl2).
NanoBiT Assays for Quantifying β-arrestin1 Recruitment to
CCR2 in Live Cells. Twenty-four hours post-transfection of HEK293t
cells with CCR2b-SmBiT and LgBiT-β-arrestin1-EE plasmids, the
cells were detached with PBS/EDTA (0.2 mM), centrifuged, and
resuspended in assay buffer (1× HBSS, 5 mM HEPES pH 7.2, 0.05%
BSA) to a concentration of 1.3 × 106 cells/mL. The cells were
aliquoted into a conical-bottom 96-well plate (Greiner Bio-One,
#651201) at 120,000 cells per well in 90 μL of assay buffer. Serial
dilutions of test compounds in assay buffer, at 10 × final
concentrations, were prepared from 10 mM DMSO stocks. Then,
10 μL of assay buffer (unwashed wells) or diluted test compounds
(washout wells) was added to the plate. After incubating the plate at
room temperature for at least 20 min, cells were washed twice by
spinning the plate at 400g for 5 min and discarding the supernatant.
Afterward, cells were resuspended in 80 μL of assay buffer and
transferred to a Falcon 96-well black/clear flat-bottom tissue-culture
treated plate (Corning, NY, USA). Ten microliters of assay buffer or
10 μL of test compounds was added to washout and unwashed wells,
respectively. Ten microliters of coelenterazine (CTZ-n, Fisher
Scientific, Waltham, MA, USA), diluted in assay buffer from a 5
mM stock in ethanol, was added to the plate to achieve a final
concentration of 10 μM in a 100 μL final volume in each well. The
plate was then incubated at room temperature for approximately 90
min, protected from light. Backing tape (PerkinElmer, #6005199) was
applied to the bottom of the plate, after which basal luminescence
from each well was read using a PerkinElmer Victor X Light 2030
apparatus (1 s, no filter). Next, 10 μL of 2 μM CCL2 in assay buffer
was added to each well and the cells were further incubated at room
temperature for 10 min, protected from light, after which the plate
was read again for endpoint luminescence generated by structural
complementation.
Molecular Modeling and Ligand Docking. Generation of a
Model of WT CCR2. The crystal structure of engineered human CCR2
in complex with an orthosteric and an allosteric antagonist (PDB
5T1A20) was used as a template for the generation of the CCR2
model. All modeling and docking was performed in ICM v3.8-7a
(Molsoft LLC, San Diego, CA61). The T4 lysozyme (T4L) fusion
protein present in the structure was removed, the mutated residues in
the intracellular part of TM6 was reverted to WT, and the intracellular
loop 3 (ICL3) was rebuilt. For rebuilding ICL3, a peptide containing
residues 223:243 of CCR2 was built ab initio as follows: the
backbones of residues 223:231 and 236:243 and the side chains of
residues 223:226 and 241:243 were tethered to their respective
positions in the crystal structure, the receptor converted into a set of
three-dimensional (3D) grid maps representing van der Waals,
electrostatic, hydrogen bonding, and surface energy potentials, and
the peptide conformation was optimized using the biased probability
Monte Carlo (BPMC) sampling in internal coordinates as
Washout Assays with [3H]-CCR2-RA-[R]. For washout assays,
membrane homogenates (80−100 μg) were preincubated in 1.5 mL
Eppendorf tubes with a single concentration of compounds 7 and 14
in a final volume of 300 μL of assay buffer (50 mM Tris-HCl pH 7.4,
5 mM MgCl2, 0.1% CHAPS). In the case of washout assays using
U2OS-CCR2 membranes, a 10×IC50 concentration was chosen for 7
(90 nM) and 14 (12 nM). In the case of washout assays using CHO-
CCR2 mutant receptor membranes, concentrations of 200 and 60 nM
were used for 7 and 14, respectively, to ensure a saturating
concentration in all mutants despite changes in affinity. For the triple
mutant, the concentration of 14 was increased to 160 nM to account
for the loss in affinity. In the case of washout assays with IA, U2OS-
CCR2 membranes were pretreated with 2 mg/mL IA for 30 min at
room temperature and protected from light prior to incubation with
ligands. After incubation for 2 h at 25 °C with shaking at
approximately 800 rpm, the mixture was centrifuged at 13,000 rpm
for 5 min at 4 °C and the supernatant containing unbound ligand was
removed. The remaining pellet was resuspended in 1 mL assay buffer
(50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 0.1% CHAPS) and
incubated for an extra 20 min before another cycle of centrifugation
and washing. After four cycles, the final membrane pellet was
resuspended in 300 μL of assay buffer, transferred to test tubes, and
incubated with 100 μL of ∼6 nM [3H]-CCR2-RA-[R] for 2 h at 25
°C while shaking. Incubations were terminated by rapid filtration
through a pre-wetted Whatman GF/B filter using a Brandel harvester
24 (Brandel, Gaithersburg, MD, USA). Filters were washed three
times with 2 mL of ice-cold wash buffer (50 mM Tris-HCl pH 7.4, 5
mM MgCl2, 0.01% CHAPS) and transferred to polyethylene Pony
vials (PerkinElmer) before measurement of filter-bound radioactivity
in a Tri-Carb 2810TR liquid scintillation analyzer (PerkinElmer).
[35S]GTPγS Binding Assays. [35S]GTPγS binding assays in CCR1,
CCR2 and CCR5 were performed as previously described.48,52 Briefly,
U2OS-CCR1 or U2OS-CCR2 membrane homogenates (10 μg) were
diluted in assay buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100
mM NaCl, 1 mM EDTA, and 0.05% BSA) supplemented with
saponin (0.5 mg/mL) and 10 μM GDP to a total volume of 100 μL.
In the case of U2OS-CCR5, 10 μg of membrane homogenates was
diluted in assay buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100
mM NaCl, 1 mM EDTA, and 0.05% BSA) supplemented with 0.25
mg/mL saponin and 5 μM GDP. To determine the (p)IC50 values of
7 and 14, membranes were preincubated for 30 min at 25 °C with
increasing concentrations of ligand in the presence of an EC80
concentration of CCL2 (20 nM) or CCL3 (8 nM for CCR1 and
25 nM for CCR5), as previously determined.48,52 Basal activity was
determined in the absence of any ligand or chemokine; maximal
activity in the presence of CCL2 or CCL3 only. After addition of 20
μL of [35S]GTPγS (0.3 nM), the mixture was incubated for 90 more
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implemented in ICM.61 The simulation simultaneously optimized the
intramolecular energy of the peptide and its interaction with the
context in the form of potential grid maps. The best scoring
conformation of the peptide was then merged with the rest of the
receptor coordinates, and the system was minimized in its full-atom
representation, with harmonic restraints of gradually decreasing
strength imposed between the model and either the X-ray coordinates
or the best prediction conformations of the ab initio modeled ICL3.
Toward the end of the optimization, the restraints were released
entirely. The simulations were performed in the presence of
crystallographic ligands.
Generation of a Model of CCR2:14 Complex. Alternative
conformers of the allosteric pocket in CCR2 were generated by
systematic sampling of aliphatic side-chain rotamers and translations
of the intracellular ends of TM helices 6 and 7. The conformer that
best predicted the binding poses of the sulfonamide series of allosteric
CCR2 antagonists (analogous to 14) was chosen. The binding pose of
14 was predicted by covalent docking in ICM under the assumption
that the covalent attachment residue is C75 due to its proximity. For
docking, the receptor atoms in the 4 Å vicinity of the allosteric pocket
were represented as 3D grid potential maps as above, with the
exception of C75 for which the explicit representation was used. The
covalent bond between 14 and C75 was imposed, and the system was
sampled as described above to generate a ranked list of alternative
conformations for the ligand. The top 10 conformations were merged
with the full-atom model of the receptor and re-scored using the ICM
ligand scoring function previously optimized for ligand geometry
prediction on a diverse benchmark of crystallographic protein−ligand
complexes.62 The top-scoring pose was selected.
La Jolla, California 92093, United States;
Email: ikufareva@health.ucsd.edu
Laura H. Heitman − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands; Oncode Institute, 2333 CC Leiden, The
Netherlands; Email: l.h.heitman@lacdr.leidenuniv.nl
Authors
Natalia V. Ortiz Zacarías − Division of Drug Discovery and
Safety, Leiden Academic Centre for Drug Research, 2333 CC
Leiden, The Netherlands; Oncode Institute, 2333 CC Leiden,
The Netherlands
Kirti K. Chahal − Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego,
La Jolla, California 92093, United States
̌
́
Tereza Simkova − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands
Cas van der Horst − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands
Yi Zheng − Skaggs School of Pharmacy and Pharmaceutical
Sciences, University of California San Diego, La Jolla,
California 92093, United States
Asuka Inoue − Graduate School of Pharmaceutical Sciences,
Tohoku University, Sendai, Miyagi 980-8578, Japan
Emy Theunissen − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands
Lloyd Mallee − Division of Drug Discovery and Safety, Leiden
Academic Centre for Drug Research, 2333 CC Leiden, The
Netherlands
Daan van der Es − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands
Julien Louvel − Division of Drug Discovery and Safety, Leiden
Academic Centre for Drug Research, 2333 CC Leiden, The
Netherlands
Adriaan P. IJzerman − Division of Drug Discovery and Safety,
Leiden Academic Centre for Drug Research, 2333 CC Leiden,
The Netherlands; orcid.org/0000-0002-1182-2259
Data Analysis. Data analyses were performed using Prism 7.00
(GraphPad software, San Diego, CA, USA). (p)IC50 values from
radioligand displacement assays, [35S]GTPγS binding assays, and
NanoBiT assays were obtained by non-linear regression curve fitting
into a sigmoidal concentration−response curve using the equation: Y
= Bottom + (Top − Bottom)/(1 + 10^(X − LogIC50)). pKi values
were obtained from pIC50 values using the Cheng−Prusoff equation.63
Data are shown as mean standard error of the mean (SEM) of at
least three individual experiments performed in duplicate. Statistical
analyses were performed as indicated. If p-values were below 0.05,
observed differences were considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01137.
■
sı
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01137
Washout-radioligand experiments in presence of N-
ethylmaleimide (Figure S1); washout-radioligand experi-
ments in CCR2-C75A mutant (Figure S2); selectivity
assessment of compounds (Figure S3); HPLC traces of
final compounds 7 and 14 (Figure S4); HPLC traces of
final compounds 11, 12, and 13 (Figure S5); and
potency of compounds 7 and 14 in U2OS-CCR1 and
U2OS-CCR5 membranes using [35S]GTPγS binding
assays (Table S1) (PDF)
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
The authors will release the atomic coordinates of the docking
models upon article publication.
Molecular formula strings (CSV)
Homology model of CCR2b with docking of com-
pounds 7 and 14 (PDB, PDB)
ACKNOWLEDGMENTS
■
AUTHOR INFORMATION
Corresponding Authors
This work is supported by NIH grants R01 AI118985, R01
GM117424, R01 GM136202, and R21 AI149369 to I.K. and
T.M.H. K.K.C. is supported by a Training Fellowship from the
Cancer Cell Mapping Initiative Consortium U54 CA209891.
L.H.H. and N.V.O.Z. were also supported by funding from
Oncode Institute, The Netherlands. A.I. was funded by the
LEAP JP19gm0010004 from the Japan Agency for Medical
Research and Development (AMED).
■
Tracy M. Handel − Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego,
La Jolla, California 92093, United States; Email: thandel@
health.ucsd.edu
Irina Kufareva − Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego,
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ABBREVIATIONS
■
BCA, bicinchoninic acid; BPMC, biased probability Monte
Carlo; BSA, bovine serum albumin (BSA); CCR2, CC
chemokine receptor 2; CCR2a, CCR2 isoform A; CCR2b,
CCR2 isoform B; CHO, Chinese hamster ovary; CTZ-n,
coelenterazine; FCS, fetal calf serum; GDP, guanosine 5′-
diphosphate; GPCRs, G protein-coupled receptors; [35S]-
GTPyS, guanosine 5′-O-[γ-thio]-triphosphate; IA, iodoaceta-
mide; ICL3, intracellular loop 3; NanoBiT, NanoLuc binary
technology; NAM, negative allosteric modulator; PEI,
polyethyleneimine; SEM, standard error of the mean; T4L,
T4 lysozyme; U2OS, human osteosarcoma cells; WT, wild-
type
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