Selection, preparation, and evaluation of small-molecule inhibitors of toll-like receptor 4

Article abstract of DOI:10.1021/ml100041f

Toll-like receptor 4 (TLR4), a membrane-spanning receptor protein that functions in complex with its accessory protein MD-2, is an intriguing target for therapeutic development. Herein, we report the identification of a series of novel TLR4 inhibitors and

Full text of DOI:10.1021/ml100041f

pubs.acs.org/acsmedchemlett
Selection, Preparation, and Evaluation of Small-Molecule
Inhibitors of Toll-Like Receptor 4
Douglas E. Bevan,† Alexander J. Martinko,† Lisa C. Loram,‡,§ Joshua A. Stahl,†
Frederick R. Taylor,‡ Sampada Joshee,† Linda R. Watkins,‡,§ and Hang Yin*,†,§
†Department of Chemistry and Biochemistry, ‡Department of Psychology and Neuroscience, and §The Center for Neuroscience,
University of Colorado at Boulder, Boulder, Colorado 80309
ABSTRACT Toll-like receptor 4 (TLR4), a membrane-spanning receptor protein
that functions in complex with its accessory protein MD-2, is an intriguing target
for therapeutic development. Herein, we report the identification of a series of
novel TLR4 inhibitors and the development of a robust, enantioselective synthesis
using an unprecedented Mannich type reaction to functionalize a pyrazole ring. In
silico and cellular assay results demonstrated that compound 1 and its analogues
selectively block TLR4 activation in live cells. Animal model tests showed that 1 and
its derivatives could potentiate morphine-induced analgesia in vivo, presumably
by attenuating the opioid-induced TLR4 activation.
KEYWORDS Toll-like receptor, glial cells, signal transduction, protein-protein
interactions, synthesis
he regulation of protein-protein interactions using
small-molecule agents is a fast-evolving field in med-
icinal chemistry and chemical biology.1,2 One of the
silico screening that targets the MD-2 binding region on the
TLR4 surface. The next step was to develop a synthesis
that would be conducive to structure-activity relationship
(SAR) studies.
When considering a retrosynthesis for 1 (Figure 1), the
most logical disconnection was that of the β-amino alcohol,
giving epoxide 2 and tetra-substituted pyrazole 3. Epoxide 2
could be traced back to epichlorohydrin (4) and phenol 5.
The pyrazole portion 3 can be dismantled via a Mannich
type reaction and SN2 displacement of benzylchloride 7 by
pyrazole 6. This retrosynthesis gave us the ability to easily
alter four parts of the molecule independently to aid in
SAR studies.
In the forward direction (Scheme 1), the construction of
the top piece commenced with the treatment of 3,5-di-
methylpyrazole (6) with KOH and the 2-chloro benzyl chlor-
ide (7) to give N-aryl pyrazole 8 in a quantitative yield. The
next reaction to append the methylene methylamine was
unprecedented in the literature to the best of our knowledge.
With guidence from a number of sources,13-16 we were able
to develop a robust and high-yielding Mannich type reaction
for the synthesis. By treating pyrazole 8 with a 1:2 mixture of
methyl amine and paraformaldehyde in refluxing ethanol,17
we were able to obtain the desired amine 3 in a 93% yield.
The synthesis of the bottom half of the molecule involved
stirring 4-ethoxy phenol (5) with epichlorohydrine (4) and
potassium carbonate in refluxing acetone18,19 to yield 83%
of desired product 2. Coupling of the two pieces was best
T
mechanisms by which the innate immune system senses the
invasion of pathogenic microorganisms is through the toll-like
receptors (TLRs). TLRs are type I integral membrane glyco-
proteins3 that recognize specific molecular patterns present in
microbial components.4 Stimulation of different TLRs induces
distinct patterns of gene expression, which not only leads to the
activation of innate immunity but also instructs the develop-
ment of antigen-specific acquired immunity. TLR4 detects
lipopolysaccharide (LPS, a component of Gram-negative bac-
terial cell walls).5 Most recently, Watkins et al. found that TLR4
plays an essential role in microglial activation that contributes to
the development of morphine tolerance and thereby compro-
mises the analgesic effects of morphine.6 This exciting discov-
ery provides a novel avenue for therapeutic development to
attenuate morphine tolerance by blocking TLR4 signal trans-
duction in glial cells.7
The structure of TLR4 has recently been solved,8,9 render-
ing opportunities for the use of structure-based drug design
technology to develop potential inhibitors. TLR4 forms a
heterodimer with MD-2, regulating a number of critical cell-
signaling pathways.10 The protein-protein interactions be-
tween TLR4 and MD-2 are essential for TLR4 signaling and
are, therefore, intriguing targets for therapeutic develop-
ment specifically for TLR4 over other TLRs since MD-2
interacts primarily with TLR4 among TLR family proteins.11
To identify novel, druglike small molecule inhibitors for
TLR4, we employed a recently developed high-throughput in
silico screening method.12 A low molecular weight, druglike
lead structure, T5342126 (1), has been idenfitied from the in
Received Date: February 21, 2010
Accepted Date: May 10, 2010
Published on Web Date: May 14, 2010
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Table 1. Results of the SEAP Reporter Gene Activation Assaya
compound
R1
R2
4-OEt
inhibition (%)
1
(-)-1
(þ)-1
11
2-Cl
2-Cl
2-Cl
4-OMe
H
52
61
60
4
4-OEt
4-OEt
4-OEt
H
12
1
13
2-Cl
2-Cl
2-Cl
2-F
4-(C2H4)OMe
2-OMe
4-Cl
32
36
99
40
35
31
14
Figure 1. Retrosynthesis of 1.
Scheme 1. Preparation of 1
15
16
4-OEt
4-OEt
4-OEt
17
2-OMe
2-Me
18
a The percent inhibition was determined by measurement of
LPS-induced TLR4 activation in HEK293 cells in the presence of 50 μM
drug as compared to blank control.
with 3 to give access to both enantiomers of 1. Using this
method, both enantiopure isomers of 1 were synthesized
with 99% enantiomeric excess (ee).
Using this robust, modular synthesis, we also prepared a
focused library of analogues of lead compound 1 (Table 1).
All of these analogues were made racemically using the
same sequence as the parent compound and in comparable
yields. This first round of analogues was meant to apply
changes only to the periphery of the parent compound.
With these analogues in hand, we tested the in vitro
inhibitory effects for TLR4 signaling in the HEK 293 cells
using a previously described SEAP assay.21 Compound 15
with 2-Cl and 4-Cl substitutes on the top and bottom aro-
matic rings has been identified with the highest potency to
block TLR4 signaling in live cells. At a concentration of
50 μM, racemic 15 showed a complete inhibition of
LPS-induced TLR4 activation in HEK 293 cells. By contrast,
analogue 12 with no substitution on the aromatic rings
showed negligible inhibition, indicating the critical impor-
tance of the functional groups. Both enantiomers of 1
showed comparable inhibitory potency, suggesting that its
stereocenter does not involve in the recognition of TLR4.
These SAR results also demonstrated that the derivatives of 1
inhibit TLR4 activation in a specific manner, ruling out the
possibility of nonspecific artifacts by the backbone scaffold.
Furthermore, none of these compounds showed any detect-
able cytotoxicity at concentrations up to 300 μM in various
cell lines (e.g., RAW264.7, HEK 293, and HeLa).
accomplished by stirring amine 3 and epoxide 2 in a 1.2:1
ratio in refluxing 2 M ethanol, to give the target compound 1
in an 89% yield, with an 82% overall yield from commer-
cially available 3,5-dimethylpyrazole.
To test the stereospecificity of the binding of 1 to TLR4, we
made the enantiopure forms of both enantiomers at the
single alcohol stereocenter of the molecule. To accomplish
this, racemic epoxide 2 was treated with Jacobsen's hydro-
lytic kinetic conditions20 (Scheme 2) to access either the
pure (þ)- or the (-)-enantiomers. Because both the (R,R)-
and the (S,S)-Jacobsen precatalysts are commercially avail-
ible, either enantiomer of 2 could be readily produced. This
material could then be used in the final coupling reaction
Next, we carried out dose dependence studies for the
representative inhibitors. Using the SEAP assay, the EC50
values for racemic compounds 15 and 1 were determined to
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Figure 2. Effect of 15 on TLR-ligand-induced NO production in RAW264.7 cells. LPS, poly(I:C) (polyinosinic-polycytidylic acid), FSL-1 [(S,R)-(2,3-
bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe], Pam3CSK4 {N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-[R]-cystei-
nyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine 3HCl}, and R848 {4-amino-2-(ethoxymethyl)-R,R-dimethyl-1H-imidazo[4,5-c]quinoline-1-
3
ethanol} were used to selectively activate TLR4, TLR3, TLR2/6, TLR2/1, and TLR7, respectively. Compound 15 (25.0 μM) selectively inhibited
the NO production induced by LPS but not ligands of other TLRs. Data are means from three independent experiments.
Scheme 2. Synthesis of Enantiopure (þ)-1
be 18.7 ( 3.2 and 48.1 ( 2.1 μM, respectively (figure in the
Supporting Information). Unfortunately, complete inhibition
plots for other less potent analogues were not achieved due
to the limitation of the cellular assay. TLR4 is an innate immune
receptor and can recognize an array of exogenous ligands.22
The addition of a higher dose of inhibitors activates TLR4's
signal transduction, which in turn compromises the inhibitory
effect analyses. Nonetheless, these results have demonstrated
that the inhibitory effects of these analogues are dose-depen-
dent and that the functional groups on the benzene rings
are crucial.
In an effort to understand the specificity of our small-
molecule inhibitors between different TLRs, we investigated
the selectivity of 15 by measuring nitric oxide (NO) produc-
tion in RAW cells. RAW cells express all TLRs, and each speci-
fic TLR can be individually activated by treatment with a
receptor-specific ligand.23 The activation of TLRs results in
downstream signaling and production of pro-inflammatory
mediators such as NO. Compound 15 (27 μM) inhibited
TLR4-mediated NO production but showed negligible effects
on the signaling of TLR3, TLR 2/6, TLR 2/1, and TLR7 (Figure
2). These results demonstrated that 15 selectively inhibits
LPS-induced TLR4 activation without affecting other homo-
logous toll-like receptors.
To obtain insights into the possible binding modes of the
lead inhibitors, we conducted in silico docking simulations
for compounds 1 and 15. Figure 3 shows the top-ranked
binding mode of 15 predicted by the AutoDock 4.0 Simula-
tion Suite, implying that 15 recognizes the same cleft on the
surface of TLR4 to which a protruding loop region of MD-2
binds. These results suggest that 15 may inhibit the TLR4-
MD-2 complex formation by competing with MD-2's binding
to TLR4. Docking results for compound 1 suggested a similar
binding mode to 15 (data not shown), lending further
support that the in silico prediction is consistent and valid.
An established rat model was employed to test whether
the newly discovered TLR4 signaling antagonist was able to
potentiate the analgesic effect of morphine in vivo.23 The
Hargreaves test was employed to measure the time taken to
observe radiant heat-induced withdrawal responses by the
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Figure 3. Results of the molecular-docking simulation predict that 15 disrupts the TLR4-MD-2 interaction by competing with MD-2
binding to TLR4. (A) Side and (B) top views of the top-ranked binding modes of 15 (shown in ball-and-stick representation) are shown in
complex with full-length TLR4 (orange), competing with MD-2 (red) for the same binding side by mimicking a protruding loop region of
MD-2. The structure of TLR4 was taken from the complex with the lymphocyte antigen 96 (PDB ID: 2Z65; resolution, 2.70 Å).
of cerebrospinal fluid (CSF) in the rat model that we em-
ployed has been determined to be around 100 μL,24 and
intrathecally administrated morphine, 15, and 1 (injection
volume= 1 μL) are estimated to be present at concentra-
tions of 530, 10, and 300 μM, respectively, in the rat CSF,
which is consistent with the previously established assay
conditions.24
In conclusion, we have accomplished our goal of develop-
ing and executing an easily modified and scalable synthesis
(5 g scale to date) of derivatives of the lead compound 1. In
the process, we developed a reliable and previously unre-
ported Mannich type reaction. We have shown that the
substituents on the benzene rings of 1 affect the in vitro
activity of the molecule and that one of the enantiomers at
the alcohol position is more active then the other. Finally, we
Figure 4. Potentiating intrathecal morphine analgesia by in-
have shown 1 and its analogues to be effective at blocking
trathecal 15. Following predrug (baseline) assessment of respon-
TLR4 activation in live cells and at potentiating morphine
sivity to radiant heat (Hargreaves test), rats received intrathecal
analgesia in live animal studies.
morphine (1 μL, 15 mg/mL), 15 (1 mM, 1 μL), or the combination of
these same morphine and small-molecule doses. While 15 or
vehicle alone (5ꢀ PBS) had no effect on pain sensitivity, 15 en-
hanced morphine analgesia. The asterisk denotes the significant
difference between morphine þ vehicle and morphine þ 15 (P<
0.001) from 85 to 125 min. Error bars show the SEM.
SUPPORTING INFORMATION AVAILABLE Experimental
procedures and characterization of novel compounds, biological
testing procedure, and results. This material is available free of
charge via the Internet at http://pubs.acs.org.
tails of unrestrained rats. Prior to drug administration, two
readings were recorded. Following these baseline measure-
ments, drugs were injected intrathecally, and the rats' re-
sponses to radiant heat were reassessed across a 3 h time
course (Figure 4). While 15 had no effect on pain responsiv-
ity in the absence of coadministered morphine, it robustly
potentiated the acute analgesic effects of morphine such
that the rats exhibited the maximum analgesia recordable
on the test. Using the same assay, compound 1 at an elevated
dose (injection of 1 μL of 30 mM solution) was shown to have
similar effects to potentiate and prolong morphine-induced
analgesia, suggesting that the analogues of 1 consistently
inhibit morphine-induced TLR4 activation in vivo. It is noti-
ceable that the moderate in vitro inhibitory potency of 1
(EC50=48.1 ( 2.1 μM) and 15 (EC50=18.7 ( 3.2 μM) is rele-
vant under the physiological conditions in vivo. The volume
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. E-mail: Hang.Yin@colorado.edu.
Funding Sources: We thank the National Institutes of Health
(DA026950, DA025740, NS067425, RR025780, and DA029119 to
H.Y. and DA023132, DA024044, NS067807, and DE017782 to
L.R.W.) for financial support of this work.
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