A novel p38 MAPK docking-groove-targeted compound is a potent inhibitor of inflammatory hyperalgesia

Article abstract of DOI:10.1042/BJ20130172

TheMAPK(mitogen-activated protein kinase) p38 is an important mediator of inflammation and of inflammatory and neuropathic pain. We have described recently that docking-groove-dependent interactions are important for p38 MAPK-mediated signal transduction. Thus virtual screening was performed to identify putative docking-groove-targeted p38 MAPK inhibitors. Several compounds of the benzo-oxadiazol family were identified with low micromolar inhibitory activity both in a p38 MAPK activity assay, and in THP-1 human monocytes acting as inhibitors of LPS (lipopolysaccharide)-induced TNFa (tumour necrosis factor a) secretion. Positions 2 and 5 in the phenyl ring are essential for the described inhibitory activity with a chloride in position 5 and a methyl group in position 2 yielding the best results, giving an IC₅₀ value of 1.8 μM (FGA-19 compound). Notably, FGA-19 exerted a potent and long-lasting analgesic effect in vivo when tested in a mouse model of inflammatory hyperalgesia. A single intrathecal injection of FGA-19 completely resolved hyperalgesia, being 10-fold as potent and displaying longer lasting effects than the established p38MAPKinhibitor SB239063. FGA-19 also reversed persistent pain in a model of post-inflammatory hyperalgesia in LysM (lysozyme M)-GRK2 (G-protein-coupledreceptor kinase)^+/- mice. These potent in vivo effects suggested p38 MAPK docking-site-targeted inhibitors as a potential novel strategy for the treatment of inflammatory pain. The Authors Journal compilation

Full text of DOI:10.1042/BJ20130172

Biochem. J. (2014) 459, 427–439 (Printed in Great Britain) doi:10.1042/BJ20130172
427
A novel p38 MAPK docking-groove-targeted compound is a potent inhibitor
of inflammatory hyperalgesia
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Hanneke L. D. M. WILLEMEN*1, Pedro M. CAMPOS†1,2, Elisa LUCAS†‡, Antonio MORREALE§ , Rube´n GIL-REDONDO§ ,
5
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Juan AGUT¶, Florenci V. GONZALEZ¶, Paula RAMOS†‡, Cobi HEIJNEN*ꢀ, Federico MAYOR, Jr†‡, Annemieke KAVELAARS*ꢀ and
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Cristina MURGA†‡
*Laboratory of Neuroimmunology and Developmental Origins of Disease, University Medical Center Utrecht, Utrecht, The Netherlands
†Departamento de Biolog´ıa Molecular and Centro de Biolog´ıa Molecular “Severo Ochoa” UAM-CSIC, Madrid, Spain
‡Instituto de Investigacio´n Sanitaria La Princesa, Madrid, Spain
§Centro de Biolog´ıa Molecular “Severo Ochoa”, UAM-CSIC, Madrid, Spain
¶Departament de Qu´ımica Inorga`nica i Orga`nica, Universitat Jaume I, Castello´, Spain
ꢀDepartment of Symptom Research, University of Texas MD Anderson Cancer Center, Houston, TX 77030, U.S.A.
The MAPK (mitogen-activated protein kinase) p38 is an important
mediator of inflammation and of inflammatory and neuropathic
pain. We have described recently that docking-groove-dependent
interactions are important for p38 MAPK-mediated signal
transduction. Thus virtual screening was performed to identify
putative docking-groove-targeted p38 MAPK inhibitors. Several
compounds of the benzo-oxadiazol family were identified with
low micromolar inhibitory activity both in a p38 MAPK activity
assay, and in THP-1 human monocytes acting as inhibitors of
LPS (lipopolysaccharide)-induced TNFα (tumour necrosis factor
α) secretion. Positions 2 and 5 in the phenyl ring are essential
for the described inhibitory activity with a chloride in position
5 and a methyl group in position 2 yielding the best results,
giving an IC50 value of 1.8 μM (FGA-19 compound). Notably,
FGA-19 exerted a potent and long-lasting analgesic effect in vivo
when tested in a mouse model of inflammatory hyperalgesia.
A single intrathecal injection of FGA-19 completely resolved
hyperalgesia, being 10-fold as potent and displaying longer lasting
effects than the established p38 MAPK inhibitor SB239063. FGA-
19 also reversed persistent pain in a model of post-inflammatory
hyperalgesia in LysM (lysozyme M)-GRK2 (G-protein-coupled-
receptor kinase)+ / − mice. These potent in vivo effects suggested
p38 MAPK docking-site-targeted inhibitors as a potential novel
strategy for the treatment of inflammatory pain.
Key words: docking, inflammatory hyperalgesia, pain, persistent
pain, p38 mitogen-activated protein kinase (p38 MAPK).
controls the activation state of downstream kinases, such as
MAPKAPK (MAPK-activated protein kinase) 2 and 3, that, in
turn, phosphorylate HSP27 (heat-shock protein 27) and other
cellular proteins. A p38-dependent route regulates the stability
and expression of mRNAs coding for cytokines such as TNFα
(tumour necrosis factor α), IL (interleukin)-1β, IL-6 and IL-8 [4].
In the context of pain, p38 MAPK is activated in the spinal cord in
several models of chronic pain, and pain hypersensitivity can be
transiently blocked by administration of p38 MAPK inhibitors [5–
8]. These findings indicate that p38 MAPK may be an interesting
target for the development of therapeutic drugs to treat chronic
inflammatory pain. However, so far human clinical trials using
p38 MAPK inhibitors have not yielded the expected results [9].
p38 MAPK is able to specifically interact with upstream
regulators and also with downstream substrates by virtue of a
specific site called docking domain that is situated opposite the
activation loop [2]. The specific features of these interactions
have been characterized in detail and the structure of p38α co-
crystallized with peptides derived from the interacting motifs in
MKK3b and MEF2A has been resolved [10]. Interestingly, the
INTRODUCTION
The MAPK (mitogen-activated protein kinase) p38 is a key
signalling protein involved in regulating the production of
multiple inflammatory mediators. Therefore p38 MAPK has
been the target of intensive research in academic groups and
in many different pharmaceutical companies [1]. p38 belongs
to the MAPK family of serine/threonine kinases that share a
common overall structure and a similar mode of activation via
phosphorylation at the activation loop by upstream activators,
including MKK3 (MAPK kinase 3) and MKK6 for p38 MAPK
(for a review, see [2]). Of the four p38 MAPK isoforms,
p38α and p38β are ubiquitously expressed and in inflammatory
cells p38α is the most prominent. Many different extracellular
stimuli activate p38 MAPK, among them inflammatory cytokines,
LPS (lipopolysaccharide) and other bacterial products, and
different stressors such as thermal, mechanical or hypoxic insults
[3]. In turn, p38 MAPK directly phosphorylates important
transcription factors including MEF2 (myocyte enhancer factor
2) and ATF2 (activating transcription factor 2). p38 MAPK also
Abbreviations: ERK, extracellular-signal-regulated kinase; GB, Generalized Born; GRK2, G-protein-coupled-receptor kinase 2; GST, glutathione
transferase; H2L, hit-to-lead; HSP27, heat-shock protein 27; IL, interleukin; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; LysM, lysozyme
M; MAPK, mitogen-activated protein kinase; MAPKAPK, MAPK-activated protein kinase; MBP, myelin basic protein; MEF2, myocyte enhancer factor 2;
MKK, MAPK kinase; MM, molecular mechanics; Nbd-Cl, 4-chloro-7-nitrobenzofurazan; PI, propidium iodide; RoF, rule of five; SA, surface area; SAPK,
stress-activated protein kinase; SASA, solvent accessible SA; TNFα, tumour necrosis factor α; VS, virtual screening; WT, wild-type.
1
These authors contributed equally to this work.
Present address: Allinky Biopharma, Parque Cient´ıfico de Madrid, Madrid, Spain.
Present address: Repsol, Technology Center, Ctra. de Extremadura, A-5, km 18, 28935 Mo´stoles, Madrid, Spain.
Present address: SmartLigs Bioinforma´tica S.L., Fundacio´n Parque Cient´ıfico de Madrid, Campus de Cantoblanco UAM, Madrid, Spain.
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Correspondence may be addressed to either of these authors (email akavelaars@mdanderson.org or cmurga@cbm.uam.es).
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H. L. D. M. Willemen and others
interactions mediated by this docking domain can be regulated by
means of phosphorylation of either the substrate or p38 MAPK
itself [11]. This characteristic provides a feasible mechanism for
intervention into the p38 MAPK route by providing a target
for therapeutic drugs that is potentially more specific for p38
MAPK than its currently most widely targeted domain, the
ATP-binding site. The site shares a high overall homology with
that of other protein kinases [12], and this probably underlies
the failure of the first and second generation ATP-site targeted
compounds tested during human clinical trials [1]. Consequently,
the need for novel strategies utilizing unexplored interactive
domains in p38 MAPK is an avenue that certainly deserves
further research. Previously, we have described that p38 MAPK
can be phosphorylated at the docking groove resulting in reduced
binding of upstream kinases and downstream targets and therefore
impairing its activity [11]. On the basis of those findings, we
performed a virtual screening of a chemical library of compounds
to search for potential novel p38 MAPK inhibitors targeting the
docking groove.
[19], mainly from ChemBridge and ASINEX, in SMILES format
[20]. Multiple protonation states and tautomeric forms were con-
sidered as implemented by default in ZINC. The molecules were
then processed within VSDMIP as: (i) conversion from SMILES
to 3D MOL2 using CORINA (http://www.molecular-networks.
com/products/corina); (ii) atomic charge calculations with
MOPAC [22] (MNDO ESP method) on every single structure
provided by CORINA; and (iii) atom type assignment according
to the AMBER ff99 force field and conformational analysis using
ALFA [23].
Filter 1
An initial filter was performed with the docking program DOCK
to quickly discard those molecules that do not geometrically fit
within the binding site. The spheres needed by DOCK were those
generated previously with GAGA. We used DOCK contact as
the scoring function, normalizing the score values (scorei) by
converting them into ZScores using mean (average score) and S.D.
(σ) values [ZScorei = (scorei − average score)/σ]. Only those
molecules with a ZScore beyond a cut-off value of 2.5 were
selected (out of 45488) and passed on to the next step.
MATERIALS AND METHODS
VS (virtual screening)
Filter 2
All VS calculations were performed within the automated
platform VSDMIP [13,14]. For the sake of clarity, below we
briefly describe the main steps comprising the protocol.
Selected molecules from the previous step were docked with the
more accurate docking algorithm CDOCK. CDOCK exhaustively
docks each molecule within the binding site of the receptor
using the interaction energy grids calculated previously with
CGRID. This was achieved by an exhaustive exploration of
the location and orientation of each molecule by positioning its
centre of mass on grid points and performing discrete rotations
of 27◦ on each axis (poses). Finally, the energy of the poses was
evaluated by the MM (molecular mechanics) force-field scoring
function implemented in CDOCK, which, beside including a
12-6 Lennard–Jones term and an electrostatic term modelled
with a sigmoidal dielectric screening function, also accounts for
ligand and receptor desolvations as well as for hydrogen-bonding
interactions [24].
Receptor preparation
The 3D structure of p38 MAPK, taken from a complex with the
MEF2A peptide (PDB code 1LEW) [10], was used as the receptor.
All atoms other than those from the receptor were removed.
AMBER ff99 force field [15] was then used to assign atom
types and charges for each atom in the receptor. The addition of
missing hydrogen atoms and computation of the protonation state
of ionizable groups at pH 6.5 were carried out using the H + +
Web server [16], which relies on AMBER parameters and finite
difference solutions to the Poisson–Boltzmann equation. A salt
concentration of 0.15 M and an internal and external dielectric
constant of 4 and 80 respectively were used.
MD simulations and MM-GBSA calculations
The top-ranked 200 molecules according to CDOCK’s scoring
function were subjected to better binding free energy estimation
by a combination of MD trajectories and MM-GBSA [25]
calculation on these trajectories. The 200 complexes were
hydrated by using boxes containing explicit water molecules
with added counter ions to maintain electroneutrality, energy
minimized, heated (20 ps) and equilibrated (100 ps). After
equilibration, MD trajectories were continued for 200 ps. From
this last part, structures were homogenously sampled each 10
ps and stored for post-processing. All the simulations were
performed at constant pressure and temperature [1 atm (1
atm≡101.325 kPa) and 300 K] with an integration time step of
2 fs. SHAKE [26] was used to constrain all the bonds involving
hydrogen atoms at their equilibrium distances. Periodic boundary
conditions and the Particle Mesh Ewald methods were used to treat
long-range electrostatic effects [27]. AMBER ff99 and TIP3P [28]
force fields were used in all cases. Finally, the effective binding
free energies were qualitatively estimated using the MM-GBSA
approach, which calculates the free energy of binding as a sum of
a MM interaction term, a solvation contribution thorough a GB
(Generalized Born) model and a SA (surface area) contribution to
account for the non-polar part of desolvation. A 12-6 Lennard–
Jones term was used to model the MM contribution. For the
Binding site definition and characterization
To delimit the binding site we selected MEF2A as the core around
which to build the docking box by adding a 5.0 Å cushion to the
maximum dimensions of the peptide. A equally spaced grid of 0.5
Å was then built, and CGRID [17] was used to calculate the protein
interaction fields (a 12-6 Lennard–Jones term and an electrostatic
term modelled with a sigmoidal dielectric screening function)
using common atom probes (C, N, O, S, P, H, F, Cl, Br and I) at
each grid point. Next, benzene, water and methanol probes were
docked using CDOCK [17] to generate intermolecular interaction
energy maps aimed at capturing the most probable areas of interest
for hydrophobic, hydrophilic and hydrogen bond interactions
respectively. These areas were further compressed into Gaussian
functions using the GAGA algorithm [18] producing a negative
image of the interaction site. The putative active ligands in the
library must conform to this approximate shape.
Chemical library preparation
Ligands for VS consisted of a selection of ∼106 non-redundant
molecules obtained from the publicly available ZINC database
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Figure 1 VS and identification of a lead compound
(A) Schematic representation of the computational protocol employed in the present study (left- and right-hand panels) and the drug-design cycle established with the experimental counterpart
(middle panel). (B) Structural pathway from the identified virtual hit 978604 to the hit candidate 5380 and to the lead compounds FGA-19 and FGA-29 after two rounds of optimization. (C) Chemical
reaction for the preparation of FGA-19 starting from Nbd-Cl and 5-chloro-2-methylanilin. DMF, N,N-dimethylformamide. (D) Proposed binding modes for FGA-19 and FGA-29. The receptor is
represented in cartoons (white in FGA-19 complex and black in FGA-29 complex), whereas the ligands and main interacting residues (see text) are shown in sticks. Hydrogen atoms have been
omitted for clarity. Thr123, that is targeted by GRK2, is also indicated.
GB model, the solute dielectric constant was set to 4, whereas
that of the solvent was set to 80, and the dielectric boundary
was calculated using a solvent probe radius of 1.4 Å. The SA
contribution was approximated as a linear relationship to the
change in SASA (solvent accessible SA) ꢀGnp = a + bꢀSASA,
where a is 0.092 kcal/mol, b is 0.00542 kcal·mol− 1·Å− 2, ꢀGnp is
the SA contribution, and the change in SASA refers to the complex
SASA minus the sum of that of the protein and the ligand alone. In
addition, interaction energy analysis between the ligands and the
more relevant residues in the binding site were computed (with
MM-GBSA). All the trajectories and analysis were performed
using the AMBER 8 program and associated modules [29].
compounds, and upon visual examination, 18 candidates were
finally selected, purchased (see below) and tested experimentally.
All visualizations were done within the molecular graphics
program PyMOL (http://www.pymol.org). Unfortunately, not all
the compounds were available from the vendors. In these cases
similarity search was performed employing two complementary
strategies based on: (i) the 2D topological representation of the
molecules and the search/compare engine implemented in ZINC;
and (ii) the 3D structure of the molecules, the shape-matching
algorithm implemented in the program ROCS [31], and the ZINC
database as implemented in VSDMIP. The similarity between
structures was assessed by the Tanimoto coefficient (Tc), selecting
only those candidates with a Tcꢀ0.9 to be docked into the
receptor and for further analysis. The final goal was to choose
one or two purchasable analogues for each not-found compound
presenting as similar interactions as possible when compared with
the originally selected molecules.
Selection of candidates
Averaged structures along the trajectories were obtained and
energy-minimized in a vacuum with the ff99 force field,
without periodic boundary conditions and during 1000 steps
(the first 500 with the steepest descent method and the rest
with the conjugated gradient) solely to alleviate the possible
clashes that may be originated by averaging the co-ordinates.
These structures were used for graphical representation and
comparison of the binding modes. From the highest-scoring
H2L (hit-to-lead) optimization
The scheme for the H2L optimization cycle is shown in Figure 1.
A typical optimization step involves a finer docking study
of the best compounds from the experimental assays over an
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Table 1 Yield and physical state of the final chemical synthesis products
of Utrecht, The Netherlands or of the Universidad Autonoma de
Madrid, Spain. Experiments were performed in accordance with
international guidelines and approved by the experimental animal
committee of each institution.
Compound
Physical state
Yield (%)
FGA-17
FGA-19
FGA-20
FGA-23
FGA-29
FGA-32
FGA-34
Red crystals
Red crystals
Yellow solid
Orange solid
Deep red solid
Orange solid
Orange solid
67
50
55
64
58
54
52
Mice received an intraplantar injection in the hind paw
of 20 μl of λ-carrageenan [2% (w/v); high dose; Sigma–
Aldrich] or 5 μl of λ-carrageenan [1% (w/v); low dose;
Sigma–Aldrich] diluted in saline. Heat withdrawal latency times
were determined using the Hargreaves test (IITC Life Science) as
described in [34]. Paw thickness was measured using a Digimatic
micrometer (Mitutoyo). Different concentrations of FGA-19 were
applied intrathecally (5 μl/mouse) while the animals were under
light isoflurane anesthaesia. All behavioural experiments were
performed by an experimenter blinded to the treatment and mice
were assigned to the treatment groups in a randomized fashion.
energy-minimized MD-averaged structure followed by MD
simulation and MM-GBSA analysis. In brief, for every docked
molecule we: (i) selected the 100 best docking solutions; (ii)
clustered them according to their structural resemblance; (iii)
selected a representative structure from each of the cluster based
on its CDOCK energy; (iv) ran a MD simulation for a period
of up to 10 ns; (v) estimated their free energy of binding using
the MM-GBSA method; (vi) selected the best pose based on the
MM-GBSA energy; and (vii) visually inspected and analysed
the structures from (vi). In this last analysis, the essential ligand–
receptor interactions were evaluated to define possible chemical
modifications to enhance binding towards the receptor. These
suggestions represented new candidates for synthesis. Following
synthesis, the compounds are experimentally assayed and the
theoretical models revised. This process (i)–(vi) was performed
twice resulting in two final lead compounds.
Fluoro-Jade B staining
At 2 days after intrathecal FGA-19- or vehicle-treated mice
were deeply anaesthetized with sodium pentobarbital (50 mg/kg
of body mass; intraperitoneal) and perfused intracardially with
4% paraformaldehyde in PBS. Spinal cords were removed and
paraffin-embedded. Spinal cord sections were deparaffinized, pre-
treated with 0.06% potassium permanganate and stained with
0.001% Fluoro-Jade B solution (Millipore Bioscience Research
Reagents) in 0.1% acetic acid to visualize the damaged neurons.
TNFα determination in tissue lysates
Spinal cords were homogenized in PBS and centrifuged at
13000 g for 15 min at 4◦C. The TNFα content in the supernatant
was quantified by ELISA (R&D systems).
Chemical synthesis
N-(5-chloro-2-methylphenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-
amine (FGA-19) was synthesized as follows: a solution of Nbd-Cl
(4-chloro-7-nitrobenzofurazan; 798.2 mg; 4 mM) and 5-chloro-
2-methylaniline (1.26 g; 8 mM) in N,N-dimethylformamide
(50 mM) was refluxed for 24 h. The reaction mixture was then
allowed to warm up to room temperature (25◦C), quenched with
a saturated aqueous sodium bicarbonate solution, extracted with
ethyl ether (3×30 ml), and the combined organic layers were
washed with a saturated aqueous sodium chloride solution, dried
(sodium sulfate), filtered and concentrated to give a crude oil
which was purified using chromatography over silica gel and
hexanes/ethyl acetate (7:3) as eluent. The desired compound
was obtained as a red oil (608 mg; yield = 50%), which was
crystallized [MeOH/H2O (1:1)] to afford pure compound FGA-19
as red crystals.
Compounds FGA-17, FGA-20, FGA-23, FGA-29, FGA-32
and FGA-34 were synthesized as follows: a solution of Nbd-
Cl (798.2 mg; 4 mM) and the corresponding aniline (8 mM) in
ethyl acetate (50 ml) was refluxed for 48 h. The reaction mixture
was then allowed to warm up to room temperature and worked up
and purified as described above. The yields and physical state of
each one of these final products are specified in Table 1.
Electrophoresis and Western blotting
Proteins were resolved by SDS/PAGE (7.5% gel), and
transferred on to nitrocellulose membranes for Western blotting
analysis. The antibodies used were: anti-p-p38 (Thr180/Tyr182
)
(catalogue number 9215), anti-p38 (catalogue number #9212),
anti-p-MAPKAPK2 (Thr334) (catalogue number 3041), anti-
MAPKAPK2 (catalogue number 3042), anti-p-HSP27 (catalogue
number 2405), anti-MEF2A (catalogue number 9736), anti-p-
ERK1/2 (catalogue number 9101; where ERK is extracellular-
signal-regulated kinase), anti-SAPK (stress-activated protein
kinase)/JNK (c-Jun N-terminal kinase) (catalogue number
9252) and anti-p-SAPK/JNK (Thr183/Tyr185) (catalogue number
9251) from Cell Signaling Technology; anti-p-MEF2A (Thr312
)
(catalogue number ab30644) from Abcam; and anti-ERK1 (C-
16) (catalogue number sc-93) and anti ERK2 (C-14) (catalogue
number sc-154) were mixed for blotting and purchased from Santa
Cruz Biotechnology.
In vitro inhibition of p38 MAPK activation and activity by FGA-19
Purified p38α (75 nM) was pre-incubated for 15 min at 30◦C
with FGA-19 at concentrations between 0.5 and 50 μM in buffer
[25 mM Tris/HCl (pH 7.5), 50 μM EGTA, 50 μM NaVO4 and
0.05% 2-mercaptoethanol]. Constitutively active MKK6 (10 nM;
Millipore), magnesium acetate (2.5 mM) and ATP (25 μM)
were added and a kinase reaction was performed for 15 min
at 30◦C. The reaction was terminated by adding Laemmli
buffer and proteins were resolved by SDS/PAGE (7.5% gel)
to detect p-p38 (pTGY) by Western blot analysis. In vitro
activity assays were performed using recombinant activated
p38α (1 nM; ProQinase), which was pre-incubated for 15 min
Animals
Female C57BL/6 mice (12–14 weeks old) and female mice with
a cell-specific reduction of GRK2 (G-protein-coupled-receptor
kinase 2) in LysM (lysozyme M)-positive macrophages (LysM-
GRK2+ / − mice) were used [32]. LysM-Cre mice and GRK2-fLox
mice were obtained from Jackson Laboratories to generate LysM-
GRK2+ / − and control LysM-GRK2+ / + mice [WT (wild-type)
littermates]. Peritoneal macrophages were collected from global
GRK2 hemizygous male mice [33] as described in [11]. All mice
were bred and maintained in the animal facility of the University
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at 30◦C with the small molecule in the same buffer. Full-length
GST (glutathione transferase)–MEF2A (10 nM; purified in our
laboratory), magnesium acetate and ATP were added and the
kinase reaction was allowed to proceed. p-MEF2A was detected
by Western blot analysis.
of p38 MAPK). For this purpose we used the space occupied by the
MEF2A peptide co-crystalized with p38 MAPK and the GAGA
algorithm to obtain a negative image of the binding site. Although
different X-ray structures were available to be used as the receptor
for the VS, we selected the one contained in the PDB code 1LEW
because of its high resolution, in particular in the area where
the docking was to be performed. Moreover, superimposition of
several structures (PDB codes 1LEW, 3P4K, 1P38, 2FSL, 2FSM,
2FSO and 2FST) showed very low RMSD values considering
equivalent Cα carbon atoms, supporting the use of a single rigid
receptor structure in the docking process.
Pull-down assay
To examine the interaction between p38 MAPK and its substrate
MAPKAPK2 in the presence or absence of FGA-19, full-
length mouse p38α–GST and full-length MAPKAPK2–His
were produced in bacteria and purified using the ProfiniaTM
Purification Protein system (Bio-Rad Laboratories) according
to the manufacturer’s protocols. p38–GST (150 nM) was pre-
incubated with 25 μM FGA-19 or vehicle (0.05% DMSO) in
binding buffer [25 mM Tris/HCl (pH 7.5), 0.25 M NaCl, 10 mM
MgCl2 and 5 mM NaF] at 30◦C for 30 min. Then 150 nM
MAPKAPK2–His was added for 15 min and the reaction was
incubated with glutathione–Sepharose 4B (GE Healthcare) at
30◦C for 15 min. Beads were washed twice with washing buffer
[25 mM Tris/HCl (pH 7.5), 0.25 M NaCl, 10 mM MgCl2, 5 mM
NaF and 0.5% Triton X-100]. GST–p38-bound proteins were
analysed by SDS/PAGE (7.5% gel) and Western blot analysis.
Upon characterizing the binding site, a library of around ×106
compounds was first screened using DOCK and the negative
image of the binding site. After applying a Z-Score cut-off value
of 2.5 on the DOCK ranked list, 45488 molecules passed on to the
next step. These molecules were then re-docked with CDOCK and
scored with its MM energy function, which explicitly included
solvent and hydrogen-bonding terms. The 200 highest-scoring
compounds were submitted to MD simulation in explicit solvent
and their binding energies estimated and pair-wise decomposed
by MM-GBSA calculation over a large collection of snapshots
homogeneously sampled along the trajectories.
Finally, from the top scoring compounds, and upon visual
examination, 18 candidates were selected, purchased and tested
experimentally. The most promising candidate, the highly
symmetrical compound 978604, was not commercially available.
In addition, and according to Lipinski’s RoF (rule of five) [35],
this compound presented a very high logP value [36] (∼8.00).
Therefore a similarity search was performed resulting in a new
compound, 5380, that was found to be structurally equivalent
to one half of the original one due to its symmetry. On the
one hand, 5380, due to its smaller size, is better suited for
chemical modifications, but, on the other hand, its logP value
was drastically reduced to within the RoF (5.00) criteria. This
compound was purchased, experimentally tested and submitted to
two optimization rounds. From the first optimization round a lead
candidate was obtained, FGA-19, with enhanced pharmacological
profile and logP value (4.35). Upon studying the binding mode
of FGA-19, a second optimization cycle led to the discovery of
a second lead compound, FGA-29, in which the methyl group
of FGA-19 was replaced by a hydroxy moiety, thus further
reducing its logP value (3.68).
Inhibition of TNFα secretion by FGA-19 and the structurally similar
compounds FGA-23 and FGA-29
THP-1 cells, growing in the exponential phase, were resuspended
in RPMI 1640 medium to a final concentration of 2×106 cells/ml
and distributed in 24-well plates. FGA molecules were added
to the wells to a final concentration ranging from 10 nM to
100 μM and plates were incubated at 37◦C and 5% CO2 in a
humidified atmosphere. Cells were stimulated 1 h later with LPS
to a final concentration of 0.5–2 μg/ml and incubated for 3 h
followed by centrifugation (2000 g for 10 min at 4◦C) to pellet
the cells. Supernatants were collected and stored at − 20◦C for
further analysis. TNFα secretion was measured by ELISA (GE
Healthcare) following the manufacturer’s instructions.
Cell culture and viability analysis
THP-1 human monocytic cells were maintained in suspension
in RPMI medium containing 10% FBS, 1 mM pyruvate, 2 mM
glutamine and antibiotics. The potential in vitro toxicity of FGA-
19 was quantified using PI (propidium iodide), which specifically
labels dying cells. Cells were resuspended in buffer (PBS, 1%
BSA, 1% FBS, 0.01% NaN3 and 1 μg/ml PI), kept on ice for 30–
60 min and then analysed by flow cytometry using a FACSCalibur
cytometer (Becton Dickinson). The data were analysed with Cell
Quest Pro Software (Becton Dickinson).
Owing to their structural similarities FGA-19 and FGA-29
display a common interaction pattern with Ile116, Leu122 and
Leu130, or His126, Pro129, Val158 and Cys161. In addition, the
interaction between Gln120 and FGA-29 (not present in FGA-19)
is of the same magnitude as the interaction between Gln133 and
FGA-19 (not present in FGA-29), counterbalancing each other.
Finally, there are some subtle differences in their binding modes
(Figure 1D). In particular, the interactions with Asn159 and Glu160
,
which are geometrically optimized in FGA-29 compared with
FGA-19, are mainly hydrophobic in nature and account for a total
energy difference of approximately 2 kcal/mol. However, this
magnitude is within the margin of error for these calculations and,
therefore, no firm conclusions can be drawn regarding possible
differences in their potential activities, which is in agreement with
the experimental observations.
Statistical analysis
+
All results are means S.E.M. Measurements were compared
using Student’s t test or one-way ANOVA followed by Tukey’s
post-hoc test, or two-way ANOVA with Bonferroni’s post-hoc
tests using Prism 5 software.
−
RESULTS
VS and identification of a lead compound
In vitro effects of FGA-19 on p38 MAPK activation and inhibition of
the p38 MAPK route in human monocytic cells
The VS protocol used in the present study is summarized in
Figures 1(A) and 1(B) and is briefly described in the Materials
and methods section. An essential part of the procedure comprises
characterization of the shape of the active site (the docking groove
We next set out to investigate the possible effects of the FGA-
19 compound on p38 MAPK in an in vitro kinase activity
assay using purified p38 MAPK-dependent phosphorylation of a
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Figure 2 Effects of FGA-19 on the activity of p38 MAPK in vitro and on the p38/MAPKAPK2/HSP27 signalling route
(A) Recombinant active p38 MAPK (1 nM) and MEF2A (10 nM) were incubated with different concentrations of FGA-19 in kinase buffer for 5 min. Proteins were resolved by SDS/PAGE and p38
MAPK activity was measured as phosphorylated MEF2A using a p-MEF2A (Thr312)-specific antibody. Average results are plotted as percentage phosphorylation S.E.M. relative to controls (DMSO)
+
−
of two independent experiments performed in duplicate. (B) Autoradiogram of a representative experiment. (C and D) THP-1 cells were pre-incubated with indicated doses of FGA-19 for 1 h and
stimulated with LPS for 30 or 60 min. Cell lysates were resolved by SDS/PAGE, the activation status of the p38 MAPK signalling route and the JNK and ERK1/2 pathways was detected with specific
+
antibodies, and films quantified by densitometric analysis. Average results of two independent experiments with duplicates are plotted in (C) as percentage phosphorylation S.E.M. relative to
−
cells with vehicle at the 60 min time point. Autoradiograms of a representative experiment are shown. (E) Effect of FGA-19 on p38 MAPK interaction with its substrate MAPKAPK2 (MK2). p38–GST
(150 nM) was pre-incubated with 25 μM FGA-19 or DMSO. Then 150 nM His–MAPKAPK2 and glutathione–Sepharose beads were added. Bound fusion proteins were detected by Western blotting
+
using specific antibodies. Results are means S.E.M. (n = 3). *P < 0.05.
−
well-characterized docking-dependent p38 MAPK substrate, the
MEF2A protein. When adding increasing doses of the FGA-19
molecule to the reaction mix containing the active kinase and
a saturating amount of substrate in the presence of ATP, we
observed a dose-dependent inhibition of p38 MAPK-mediated
phosphorylation of MEF2A at the specific site targeted by this
kinase (Thr312) (Figures 2A and 2B). Densitometric analysis of
the Western blotting data of MEF2A phosphorylation in the
presence of increasing amounts of FGA-19 in several experiments
revealed an estimated mean inhibitory concentration (IC50) of
phosphorylation at the activation loop by upstream kinases.
FGA-19 also inhibited the activity of p38 MAPK in THP-1
cells as measured by a decrease in the phosphorylation of the
downstream substrates MAPKAPK2 and HSP27. Quantification
of these inhibitory effects of FGA-19 revealed a ∼80% inhibition
for phosphorylation of p38 MAPK and ∼100% inhibition for
MAPKAPK2 phosphorylation at the 5 μM dose of FGA-19
(Figures 2C and 2D). Interestingly, no feedback activation effects
were detected at the 1 μM dose for the JNK or ERK1/2 pathways
(Figure 2D, lower panel) and, in fact, both kinases appear to be
inhibited to a certain extent by FGA-19 at the highest dose tested
(5 μM), although with a much lower efficacy compared with the
effect observed on the p38 MAPK pathway
+
6.31 2.32 μM. An almost complete inhibition of p38 MAPK
−
activity towards MEF2A was observed at the 50 μM dose
(Figures 2A and 2B).
Next we analysed the effects of the FGA-19 compound
on the p38 MAPK signalling route in the human monocytic
cell line THP-1. As shown in Figures 2(C) and 2(D), in the
presence of FGA-19 we detected clear inhibition of p38 MAPK
Interestingly, as shown in Figure 2(E), FGA-19 was capable
of impairing the in vitro interaction between p38 MAPK and
the docking-dependent substrate MAPKAPK2, which was almost
absent from p38 MAPK sediments in the presence of FGA-19,
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whereas it was clearly detected in the vehicle (DMSO) control
at position 5 and a methyl group at position 2 are the most effective
chemical groups among those tested so far.
samples. These results suggest that competitive inhibition towards
docking-dependent partners is a probable mechanism for the
actions of FGA-19 in agreement with the kinetic constants of
the phosphorylation of MEF2 by constitutively active p38 MAPK
in the presence or absence of FGA-19 (Supplementary Table S1
at http://www.biochemj.org/bj/459/bj4590427add.htm)
In vivo effect of FGA-19 on inflammatory hyperalgesia
To determine the in vivo efficacy of FGA-19, we examined
its effect in the high-dose carrageenan model of persistent
inflammatory hyperalgesia in C57BL/6 mice. In response to
intraplantar injection of 20 μl of 2% carrageenan, mice developed
thermal hyperalgesia that lasted at least 11 days (Figure 4A).
We injected increasing doses of FGA-19 intrathecally at day 6
after intraplantar carrageenan. The data show that hyperalgesia
completely resolved in response to a single injection of 1 μg of
FGA-19. A lower dose of 0.5 μg of FGA-19 transiently inhibited
carrageenan-induced hyperalgesia (Figure 4A). For comparison,
we used the established p38 MAPK inhibitor SB239063. At the
maximal dose that could be injected using 20% DMSO as a
solvent, i.e. 5 μg of SB239063, carrageenan-induced hyperalgesia
was only transiently attenuated (Figure 4A). Thermal sensitivity
was not affected by FGA-19 in the control saline-treated mice
(Figure 4B). The structurally similar FGA-23 compound, that
in vitro has an IC50 value of >100 μM, did not have any effect
on the course of carrageenan-induced hyperalgesia (Figure 4B).
To determine whether the inhibition of hyperalgesia by FGA-
19 was associated with cellular damage in the spinal cord,
we analysed the effect of FGA-19 on staining with Fluoro-
Jade B, a widely used marker of neurodegeneration. We did
not observe an increase in Fluoro-Jade B-positive cells in
the spinal cord at 48 h after injection of 1 μg of FGA-19,
indicating that there was no effect of this dose of FGA-19 on
neuronal cell death (Figure 4C). Our cell culture experiments
showed that FGA-19 inhibits LPS-induced TNFα production
(Figure 2C), and spinal cord production of pro-inflammatory
cytokines such as TNFα is known to contribute to the persistent
hyperalgesia in the carrageenan model of inflammatory pain
[38,39]. To investigate whether in vivo FGA-19 treatment affects
TNFα levels, we injected carrageenan or saline intraplantarly,
followed by intrathecal injection of FGA-19 or vehicle on day
6, and then isolated the lumbar spinal cord 2 days later for
analysis of TNFα levels by ELISA. As anticipated, intraplantar
injection of carrageenan caused a significant increase in
spinal cord TNFα (Figure 4D). Intrathecal FGA-19 treatment
significantly inhibited this carrageenan-induced rise in spinal
cord TNFα levels. FGA-19 alone did not have any effect on
TNFα production in the spinal cord (Figure 4D). At 6 days after
intrathecal FGA-19 treatment the mice still showed increased
paw thickness and redness, indicating that intrathecal FGA-19
treatment did not directly affect peripheral inflammatory activity
(Figure 4E).
A broad specificity analysis was performed using a commer-
cially available kinase screen (MRC-PPU Express Screen; Inter-
national Centre for Kinase Profiling, University of Dundee, Dun-
dee, U.K.). As shown in Supplementary Table S2 (http://www.
biochemj.org/bj/459/bj4590427add.htm), FGA-19, at a dose of
10 μM, has little or no effect on a variety of kinases representative
of the different groups comprising the human kinome. In
agreement with its docking-groove-targeting design, FGA-19 had
no inhibitory activity on p38α in this screening, since this assay
is performed using MBP (myelin basic protein) as a substrate, a
protein that apparently lacks a functional docking site [37].
The effects of FGA-19 were then analysed in the well-
established cellular system of cytokine secretion by THP-1
cells stimulated with LPS [4]. Secretion of TNFα by these
cells is p38 MAPK-dependent, and thus this system has been
broadly utilized to study the effects of different p38 MAPK
inhibitors. As shown in Figure 2(C), FGA-19 has a dose-
dependent inhibitory effect on TNFα secretion in this cell system
+
with an IC50 of 1.8 0.006 μM reaching an almost maximal
−
inhibition at the 10 μM concentration. For comparison, the
IC50 value for SB203580 (a commercially available p38 MAPK
inhibitor targeting the ATP-binding site) in the same system was
1 μM (results not shown), and the effect of SB203580 at the
10 μM dose was similar to that obtained for FGA-19. Of note,
we did not detect significant cell death, as determined by FACS
analysis of PI-stained cells, at concentrations up to 100 μM FGA-
19, indicating that there was no cellular toxicity (Figure 3A).
Identification of the structural determinants of the inhibitory effect
Comparative analysis of FGA-19 and several structurally related
compounds, all belonging to the benzo-oxadiazol family, allowed
us to conclude that the substituents in positions 2 and 5 of the
phenyl ring are essential for the inhibitory activity towards p38
MAPK. When the substituents in positions 2 and 5 present in the
FGA-19 molecule (a chloride and a methyl group respectively)
are located in other positions of the phenyl ring (as in the FGA-17,
FGA-20 and FGA-23 compounds), the IC50 value for inhibition of
monocyte TNFα secretion decreased by at least one logarithmic
unit (Figures 3B and 3C, and Table 2). This was the case for Cl and
CH3 located in positions 4 and 5 (FGA-17), or 4 and 6 (FGA-20),
with the IC50 value changing from 1.8 μM to 17 μM and 28 μM
respectively, or for OH and CH3 at positions 2 and 4 (FGA-23;
IC50 >100 μM). The presence of a single substituent at different
positions of the ring also resulted in an IC50 value >100 μM in
at least three other compounds (results not shown). On the other
hand, when other substituents are placed in the same positions of
the ring where the chloride and the methyl group are located in the
FGA-19 molecule, an IC50 value similar to that obtained for FGA-
19 was detected. This is the case for the substitution at positions
2 and 5 by OH and Cl (in FGA-29), by Cl and OCH3 (in FGA-
34), and by F (FGA-32). These three compounds yielded IC50
values in the low micromolar range (3.5 μM, 3.2 μM and 2.8 μM
respectively), which is comparable with the IC50 value of FGA-19
(1.8 μM) (Table 2). Taken together, these results establish that a
substituent at positions 2 and 5 in the phenyl ring is essential for
the described inhibitory activity and, furthermore, that a chloride
Since p38 MAPK can be phosphorylated by GRK2 at the
docking groove [11], it was tempting to hypothesize that cells
with decreased levels of GRK2 and lower amounts of docking-
site-phosphorylated p38 MAPK protein would display differential
susceptibility to inhibition via docking-targeted compounds. For
instance, inflammatory cells from patients with autoimmune
diseases or murine models of these diseases contain decreased
levels of GRK2 [40], and this is recapitulated in hemizygous
GRK2 macrophages [11]. We thus set out to investigate whether
TNFα secretion by macrophages isolated from GRK2+ / − mice
was differentially inhibited by FGA-19 when compared with
macrophages from WT mice. Interestingly macrophages collected
from GRK2+ / − mice were found to be more sensitive to FGA-
19-dependent blockade of TNFα secretion than WT macrophages
(Figure 5A).
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Figure 3 Inhibition of TNFα secretion by FGA-19, FGA-23 and FGA-29
(A) THP-1 cells were pre-incubated with the indicated doses of FGA-19 for 1 h and stimulated with LPS for 3 h. Cells were stained with PI and surviving cells were counted with a FACSCalibur
cytometer. TNFα secretion in cell supernatants was quantified using a TNFα ELISA kit and referred to the control (DMSO). Results are average of three independent experiments each in duplicate. (B)
THP-1 cells growing in the exponential phase were pre-incubated for 1 h with indicated doses of the different compounds or with 10 μM SB203580 (SB) as a control, and then stimulated with LPS
+
for an additional 3 h to analyse the supernatants for TNFα secretion by ELISA. Results are means S.E.M. for two independent experiments performed in duplicate and are referred to the controls
with DMSO (vehicle). (C) FGA-19 and structurally-related compounds.
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Table 2 Comparative analysis of the effects on TNFα secretion of FGA-19
and several structurally related compounds belonging to the benzo-oxadiazol
family
in response to an intraplantar injection of a low dose of carr-
ageenan (5 μl of a 1% solution) [11]. In this model of transient
hyperalgesia, the pain response in WT mice resolves within
2 days [38]. The LysM-GRK2+ / − mice can be considered as a
model of persistent post-inflammatory hyperalgesia as we do not
observe ongoing peripheral inflammation (no redness or thick
paws and no detectable increase in inflammatory cytokines in
comparison with control paws [32]). As shown in Figure 5(B),
intrathecal administration of FGA-19 also completely attenuated
hyperalgesia in this model of post-inflammatory hyperalgesia.
The lowest effective dose providing a long-lasting analgesic
effect was 0.5 μg of FGA-19, which is lower than the 1 μg
dose found in the high-dose carrageenan model in WT mice
(Figure 4A).
THP-1 cells were pre-incubated with the indicated doses of several benzo-oxidiazol-derived
compounds for 1 h and stimulated with LPS for 3 h. TNFα secretion in cell supernatants was
quantified using a TNFα ELISA kit and referred to the control (DMSO). Values were used to
graphically approximate an IC50 value in micromolar units. When the substituents in positions
2 and 5 present in the FGA-19 molecule (namely a chloride and a methyl group) are located
in other positions of the phenyl ring, the IC50 value for the inhibition of TNFα secretion by
monocytic cells decreases.
Compound
2D molecular structure
IC50 (μM)
FGA-17
17
DISCUSSION
The search for clinically applicable inhibitors of p38 MAPK
has rendered, so far, no fruitful therapeutic results. The reasons
underlying this lack of success include low efficacy in some
cases and the identification of certain toxicities during clinical
trials that were not uncovered in surrogate animal models of the
different diseases tested [41]. These toxicities can have several
aetiologies. On the one hand, first and second generation p38
MAPK inhibitors were designed to target the ATP-binding pocket
of p38 MAPK, thus exploiting a common domain found in many
different kinases, probably triggering off-target effects [1]. On
the other hand, even when new allosteric site inhibitors were
developed to overcome these problems, it became apparent that
a strong down-regulation of the p38 MAPK pathway can trigger
the up-regulation of other signalling pathways by virtue of the
concomitant stimulation of interconnecting feedback mechanisms
(see below). These final undesired consequences were apparently
underestimated [3]. Lastly, the systemic administration of p38
MAPK inhibitors has been proven to induce toxicities in organs
other than those important for the disease they were meant to
combat, including the liver and brain [41]. For all these reasons,
in the present study we set out to characterize a novel family
of inhibitors that take advantage of the existence of docking
domains in the p38 MAPK protein providing the possibility of
modulating the activity and activation of this kinase independently
of its intrinsic catalytic activity in a more subtle, but possibly
more efficacious, manner. Our strategy relied on our previous
results showing that p38 MAPK can be phosphorylated at the
docking domain, specifically at Thr123 of murine p38α. This
phosphorylation of Thr123 causes the loss of interaction with
different p38-binding partners and reduced activation and activity
of p38 MAPK [11]. On this basis, we performed a VS of a
chemical library of compounds over a model based on pre-existing
structures of p38 MAPK bound to peptides derived from upstream
regulators or downstream substrates [10]. This search yielded
the identification of compounds of different chemical nature that
were analysed for their in vitro inhibitory capacity towards p38
MAPK activity or towards cytokine secretion induced by LPS in a
human monocytic cell line. Only some molecules were inhibiting
these events with IC50 values in the micromolar range. Of these,
the family of benzo-oxadiazol-based compounds described in the
present study, with FGA-19 as a lead candidate, was found to
have a biological effect both in cells and in two animal models of
hyperalgesia. The fact that FGA-19 contains several putative sites
susceptible for chemical modifications highlights this molecule
as a good lead compound for further drug development strategies.
In vitro experiments support the notion that the biochemical
mechanism of action of FGA-19 involves its ability to interfere
FGA-32
FGA-20
2.8
28
FGA-23
FGA-19
>100
1.8
FGA-29
FGA-34
3.5
3.2
The next question we addressed is whether FGA-19 treatment
also reverts the persistent hyperalgesia that develops in mice
with a targeted deletion of GRK2 in LysM-positive macrophages/
monocytes. We have described previously that LysM-GRK2+ / −
mice develop persistent hyperalgesia lasting at least 20 days
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Figure 4 Effect of FGA-19 treatment on high-dose carrageenan-induced persistent hyperalgesia in C57BL/6 mice
Mice received an intraplantar injection of 20 μl of 2% λ-carrageenan or saline. At day 6, carrageenan-treated mice were still hyperalgesic. At this time point, mice received an intrathecal (i.t.)
injection of (A) different doses of FGA-19 or (B) 1 μg of the inactive compound FGA-23, and the percentage change in heat withdrawal latency was determined (n = 4–8 per group) *P < 0.05 and
***P < 0.001 for 0.5 μg of FGA-19 compared with the vehicle and #P < 0.05 and ###P < 0.001 for 5 μg of SB239063 compared with the vehicle. At 2 days after intrathecal injection, lumbar
spinal cord was collected and (C) sections were stained for Fluoro-Jade B to investigate the potential neurotoxicity of FGA-19 and (D) TNFα levels were determined by ELISA (n = 4) *P < 0.05. (E)
+
As a measure for paw inflammation, the effect of FGA-19 treatment on the paw thickness was determined (n = 8). ***P < 0.001. Results are means S.E.M.
−
with binding of docking-dependent substrates to p38 MAPK, as
indicated by kinetic competition experiments and the inhibition
of the pull down of purified MAPKAPK2 by p38 MAPK.
Accordingly, FGA-19 does not efficiently inhibit the activity of
p38 towards MBP, a substrate that does not rely on a functional
docking site for its phosphorylation by members of the p38
MAPK family [37]. Moreover, 10 μM FGA-19 had little or no
effect on the activity of a variety of kinases representative of the
human kinome, supporting the notion that by targeting the docking
groove of p38, FGA-19 displays less cross-inhibition with other
kinases and thus increased specificity. However, the possibility
exists that FGA-19 might interfere with the phosphorylation of
specific docking-dependent substrates of other members of the
p38 MAPK family, or of the related MAPK family members
JNK1 or ERK1/2, and this will be the subject of future research.
The strategy utilized to identify FGA-19 aimed at interfering
with docking interactions between p38 MAPK and substrates or
activators of p38 MAPK [11]. This particular characteristic was
utilized as it could in principle yield a different mode of action
leading to a less abrupt blockade of the pathway flow and thus,
at least theoretically, less toxic effects. For instance, one of the
off-target effects described for classical p38 MAPK inhibitors
is the hyperactivation of the JNK cascade. This is thought to
happen because strong inhibition of p38 MAPK can abrogate
some negative-feedback loops that p38 MAPK maintains towards
upstream kinases such as MLK2/3 (mixed lineage kinase 2/3) or
TAK1 (transforming growth factor-β-activated kinase 1), which
also function upstream of JNK [42]. Thus abolition of the negative
feedback control exerted by the p38 MAPK branch redirects the
pathway flow towards a higher activation of the JNK pathway.
However, we did not detect increased activation of JNK in cells
in the presence of FGA-19. If anything, the JNK pathway was
inhibited, but to a lesser extent than the p38 MAPK route by
FGA-19. These findings suggest that this type of unwanted effect
may not occur for docking-based inhibitors or for inhibitors that
do not fully ablate p38 MAPK catalytic activity. The possibility
that the more refined and less potent inhibition of p38 MAPK,
such as that obtained by FGA-19, can prevent other undesired side
effects such as activation of cell death routes certainly deserves
further investigation.
The in vivo effects of FGA-19 surpass those detected for other
well-established p38 MAPK inhibitors in our mouse models of
inflammatory pain, both in potency and in persistence of the
pharmacological effect. Of note, the FGA-19 molecule is almost
10-fold as potent as the SB239063 inhibitor, since a 0.5 μg dose
of FGA-19 exerts the same transient analgesic effect as 5 μg
of SB239063 despite both molecules having similar molecular
masses (368.40 g/mol for SB239063 and 304.45 g/mol for
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Figure 5 Effect of FGA-19 treatment in GRK2+ / − macrophages and on low dose carrageenan-induced persistent hyperalgesia in LysM-GRK2+ / − mice
(A) GRK2+ / − peritoneal macrophages were stimulated with LPS in the presence of increasing FGA-19 doses and TNFα secretion was measured as described above. The inhibition observed in
GRK2+ / − macrophages was larger than that obtained for WT cells for all FGA-19 doses. Results are means S.E.M. for two independent experiments. *P < 0.01 (Student’s t test), ***P < 0.001
+
−
(Student’s t test). (B) LysM-GRK2+ / − mice received a low dose of carrageenan (5 μl of 1% λ-carrageenan) and at day 6 mice were still hyperalgesic. At this time point, the mice received an
+
intrathecal (i.t.) injection with different doses of FGA-19 and the percentage change in heat withdrawal latency was determined (n = 4). Results are means S.E.M.
−
FGA-19). Moreover, longer-lasting effects are detected in
mice treated intrathecally with FGA-19 at the 1 μg dose,
which abrogates inflammatory hyperalgesia for at least 5 days
post-injection. For comparison, the pharmacological effect of
SB239063 at the highest injectable dose did not last longer
than 6 h. These results suggest that a very promising family of
molecules could be derived from the benzo-oxadiazol structure to
treat chronic inflammatory pain. Interestingly, recent studies have
shown that mice with a reduced level of GRK2 in macrophages
develop chronic hyperalgesia in response to inflammatory
mediators that induce only transient hyperalgesia in WT mice
(for a review, see [43]). We therefore used these mice to test
whether FGA-19 also inhibits the hyperalgesia in this model of
post-inflammatory pain. We show that the persistent hyperalgesia
that develops in LysM-GRK2+ / − mice is completely reversed by
a single intrathecal injection of FGA-19. Moreover, macrophages
obtained from GRK2+ / − mice are more sensitive to FGA-
19-dependent blockade of TNFα secretion. These findings are
particularly interesting because decreased levels of GRK2 are
found in cells from patients with various inflammatory and
autoimmune diseases [34], and it is tempting to suggest that our
docking-groove-targeting inhibitors would be especially suited
for treatment of inflammation and pain in these conditions.
Collectively, our findings indicate that FGA-19 is a potential
candidate for the treatment of inflammatory hyperalgesia.
Clinical trials performed for p38 MAPK in the context of
chronic inflammation have been unsuccessful so far due to both
lack of efficacy and toxic effects [41]. However, although the
importance of p38 MAPK in regulating nociception is well-
established in mice and in humans [43], few studies have been
conducted so far to assess the efficacy of p38 MAPK inhibition in
pain therapeutics. In particular, different laboratories have shown
in rodent models that phosphorylated p38 MAPK is increased in
spinal cord microglia after nerve ligation or spinal cord injury,
which are two widely used models of chronic neuropathic pain.
More importantly, intrathecal infusion of p38 MAPK inhibitors
attenuates neuropathic pain in animal models (see references in
[44]). A small study has described a potent analgesic effect of
p38 MAPK inhibition for post-surgical dental pain in humans
[45] and several recent studies have described the contribution
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8
9
Binshtok, A. M., Wang, H., Zimmermann, K., Amaya, F., Vardeh, D., Shi, L., Brenner, G. J.,
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of p38 MAPK in animal models of neuropathic post-surgical
and cancer pain [46]. It is also well-established that increased
levels of phosphorylated p38 MAPK are detected in spinal cord
microglia in animal models of chronic inflammatory pain (see
references in [43]). In agreement with these published studies, the
results of the present study indicate that inhibition of spinal cord
p38 MAPK by intrathecal administration of specific inhibitors
efficiently reverses ongoing hyperalgesia ([47] and the present
study). However, the effect observed using these classic p38
MAPK inhibitors was much more transient than that observed
using the FGA-19 inhibitor, which at a 1 μg dose shows an effect
that lasted the 5-day duration of our experimental setup.
In conclusion, we present evidence that a novel family of
small molecules targeting the docking groove of p38 MAPK,
the benzo-oxadiazol-based compounds, are potent inhibitors of
cytokine production in human monocytic cells. In addition, we
show that a member of this family, the FGA-19 molecule, inhibits
inflammatory and post-inflammatory pain in murine models. We
suggest that this novel compound can represent the basis for
further research on similar compounds for their specificity and
potency in these models and, ultimately, for translation into a
clinical setting.
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AUTHOR CONTRIBUTION
Hanneke Willemen, Pedro Campos, Elisa Lucas and Paula Ramos performed experiments;
Juan Agut and Florenci Gonza´lez designed the synthesis protocols and synthesized the
compounds;AntonioMorrealeandRube´nGil-Redondodesignedandperformedthevirtual
screening, analysed the results and wrote the paper; Cobi Heijnen, Federico Mayor Jr,
Annemieke Kavelaars and Cristina Murga designed the experiments, analysed the results
and wrote and revised the paper.
22 Stewart, J. J. (1990) MOPAC: a semiempirical molecular orbital program. J. Comput.
Aided Mol. Des. 4, 1–105
23 Klett, J., Corte´s-Cabrera, A., Gil-Redondo, R., Gago, F. and Morreale, A. (2014) ALFA:
automatic ligand flexibility assignment. J. Chem. Inf. Model. 54, 314–323
24 Morreale, A., Gil-Redondo, R. and Ortiz, A. R. (2007) A new implicit solvent model for
protein–ligand docking. Proteins 67, 606–616
25 Kollman, P. A., Massova, I., Reyes, C., Kuhn, B., Huo, S., Chong, L., Lee, M., Lee, T.,
Duan, Y., Wang, W. et al. (2000) Calculating structures and free energies of complex
molecules: combining molecular mechanics and continuum models. Acc. Chem. Res. 33,
889–897
26 Ryckaert, J.-P., Ciccotti, G. and Berendsen, H. J. C. (1977) Numerical integration of the
cartesian equations of motion of a system with constraints: molecular dynamics of
n-alkanes. J. Comput. Phys. 23, 327–341
FUNDING
ThisworkwassupportedbytheMinisteriodeEducacio´nyCiencia[grantnumberSAF2011-
23800], the Cardiovascular Network (RECAVA) of Ministerio Sanidad y Consumo-Instituto
Carlos III [grant number RD12/0042/0012], the Comunidad de Madrid [grant number
S2010/BMD-2332 (to F.M.)], the Instituto Carlos III [grant number PS09/01208 (to
C.M.)], UAM-Grupo Santander, the Comunidad Auto´noma de Madrid [grant numbers
S-BIO-0214-2006-BIPEDD and S2010-BMD-2457-BIPEDD2 (to A.M.)] and the National
Institutes of Health [grant numbers RO1 NS074999 and RO1 NS073939 (to A.K.)]. A.M.
acknowledges financial support from Fundacio´n Severo Ochoa through the AMAROUTO
program. The work of A.K. is supported by the STARS award from the University of Texas
System.
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Received 1 February 2013/4 February 2014; accepted 11 February 2014
Published as BJ Immediate Publication 11 February 2014, doi:10.1042/BJ20130172
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Biochem. J. (2014) 459, 427–439 (Printed in Great Britain) doi:10.1042/BJ20130172
SUPPLEMENTARY ONLINE DATA
A novel p38 MAPK docking-groove-targeted compound is a potent inhibitor
of inflammatory hyperalgesia
3
4
Hanneke L. D. M. WILLEMEN*1, Pedro M. CAMPOS†1,2, Elisa LUCAS†‡, Antonio MORREALE§ , Rube´n GIL-REDONDO§ ,
5
´
Juan AGUT¶, Florenci V. GONZALEZ¶, Paula RAMOS†‡, Cobi HEIJNEN*ꢀ, Federico MAYOR, Jr†‡, Annemieke KAVELAARS*ꢀ and
5
Cristina MURGA†‡
*Laboratory of Neuroimmunology and Developmental Origins of Disease, University Medical Center Utrecht, Utrecht, The Netherlands
†Departamento de Biolog´ıa Molecular and Centro de Biolog´ıa Molecular “Severo Ochoa” UAM-CSIC, Madrid, Spain
‡Instituto de Investigacio´n Sanitaria La Princesa, Madrid, Spain
§Centro de Biolog´ıa Molecular “Severo Ochoa”, UAM-CSIC, Madrid, Spain
¶Departament de Qu´ımica Inorga`nica i Orga`nica, Universitat Jaume I, Castello´, Spain
ꢀDepartment of Symptom Research, University of Texas MD Anderson Cancer Center, Houston, TX 77030, U.S.A.
Table S1 Competition assay: effect of increasing concentrations of
substrate over FGA-19 inhibition efficacy
Usingconstitutivelyactivep38MAPK,inthepresenceorabsenceof2 μMFGA-19,theinhibitory
activity of FGA-19 was analysed over increasing concentrations of its substrate MEF2a (100,
MEF2a
200, 400 and 800 nM) and quantified by Western blotting. Vmax and Km
calculated using the graphical representation of the Lineweaver–Burk plot.
values were
Parameter
DMSO
FGA-19
Vmax (pmol/min)
KmMEF2a (nM)
0.933
70.09
1.162
650.15
1
These authors contributed equally to this work.
Present address: Allinky Biopharma, Parque Cient´ıfico de Madrid, Madrid, Spain.
Present address: Repsol, Technology Center, Ctra. de Extremadura, A-5, km 18, 28935 Mo´stoles, Madrid, Spain.
Present address: SmartLigs Bioinforma´tica S.L., Fundacio´n Parque Cient´ıfico de Madrid, Campus de Cantoblanco UAM, Madrid, Spain.
Correspondence may be addressed to either of these authors (email akavelaars@mdanderson.org or cmurga@cbm.uam.es).
2
3
4
5
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The Authors Journal compilation 2014 Biochemical Society

H. L. D. M. Willemen and others
Table S2 The effect of FGA-19 at a dose of 10 μM was assessed in a kinase screen (MRC-PPU Express Screen; International Centre for Kinase Profiling)
+
Results are the percentage of remaining activity S.D. with little or no inhibitory effect on a variety of kinases representative of the different groups comprised in the human kinome (http://www.
−
kinase-screen.mrc.ac.uk/kinase-screen-express). Of note, FGA-19 had no inhibitory activity on p38α, since this assay is performed using a substrate that appears not to have a functional docking
site [1].
Kinase
Remaining activity (%)
Kinase
Remaining activity (%)
+
+
PDK1
PKBa
SGK1
S6K1
PKA
ROCK 2
PRK2
PKCa
PKD1
MSK1
CAMKKb
CAMK1
SmMLCK
CHK2
RSK1
PLK1
Aurora B
LKB1
AMPK (human)
MARK3
CK1δ
104 10
105 16
NEK6
TBK1
PIM1
SRPK1
EF2K
HIPK2
PAK4
MST2
MLK3
TAK1
IRAK4
RIPK2
TTK
Src
Lck
BTK
JAK2
SYK
EPH-A2
HER4
IGF-1R
TrkA
p38α MAPK
p38α MAPK (25μM [ATP])
p38β MAPK
p38γ MAPK
p38δ MAPK
101
95
74
99
4
5
1
1
9
−
−
+
−
+
−
+
−
+
−
+
+
−
+
99
97
2
5
−
+
−
+
113 11
94
−
+
102 12
119 13
−
−
+
+
99 10
78
98
100
95
9
1
2
9
6
−
+
−
+
−
+
−
+
−
+
−
+
100
81
97
7
8
0
−
+
−
+
−
+
94 11
97
−
+
97
88
92
3
1
1
108 12
−
+
−
+
−
+
−
+
94
74
93
100
94
106
101
9
5
5
8
0
1
2
−
+
−
+
−
+
−
+
−
+
−
+
−
+
86 13
−
+
97
100
99
9
1
6
2
−
+
−
+
−
+
−
+
101
104 14
92 12
−
−
+
+
97 16
116 19
−
+
−
+
CK2
95
0
89 22
−
−
+
+
DYRK1A
MKK1
JNK1
VEG-FR
GSK3b
92 10
91
91
104
94
1
3
6
1
3
−
−
+
−
+
−
+
−
+
−
+
83
104
98
1
9
3
6
−
+
−
+
−
+
−
102
93
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466–479
Received 1 February 2013/4 February 2014; accepted 11 February 2014
Published as BJ Immediate Publication 11 February 2014, doi:10.1042/BJ20130172
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The Authors Journal compilation 2014 Biochemical Society