Discovery and hit-to-lead optimization of non-ATP competitive MK2 (MAPKAPK2) inhibitors

Article abstract of DOI:10.1021/ml200113y

A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-throughput screening using the affinity selection-mass spectrometry-based Automated Ligand Identification System platform. Medicinal

Full text of DOI:10.1021/ml200113y

LETTER
pubs.acs.org/acsmedchemlett
Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2
(MAPKAPK2) Inhibitors
Xiaohua Huang,*,† Gerald W. Shipps, Jr.,† Cliff C. Cheng, Peter Spacciapoli,† Xingmin Zhang,
Mark A. McCoy,‡ Daniel F. Wyss,‡ Xianshu Yang,† Abdelghani Achab,† Kyle Soucy, Donna K. Montavon,
Denise M. Murphy, and Charles E. Whitehurst*,†
Merck Research Laboratories, 320 Bent Street, Cambridge, Massachusetts 02141, United States
S Supporting Information
b
ABSTRACT:
A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-
throughput screening using the affinity selectionÀmass spectrometry-based Automated Ligand Identification System platform.
Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical
and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and
biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective
inhibitors of MK2 should be feasible by targeting the outside ATP pocket.
KEYWORDS: Mitogen-activated protein kinase-activated protein kinase 2, non-ATP-competitive inhibitors, Automated Ligand
Identification System (ALIS), saturation-transfer-difference (STD) NMR, 1H/15N-HSQC (heteronuclear single quantum coherence)
nregulated protein kinase activity is often associated with in-
flammatory diseases, such as rheumatoid arthritis, which is
required for lipopolysaccharide (LPS)-induced tumor necrosis
factor R (TNFR) and interleukin 6 (IL6) production.11À13
Furthermore, MK2-null mice were shown to have reduced joint
damage in a collagen-induced arthritis model and no induction of
asthma in a lung ovalbumin sensitization model.14,15 In contrast
to the embryonic lethality associated with knocking out the gene-
encoding p38R MAPK, MK2-null mice were fertile and healthy,
suggesting that inhibition of MK2 in inflammatory disease states
may provide efficacy comparable to direct p38R MAPK inhibition
but without debilitating side effects.
Over the past few years, many small molecule MK2 inhibitors,
either extracted from natural products16 or developed from
various research laboratories,17À21 were reported. In particular,
the ATP-competitive MK2 inhibitor from Pfizer, PF-3644022,
showed efficacy in different cell and rodent models.22 From their
study, Pfizer forecasted difficulties trying to use an ATP-compe-
titive approach to inhibiting MK2 in humans due to poor
biochemical efficiency (BE, ratio of binding affinity to target vs
cellular activity). In light of the high cellular concentrations of
ATP (ca. 2À5 mM) and affinity of ATP for P38R-activated MK2
(ATP Km = 2 μM), it was suggested that the BE increase in MK2
U
characterized by autoimmune-mediated cartilage and bone de-
struction that leads to intolerable joint pain and crippling and
afflicts about 1% of the world's population.1 Activation of the
p38/mitogen-activated protein kinase-activated protein kinase 2
(MAPKAPK2 or MK2) pathway has been implicated in promot-
ing pro-inflammatory cytokine production and related inflam-
matory diseases.2À4 However, despite numerous assessments of
potent inhibitors in early stage clinical trials, no p38 MAPK
inhibitors have progressed beyond phase II trials largely because
of dose-dependent toxicities and unimpressive efficacies.4,5 The
broad involvement of p38R MAPK and its downstream sub-
strates in diverse cellular processes may be a major cause of related
clinical trial failures.4,6,7 To minimize the apparent side effects
caused by directly targeting p38R MAPK, several discovery teams
have attempted inhibiting targets downstream of p38R MAPK
that are responsible for its pro-inflammatory cytokine-regulating
properties in macrophages.4,7 Of these MK2, a serine/threonine
protein kinase that directly associates with p38R MAPK to form a
heterodimer and is phosphorylated and activated by p38R MAPK
has emerged as the most attractive downstream target.7À10 For
example, in studies employing genetically engineered mouse
models wherein MK2, MK3, and MK5 genes were knocked out,
it was shown that MK2 is the primary MAPKAPK family member
Received: May 11, 2011
Accepted: June 24, 2011
Published: June 24, 2011
r
2011 American Chemical Society
632
dx.doi.org/10.1021/ml200113y ACS Med. Chem. Lett. 2011, 2, 632–637
|

ACS Medicinal Chemistry Letters
LETTER
inhibitors could be achieved through a different binding mode.
A recent report presented a class of MK2 inhibitors with a mainly
uncompetitive binding mechanism.23
small molecule ligands with novel binding modes from the
screening libraries. Compound 1 emerged as the leading hit
from the ALIS screening of a mixture-based combinatorial library
with the desired profile in biochemical and cell-based potency,
kinase selectivity (see the Supporting Information for kinase
abbreviations), DMPK (drug metabolism and pharmacokinetics),
and toxicity window (Figure 1). Compound 1 showed essentially
no potency shift when tested by enzyme assay at low versus high
ATP concentrations (2 and 100 μM).
To further characterize the binding mode, saturation-transfer-
difference (STD) NMR studies using ATP as an active site
marker were conducted to test the binding of 1 to MK2.30 In
these experiments, a 2 h reference data set was first acquired
using 2.5 μM MK2, 225 μM ATP, and 5 mM MgCl2. The
addition of 50 μM 1 resulted in a strong set of peaks for 1 but had
no effect on the intensity of ATP STD NMR peaks (Figure 2A,B).
These experiments demonstrated non-ATP competitive binding
of 1 to MK2. 1H/15N-heteronuclear single quantum coherence
(HSQC) studies were carried out to ensure that the observed
binding was site-specific. Comparison of 1H/15N-HSQC spectra
collected on a sample of 50 μM MK2 with and without 1
(Figure 2C) showed that 1 binds site specifically and affects
different residues than those observed for ATP binding. These
NMR data provided compelling evidence that the binding of 1 to
MK2 was site-specific and non-ATP competitive.
Determination of the mode of inhibition of MK2 by com-
pound 1 with respect to peptide substrate, Acam peptide (see the
Supporting Information), was made by analysis of MK2 activity
in the presence of saturating ATP and varying concentrations of
peptide substrate and the inhibitor (Figure 3). The data obtained
were globally fit to various kinetic models of inhibition with
goodness of fit evaluated by use of the Akaike information criteria
(AICc), which permits direct comparison of models of inhibition
of varying complexity.31 The results identified a noncompetitive
inhibition model as the most likely mechanism. Mixed inhibition,
wherein inhibitor 1 binds preferentially to the peptide substrate-
free form of MK2, is 2-fold less likely. In contrast, competitive
and uncompetitive models of inhibition were, respectively, 105
and 1011 times less likely.
In general, for kinases with relatively high ATP binding
affinities (Km = 1À20 μM), it has been difficult to develop
desirable ATP competitive inhibitors because of problems asso-
ciated with attaining sufficient general selectivity and cellular
activity in addition to required novel patent space. In contrast,
non-ATP-competitive inhibitors can provide distinct advantages
with respect to these issues. Herein, a new series of small
molecules with inhibitory activity against MK2 by virtue of a novel
non-ATP-competitive binding mode are described.
To discover novel MK2 inhibitors, we employed affinity
selectionÀmass spectrometry (AS-MS)-based high-throughput
screening using the Automated Ligand Identification System
(ALIS)24 and recombinant purified MK2 protein. We employed
a protein construct consisting of the minimal kinase domain of
MK2 from amino acid Gln41 to Thr338. In this construct, amino
and carboxy termini were truncated to both facilitate expression
and slightly decrease thermal stability of the kinase domain. It
was shown previously that the carboxy terminus of MK2 from
residues Thr334 to Arg 364 forms an R-helical peptide that
regulates MK2 nuclear localization and embeds into a substrate
binding cavity on the kinase domain surface.25À27 This interac-
tion is thought to stabilize the kinase domain;28,29 therefore, we
surmised that removing this carboxy terminal peptide region by
truncation at Thr338 would reduce thermal stability and in turn
may increase kinase domain accessibility and in the specific
context of ALIS screening may increase the probability of binding
A medicinal chemistry effort was carried out to optimize this
encouraging hit into a lead by improving biochemical and cell-based
potency, demonstrating a favorable in vitro pharmacokinetic (PK)
Figure 1. ALIS hit 1 profiling.
Figure 2. NMR characterization of binding of 1 to MK2. (A) ATP binds to the active site of MK2 and gives a large STD-NMR signal in the presence of
2.5 μM MK2. (B) The addition of 50 μM 1 results in the appearance of new STD peaks for 1 but has no effect on ATP binding. (C) A comparison of
1H/15N-HSQC spectra for apo-MK2 (black) and MK2-1 complex (red) shows that the binding of 1 affects specific MK2 residues. All NMR data were
collected at 27 °C on a Bruker Avance DRX 500 spectrometer equipped with a 5 mm TXI cryoprobe.
633
dx.doi.org/10.1021/ml200113y |ACS Med. Chem. Lett. 2011, 2, 632–637

ACS Medicinal Chemistry Letters
LETTER
profile and retaining the non-ATP-competitive binding mode. A
series of analogues, 2À8, with variation at the left-hand side (LHS)
of the molecule were prepared following Scheme 1. 5-Bromofur-
an-2-carboxylic acid was reacted with aniline to give amide.
5-Aryl-furan-2-carboxamide was obtained by Suzuki cross-cou-
pling. The removal of Boc protecting group using TFA provided
the final compound.
The structureÀactivity relationship (SAR) at LHS of this
series proved to be challenging (Table 1). While an ortho-Cl
substituent (2) led to a slight decrease in potency, the meta-Cl
analogue (3) was inactive. Adding an ortho substitutent with
either a Cl (4) or a Me group (5) to the para-Cl substitution
afforded analogues with comparable affinity to compound 1. The
replacement of a para-Cl group with either an electron-with-
drawing CF3 group (6) or electron-donating methoxy group (7)
decreased the potency by about 4-fold. The phenyl group could
be replaced with a thiophene (8) with the same level of potency.
Initial efforts to improve affinity through right-hand side
(RHS) changes to the series were not encouraging (Table 2).
Whereas a fluorine substitution in the phenyl ring (9) reduced the
affinity slightly, potency loss was observed in CF3 substitution (10).
Figure 3. Enzymatic analysis of mode of inhibition by ALIS hit 1 (2.5
nM MK2, 100 μM ATP, 0À200 μM peptide substrate, Km = 35 μM).
Scheme 1. Synthesis of 5-Aryl-N-(4-(piperazin-1-yl)phenyl)furan-2-carboxamidea
aReagents and conditions: (A) HATU, DIEA, DMF, rt; (B) ArB(OH)2, Pd(PPh3)4, Dioxane/H2O (3/1), 100 °C; (C) TFA; (D) RX,
NaH, DMF.
Table 1. Variation of the 5-Aryl Group of Furan on the LHS of the Molecule
a Biochemical and cell-based data represent the average values of duplicates or triplicates. b ND, not determined.
634
dx.doi.org/10.1021/ml200113y |ACS Med. Chem. Lett. 2011, 2, 632–637

ACS Medicinal Chemistry Letters
LETTER
Table 2. Variation of N-Substitution in Furan-2-carboxamide on the RHS of the Molecule
a Biochemical and cell-based data represent the average values of duplicates or triplicates. b ND, not determined.
Table 3. Variation of Furan with Other Heterocycles
A breakthrough in potency improvement was achieved when
thesecondary amide was substituted with analkyl group(Table4).
The synthesis of analogues, 19À26, follows from Scheme 1.
Deprotonation of the secondary amide using sodium hydride and
subsequent alkylation using alkyl halide provided the tertiary
amide product, which was converted to the final compound after
the removal of Boc protecting group using TFA. An 8-fold
potency improvement in biochemical assay was observed when
a methyl group (19) was attached to the amide. However, the
cell-based potency of this compound was reduced by about
4-fold. Elongation to two carbons (20) further improved po-
tency, not only in a biochemical assay by 40-fold but surprisingly
also in cell-based activity by 2-fold. Continuing elongation to
propyl (21) or iso-propyl (22) or butyl (23) provided analogues
with less potency improvement than ethyl (20) but still better
compounds than the starting ALIS hit 1. Notably, these alkyl-
substituted analogues have shown a favorable separation between
potency and cytotoxicity. Encouraged by these results, other
substituted heterocycles such as imidazole (24) and pyridine
(25 and 26) were explored. Biochemical potency was improved
to ∼100 nM in these three analogues. Whereas the N-methyli-
midazole analogue (24) was much less potent in cell-based
activity, N-methyl-ortho-pyridine (25) and N-methyl-para-pyridine
(26) showed significant improvement in cell-based potency while
retaining more than a 10-fold toxicity window.
Compound 25 was profiled for kinase selectivity by screening
against a broad panel of 150 protein kinases at a concentration of
10 μM, and only CK1γ3 was significantly inhibited at greater
than 50%. Compound 25 bioavailability was demonstrated in an
oral rat PK experiment, where an AUC of 2300 nM h was deter-
mined after a 10 mg/pk single dose. Compound 25 also demon-
strated no inhibition against a panel of cytochrome P450 (CYP)
enzymes up to 30 μM. Consistent with specific MK2 inhibition,
compound 25 inhibited pro-inflammatory cytokine secretion
from the human THP1 acute monocytic leukemia cell line,
causing dose-dependent inhibition of LPS-stimulated TNFR and
a Biochemical and cell-based data represent the average values of
duplicates or triplicates.
Opportunistic changes to the piperazine group at the RHS of
molecule were also undertaken. Capping the distal NH group
with a methyl group (11) exhibited no improvement in potency.
Removal of the distal NH group and introduction of an exocyclic
OH group (12) resulted in a loss of potency. The anilinic N in the
piperazine ring was replaced with an sp2 carbon (13). While a
potency improvementof 2-fold was observed in 13, this compound
suffered from high toxicity. The change of N-arylated piperazine to
N-arylated lactam (14) yielded a much less potent compound.
Attempts to replace the furan ring with other heterocycles
such as thiophene (15), thiazole (16), isooxazole (17), and pyridine
(18) were undertaken (Table 3). These compounds, 15À18,
were prepared by analogy to those in Table 1 by starting with
the bromo-heterocyclic carboxylic acid. Compounds generally showed
a 1À2-fold reduction in affinity. In the cases of thiazole (16) and
isooxazole (17) replacement, high toxicity was also observed.
635
dx.doi.org/10.1021/ml200113y |ACS Med. Chem. Lett. 2011, 2, 632–637

ACS Medicinal Chemistry Letters
LETTER
Table 4. N-Alkylation of Furan-2-carboxamide
a Biochemical and cell-based data represent the average values of duplicates or triplicates.
IL6 secretion (Figure 4A,B). Compound 25 also dose depen-
dently inhibited IL1β-stimulated matrixmetalloprotease (MMP)13
secretion from the SW1353 chondrosarcoma cell line and
human primary chondrocyte cultures (Figure 4C,D), confirming a
previous report wherein MK2 activity was demonstrated to modulate
secretion of MMP13 from osteoarthritis-derived chondrocytes.32
In summary, a new series of small molecules, N-alkyl-5-aryl-
N-(4-(piperazin-1-yl)phenyl)furan-2-carboxamide, were devel-
oped to be potent inhibitors against MK2 with a non-ATP-
competitive binding mode, enabling a high degree of MK2 selec-
tivity. Hit-to-lead chemistry efforts resulted in significant im-
provements in biochemical and cellular potencies and reductions
in cell toxicities, while maintaining a high degree of MK2
selectivity versus the human kinome. Of note, given its high
degree of selectivity, our data suggest that compound 25 may be
an excellent pharmacologic tool for specifically exploring and
validating MK2 biology. We have also demonstrated another
example where AS-MS screening of mixture-based combinatorial
libraries combined with careful formulation of recombinant
protein targets can result in the discovery of pharmacologically
active compounds showing interesting and novel binding modes.
The unique non-ATP-competitive binding mode, excellent kinase
selectivity, and DMPK profile of this MK2 inhibitor series as
exemplified by compound 25 warrant further development that
may lead to a clinical candidate for inflammation or other
diseases where emerging evidence implicates MK2.
Figure 4. Dose-dependent inhibition of inflammatory cytokine and
MMP-13 production by compound 25. Treatment of LPS-stimulated
THP1 cells with compound 25 dose dependently inhibits TNFR
(A) and IL6 secretion (B). Treatment of IL1-β-stimulated SW1353
cells (C) and human primary osteoarthritis-derived chondrocytes
(D) with compound 25 dose dependently inhibits MMP-13 secretion.
For all figures, data points and error bars reflect the average and standard
error of duplicate samples.
’ ASSOCIATED CONTENT
Present Addresses
†Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston,
Massachusetts 02115.
S
Supporting Information. Experimental procedures and
b
spectroscopic characterization of key compounds, kinase counter
screen data, CYP inhibition data, and assay protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
‡Merck Research Laboratories, 2000 Galloping Hill Road,
Kenilworth, New Jersey 07033.
’ AUTHOR INFORMATION
’ ACKNOWLEDGMENT
Corresponding Author
*E-mail: xiaohua.huang@merck.com (X.H.) or charles.whitehurst@
merck.com (C.E.W.).
Many thanks to Dr. J. Richard Miller for valuable sugges-
tions regarding analysis of enzymatic mode of inhibition;
636
dx.doi.org/10.1021/ml200113y |ACS Med. Chem. Lett. 2011, 2, 632–637

ACS Medicinal Chemistry Letters
LETTER
Dr. Mingxuan Zhang and Dr. Michael Ziebell for HRMS analysis; and
Michael Starks, Jason Hill, and Mark Pietrafitta for analytical support.
T. R.; Talanian, R. V. 2,4-Diaminopyrimidine MK2 inhibitors. Part II:
Structure-based inhibitor optimization. Bioorg. Med. Chem. Lett. 2010,
20, 334.
(19) Xiong, Z.; Gao, D. A.; Cogan, D. A.; Goldberg, D. R.; Hao,
M.-H.; Moss, N.; Pack, E.; Pargellis, C.; Skow, D.; Trieselmann, T.;
Werneburg, B.; White, A. Synthesis and SAR studies of indole-based
MK2 inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 1994.
(20) Anderson, D. R.; Meyers, M. J.; Vernier, W. F.; Mahoney,
M. W.; Kurumbail, R. G.; Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz,
D. B.; Mourey, R. J. Pyrrolopyridine inhibitors of mitogen-activated
protein kinase-activated protein kinase 2 (MK-2). J. Med. Chem. 2007,
50, 2647.
(21) Lin, S.; Lombardo, M.; Malkani, S.; Hale, J. J.; Mills, S. G.;
Chapman, K.; Thompson, J. E.; Zhang, W. X.; Wang, R.; Cubbon,
R. M.; O'Neill, E. A.; Luell, S.; Carballo-Jane, E.; Yang, L. Novel 1-(2-
aminopyrazin-3-yl)methyl-2-thioureas as potent inhibitors of mitogen-
activated protein kinase-activated protein kinase 2 (MK-2). Bioorg. Med.
Chem. Lett. 2009, 19, 3238.
(22) Mourey, R. J.; Burnette, B. L.; Brustkern, S. J.; Daniels, J. S.;
Hirsch, J. L.; Hood, W. F.; Meyers, M. J.; Mnich, S. J.; Pierce, B. S.;
Saabye, M. J.; Schindler, J. F.; South, S. A.; Webb, E. G.; Zhang, J.;
Anderson., D. R. A benzothiophene inhibitor of mitogen-activated
protein kinase-activated protein kinase 2 inhibits tumor necrosis
factor R production and has oral anti-inflammatory efficacy in acute
and chronic models of inflammation. J. Pharmacol. Exp. Ther. 2010,
333, 797.
(23) Olsson, H.; Sj€o, P.; Ersoy, O.; Kristoffersson, A.; Larsson, J.;
Nordꢀen, B. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated
protein kinase-activated protein kinase 2 (MK2). Bioorg. Med. Chem.
Lett. 2010, 20, 4738.
(24) Annis, A.; Chuang, C.-C.; Nazef, N. ALIS: An Affinity Selec-
tion-Mass Spectrometry System for the Discovery and Characterization
of Protein-Ligand Interactions. In Methods and Principles in Medicinal
Chemistry; Wanner, K., H€ofner, G., Eds.; Mass Spectrometry in Medicinal
Chemistry: Applications in Drug Discovery; Mannhold, R., Kubinyi, H.,
Folkers, G., Series Eds.; Wiley-VCH Verlag GmbH & Co. KGaA:
Weinheim, 2007; Vol. 36, Chapter 3.
(25) Zu, Y.-L.; Ai, Y.; Huang, C.-K. Characterization of an auto-
inhibitory domain in human mitogen-activated protein kinase-activated
protein kinase 2. J. Biol. Chem. 1995, 270, 202.
(26) Meng, W.; Swenson, L. L.; Fitzgibbon, M. J.; Hayakawa, K.;
Haar, E. t.; Behrens, A. E.; Fulghum, J. R.; Lippke, J. A. Structure of
mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2
suggests a bifunctional switch that couples kinase activation with nuclear
export. J. Biol. Chem. 2002, 277, 37401.
(27) Engel, K.; Kotlyarov, A.; Gaestel, M. Leptomycin B-sensitive
nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.
EMBO J. 1998, 17, 3363.
(28) Kervinen, J.; Ma, H.; Bayoumy, S.; Schubert, C.; Milligan, C.;
Lewandowski, F.; Moriarty, K.; DesJarlais, R. L.; Ramachandren, K.;
Wang, H.; Harris, C. A.; Grasberger, B.; Todd, M.; Springer, B. A.;
Deckman, I. Effect of construct design on MAPKAP kinase-2 activity,
thermodynamic stability and ligand-binding affinity. Arch. Biochem.
Biophys. 2006, 449, 47.
(29) Malawski, G. A.; Hillig, R. C.; Monteclaro, F.; Eberspaecher, U.;
Schmitz, A. A. P.; Crusius, K.; Huber, M.; Egner, U.; Donner, P.; M€uller-
Tiemann, B. Identifying protein construct variants with increased
crystallization propensity—A case study. Protein Sci. 2006, 15, 2718.
(30) McCoy, M. A.; Senior, M. M.; Wyss, D. F. Screening of protein
kinases by ATP-STD NMR spectroscopy. J. Am. Chem. Soc. 2005,
127, 7978.
(31) Akaike, H. An information criterion (AIC). Math. Sci. 1976, 14, 5.
(32) Jones, S. W.; Brockbank, S. M. V.; Clements, K. M.; Le Good,
N.; Campbell, D.; Read, S. J.; Needham, M. R. C.; Newham, P. Mitogen-
activated protein kinase-activated protein kinase 2 (MK2) modulates
key biological pathways associated with OA disease pathology. Osteoar-
thritis Cartilage 2009, 17, 124.
’ REFERENCES
(1) Tobꢀon, G. J.; Youinou, P.; Saraux, A. The environment, geo-
epidemiology, and autoimmune disease: Rheumatoid arthritis. Autoim-
mun. Rev. 2010, 9, A288.
(2) Kumar, S.; Boehm, J.; Lee, J. C. p38 MAP Kinases: key signalling
molecules as therapeutic targets for inflammatory diseases. Nat. Rev.
Drug Discovery 2003, 2, 717.
(3) Schindler, J. F.; Monahan, J. B.; Smith, W. G. p38 Pathway
kinases as anti-inflammatory drug targets. J. Dent. Res. 2007, 86, 800.
(4) Schett, G.; Zwerina, J.; Firestein, G. The p38 mitogen-activated
protein kinase (MAPK) pathway in rheumatoid arthritis. Ann. Rheum.
Dis. 2008, 67, 909.
(5) Alten, R. E.; Zerbini, C.; Jeka, S.; Irazoque, F.; Khatib, F.; Emery,
P.; Bertasso, A.; Rabbia, M.; Caulfield, J. P. Efficacy and safety of
pamapimod in patients with active rheumatoid arthritis receiving stable
methotrexate therapy. Ann. Rheum. Dis. 2010, 69, 364.
(6) Dambach, D. M. Potential adverse effects associated with
inhibition of p38R/β MAP kinases. Curr. Top. Med. Chem. 2005, 5, 929.
(7) Gaestel, M; Mengel, A.; Bothe, U.; Asadullah, K. Protein kinases
as small molecule inhibitor targets in inflammation. Curr. Med. Chem.
2007, 14, 2214.
(8) Duraisamy, S.; Bajpai, M.; Bughani, U.; Dastidar, S. G.; Ray, A.;
Chopra, P. MK2: A novel molecular target for anti-inflammatory
therapy. Expert Opin. Ther. Targets 2008, 12, 921.
(9) White, A.; Pargellis, C. A.; Studts, J. M.; Werneburg, B. G.;
Farmer, B. T., II. Molecular basis of MAPK-activated protein kinase 2:
p38 assembly. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6353.
(10) Haar, E. t.; Prabhakar, P.; Liu, X.; Lepre, C. Crystal structure of
the p38R-MAPKAP kinase 2 heterodimer. J. Biol. Chem. 2007, 282,
14684.
(11) Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert, R.; Birch-
meier, C.; Volk, H.-D.; Gaestel, M. MAPKAP kinase 2 is essential for
LPS-induced TNF-R biosynthesis. Nat. Cell Biol. 1999, 1, 94.
(12) Shi, Y.; Kotlyarov, A.; Laaβ, K.; Gruber, A. D.; Butt, E.; Marcus,
K.; Meyer, H. E.; Friedrich, A.; Volk, H.-D.; Gaestel, M. Elimination of
protein kinase MK5/PRAK activity by targeted homologous recombi-
nation. Mol. Cell. Biol. 2003, 23, 7732.
(13) Ronkina, N.; Kotlyarov, A.; Dittrich-Breiholz, O.; Kracht, M.;
Hitti, E.; Milarski, K.; Askew, R.; Marusic, S.; Lin, L.-L.; Gaestel, M.;
Telliez, J.-B. The mitogen-activated protein kinase (MAPK)-activated
protein kinases MK2 and MK3 cooperate in stimulation of tumor
necrosis factor biosynthesis and stabilization of p38 MAPK. Mol. Cell.
Biol. 2007, 27, 170.
(14) Hegen, M.; Gaestel, M.; Nickerson-Nutter, C. L.; Lin, L.-L.;
Telliez, J.-B. MAPKAP Kinase 2-deficient mice are resistant to collagen-
induced arthritis. J. Immunol. 2006, 177, 1913.
(15) Gorska, M. M.; Liang, Q.; Stafford, S. J.; Goplen, N.; Dharajiya,
N.; Guo, L.; Sur, S.; Gaestel, M.; Alam, R. MK2 controls the level of
negative feedback in the NF-kB pathway and is essential for vascular
permeability and airway inflammation. J. Exp. Med. 2007, 204, 1637.
(16) Basabe, P.; Martín, M.; Bodero, O.; Blanco, A.; Marcos, I. S.;
Díez, D.; Urones, J. G. Synthesis of (+)-makassaric acid, a protein kinase
MK2 inhibitor. Tetrahedron 2010, 66, 6008.
(17) Argiriadi, M. A.; Ericsson, A. M.; Harris, C. M.; Banach, D. L.;
Borhani, D. W.; Calderwood, D. J.; Demers, M. D.; DiMauro, J.; Dixon,
R. W.; Hardman, J.; Kwak, S.; Li, B.; Mankovich, J. A.; Marcotte, D.;
Mullen, K. D.; Ni, B.; Pietras, M.; Sadhukhan, R.; Sousa, S.; Tomlinson,
M. J.; Wang, L.; Xiang, T.; Talanian, R. V. 2,4-Diaminopyrimidine MK2
inhibitors. Part I: Observation of an unexpected inhibitor binding mode.
Bioorg. Med. Chem. Lett. 2010, 20, 330.
(18) Harris, C. M.; Ericsson, A. M.; Argiriadi, M. A.; Barberis, C.;
Borhani, D. W.; Burchat, A.; Calderwood, D. J.; Cunha, G. A.; Dixon,
R. W.; Frank, K. E.; Johnson, E. F.; Kamens, J.; Kwak, S.; Li, B.; Mullen,
K. D.; Perron, D. C.; Wang, L.; Wishart, N.; Wu, X.; Zhang, X.; Zmetra,
637
dx.doi.org/10.1021/ml200113y |ACS Med. Chem. Lett. 2011, 2, 632–637