Discovery of a novel series of Chk1 kinase inhibitors with a distinctive hinge binding mode

Article abstract of DOI:10.1021/ml200249h

A novel series of CHK1 inhibitors with a distinctive hinge binding mode, exemplified by 2-aryl-N-(2-(piperazin-1-yl)phenyl)thiazole-4-carboxamide, was discovered through high-throughput screening using the affinity selection-mass spectrometry (AS-MS)-based Automated Ligand Identification System (ALIS) platform. Structure-based ligand design and optimization led to significant improvements in potency to the single digit nanomolar range and hundred-fold selectivity against CDK2.

Full text of DOI:10.1021/ml200249h

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Novel Series of CHK1 Kinase Inhibitors with a
Distinctive Hinge Binding Mode
Xiaohua Huang,*,† Cliff C. Cheng, Thierry O. Fischmann,‡ Jose S. Duca,§ Xianshu Yang,†
́
Matthew Richards,† and Gerald W. Shipps, Jr.†
Merck Research Laboratories, 320 Bent Street, Cambridge, Massachusetts 02141, United States
S
* Supporting Information
ABSTRACT: A novel series of CHK1 inhibitors with a distinctive hinge binding mode, exemplified by 2-
aryl-N-(2-(piperazin-1-yl)phenyl)thiazole-4-carboxamide, was discovered through high-throughput screen-
ing using the affinity selection−mass spectrometry (AS-MS)-based Automated Ligand Identification
System (ALIS) platform. Structure-based ligand design and optimization led to significant improvements
in potency to the single digit nanomolar range and hundred-fold selectivity against CDK2.
KEYWORDS: affinity selection−mass spectrometry (AS-MS), Automated Ligand Identification System (ALIS), CHK1 protein kinase,
structure-based drug design, thiazole-4-carboxamide
ancer is the leading cause of death globally.1 Among the
treatments used in cancer clinics, DNA-damaging chemo-
lead compounds in various classes of protein targets, including
the anti-infective target Escherichia coli dihydrofolate reduc-
tase,13 antibacterial AccC (acetyl coenzyme-A carboxylase),14
HCV NS5B (hepatitis C virus nonstructural protein 5B)
polymerase,15−17 protein kinase CDK2 (cyclin-dependent
kinase 2),18 KSP (kinesin spindle protein) in oncology,19
FABP4 (fatty acid binding protein-4)20 and the lipid
phosphatase SHIP2 (SH2 domain-containing inositol 5-
phosphatase 2)21 in diabetes, MK2 (mitogen-activated protein
kinase-activated protein kinase 2)22 and TACE (TNF-α
converting enzyme)23 in inflammation, and the GPCR (G-
protein-coupled receptor) muscarinic M2 acetylcholine recep-
tor.24 Herein, we describe another example using ALIS to
screen for novel inhibitors of the CHK1 protein and
subsequent medicinal chemistry optimization.
From the CHK1 ALIS screening of mixture-based libraries,
compound 1 emerged as a high affinity ligand (ALIS Kd <100
nM), and the series was selected for additional optimization.
Biochemical assays confirmed its inhibitory activity against
CHK1 with an IC50 value of 75 nM with a clearly detectable in-
cell activity (∼10 μM) that was hydroxyurea (HU)-dependent.
However, an initial selectivity assessment revealed only a
modest selectivity (∼3-fold) against CDK2, the inhibition of
which was anticipated to mitigate the inhibition of CHK1.25,26
Structure-based ligand design and empirical structure−activity
relationship (SAR)-driven optimization were initiated with the
minimal goals of achieving a 10-fold improvement in potency
and demonstrating a 100-fold in selectivity against CDK2.
C
therapies have received widespread use despite their severe side
effects on highly proliferative tissues and vulnerability to drug
resistance.2 There is an urgent need in cancer therapy for
improved tolerability and longer lasting efficacy. When cells are
exposed to agents that induce DNA damage, DNA repair
pathways are activated by arresting at various cell cycle check
points, G1, S, and G2/M.3 While normal cells could arrest in
the G1 phase through the tumor suppressor protein p53, most
cancer cells lack this option because of p53 mutations and will
have to rely on the S or G2/M checkpoint for DNA repair and
survival. Such reliance in p53 mutant cancer cells would offer a
significant opportunity for targeted cancer therapy.4 CHK1
(checkpoint kinase 1) is a serine/threonine kinase and the key
mediator in S and G2/M checkpoints.5 The abrogation of
CHK1 and the remaining checkpoints consequentially will
cause cancer cells with DNA damage premature entry into
mitosis and result in cell death.6 Therefore, an enlarged
therapeutic window would be expected in anticancer therapies
utilizing a combination of a DNA-damaging agent with a CHK1
inhibitor.
Over the past years, extensive research efforts in CHK1
inhibition have been undertaken in oncology. Several small
molecule CHK1 inhibitors have been discovered and entered
into clinical trials.7−11 In our efforts to discover novel small
molecule CHK1 inhibitors, the AS-MS ALIS (affinity
selection−mass spectrometry-based Automated Ligand Identi-
fication System) platform was used to identify hits from
mixture-based combinatorial libraries.12 ALIS has demonstrated
its unique capability in screening label-free mixture-based
libraries and finding hits that were subsequently developed into
Received: October 19, 2011
Accepted: January 6, 2012
Published: January 20, 2012
© 2012 American Chemical Society
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Figure 1. Affinity competition experiment. For all figures, data points and error bars reflect the average and standard error of duplicate samples.
Figure 2. X-ray of crystal structure of compound 1 bound to CHK1 protein.
The characterization of the binding mode of CHK1 to
compound 1 was analyzed using an affinity competition
experiment (Figure 1).27 In these experiments, a constant
concentration of compound 1 and serially increasing
concentrations of the titrant (compound 2, a previously
reported ATP-competitive CHK1 inhibitor with IC50 value of
14 nM11,28) were incubated together with the CHK1 protein.
The protein−ligand complexes were separated from the
unbound compounds by size-exclusion chromatography. The
protein-bound compound 1 and the titrant were dissociated
from the protein using reverse-phase chromatography and
quantified by a mass spectrometer. The AS-MS recovery of
compound 1 in the presence of the titrant suggested the
binding site. As shown in Figure 1A, compound 1 was fully
displaced from binding to the CHK1 protein by the titrant at a
higher concentration. The linear plot of titrant/1 response ratio
versus total titrant concentration in Figure 1B provided
compelling evidence of direct competition between compound
1 and the titrant for binding with CHK1.
To further establish the binding site and gain insight into the
specific interactions between compound 1 and CHK1, the X-
ray crystal structure of the complex was successfully pursued.29
The X-ray crystal structure of compound 1 bound to CHK1 at
1.85 Å (Figure 2) illuminated key interactions that were
noteworthy for kinase−ligand complexes. Compound 1
overlaps with the ATP binding pocket. As most kinase
inhibitors, it interacts with several main-chain polar atoms
from the hinge region exposed to the binding site. A first
interaction is between the 2,3-dihydrobenzofuran 6-C-H and
the Cys87 carbonyl. A second interaction is between the Glu85
main-chain carbonyl and the 5-C-H of the thiazole. In both
cases, the distances between atoms, 3.3 and 3.1 Å, respectively,
are consistent with an interaction with partial H-bond
character. This is the first reported example in a kinase30
where the C−H next to a S in a thiazole ring forms a
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Table 1. Optimization To Probe Intramolecular Hydrogen Bonding and Salt Bridge Interactions
a
b
Biochemical data represent average values of duplicates or triplicates with a standard deviation of 10%. nd, not determined.
Table 2. Optimization To Probe Interaction with Hinge Binding
a
Biochemical data represent the average values of duplicate or triplicate data sets with a standard deviation of 10%.
Table 3. Optimization To Probe Solvent Front Interactions
a
b
Biochemical data represent average values of duplicates or triplicates with a standard deviation of 10%. nd, not determined.
nonstandard hydrogen bond, whereas most of reported
examples have the C−H adjacent to N in various hetero-
cycles.31 Quantum mechanical calculations suggest that the
polarizability of the S in the thiazole ring contributes to this
“unusual” hinge interaction. It is also evident from the X-ray
crystal structure that the secondary amide NH serves as an
intramolecular H-bond bridge between two proximal N's to
confer a “U-shaped” topology to compound 1.32 Such
intramolecular hydrogen bonding may significantly preorganize
compound 1 to fit into the binding pocket with a reduced
entropic penalty. The preferred orthogonal orientation of the
piperazine with respect to the aryl group enables this
conformation. Additional key interactions are evident: a salt
bridge between Glu91 and the piperazine NH, which also
shares H-bonds with Glu134 main-chain carbonyl and a water
molecule, an H-bond between the amide oxygen and a
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a
Scheme 1. Synthesis of 2-Aryl-N-(2-(piperazin-1-yl)phenyl)thiazole-4-carboxamide
a
Reagents and conditions: (A) HATU (N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate), DIEA (N,N-
diisopropylethylamine), DMF, room temperature. (B) Pd(OAc)2, S-Phos (2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl), ArB(OH)2, K3PO4,
dioxane, 100 °C. (C) TFA (trifluoroacetic acid). (D) Pd(OAc)2, X-Phos (2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl), alkyne, Cs2CO3,
MeCN, 85 °C.
Table 4. Cell-Based Activity
a
Biochemical and cell-based data represent average values of duplicates or triplicates with a standard deviation of 10%.
conserved water,33,34 and contacts between the 2,3-dihydro-
benzofuran moiety and the solvent front. Inhibitor binding
induces a conformational rearrangement of the glycine-rich
loop. The phenol of Tyr20 at the tip of the glycine-rich loop
moves to stack itself against a hydrophobic edge of the
piperazinyl.
in a significant loss of potency and reinforced the value of the
C−H/Glu85 to the overall binding affinity.
A breakthrough in potency and selectivity against CDK2 was
achieved through the modification of the aryl group connected
to thiazole (Table 3). These analogues were prepared by Suzuki
cross-coupling of bromothiazole following Scheme 1.35 The
compound with a plain phenyl group (16) is close to
compound 1 in potency but with slightly better selectivity
against CDK2. para-MeO substitution of the phenyl group
(17) afforded a compound comparable to 1 in potency and
selectivity. Additional substitution of a methyl (18) or methoxy
(19) group at the meta position resulted in a potency decrease
of 7-fold. Bridging the two oxygen atoms at the 3- and 4-
position of the phenyl group through either one carbon (20) or
three carbons (21) gave compounds with potency 2-fold less
than compound 1. Interestingly, pronounced improvements in
potency and selectivity against CDK2 were observed when the
phenyl group was substituted with a para-phenoxy group (22).
While para-NMe2 substitution of phenyl group (23) resulted in
a slight increase in potency, insertion of a methylene bridge
between the dimethylamino and phenyl group (24) led to the
complete loss of activity.
Identifying modifications of the piperazine portion of
compound 1 which improved potency proved to be challenging
(Table 1). A significant loss of potency was observed in changes
to the tertiary aniline, such as the replacement of a nitrogen
with either an sp3 (3) or an sp2 (4) carbon or adding a methyl
(5) or carbonyl (6) group adjacent to the tertiary nitrogen.
Such sensitivity to structural change strongly suggested the
necessity of intramolecular hydrogen bonding between the
tertiary N and N−H of the secondary amide. Attempts to probe
the salt bridge interaction between the distal secondary amino
group and the Glu91 were made, and loss of potency was
observed. While adding a methyl group adjacent to the distal
nitrogen in either stereo configuration (8 and 9) gave a slight
decrease in potency, complete potency loss was observed in the
change from a basic amino group to a lactam (7). Capping with
a methyl (10) or acetyl (11) group or replacement with O (12)
yielded a much less potent compound.
The replacement of phenyl moieties with selected hetero-
cycles did not result in appreciable improvements in IC50.
While the 2-pyrrole (26) is 10-fold weaker in potency than the
3-pyrrole (25), 2-thiophene (28) is a few fold better in potency
and selectivity than the 3-thiophene (27) and compound 1 as
well. Substitution of the 2-thiophene group with thienyl (29) or
the phenyl (30) afforded a slight potency improvement. Both
Modifications to probe the interaction between the C−H of
the thiazole and the carbonyl of Glu85 in the hinge region were
attempted (Table 2), such as substitution with a methyl group
(13), different connectivity at the thiazole (14), and
replacement with isoxazole (15). All of these changes resulted
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the 3- (31) and the 4-pyrazole (32) were slightly less potent
than compound 1; however, encouraging selectivity toward
CDK2 was demonstrated by the 3-pyrazole (31). Other N-
containing heterocycles such as pyridines (33, 34, and 35) and
pyrimidine (36) showed reduced CHK1 inhibition. Replace-
ment with fused bicyclic aromatics was also explored. The
replacement of the 2,3-dihydrobenzofuran group in compound
1 with benzofuran (37) gave slight improvements in potency
and selectivity. The change of oxygen to other heteroatoms
such as NH (38) or S (39) led to further improvement in IC50
to single-digit nanomolar as well as selectivity against CDK2 to
several hundred-fold. Other connections at benzothiophene
were also explored, and 3-benzothiophene (41) was found to
be less potent and selective than the 2- (40) and the 5-
benzothiophene (39). Analogues with an alkyne group attached
to the thiazole were also prepared by employing Sonogashira
cross-coupling of bromothiazole.36 While less potency was
observed, a 10-fold improvement in selectivity against CDK2
was obtained in 42 in comparison to compound 1.
Top compounds in biochemical activity were tested in cell-
based assays using γ-H2AX as the cellular biomarker for the
DNA double-strand breaks (Table 4).6 Compounds 37−40
showed improved cell-based activity by 3−6-fold. Compound
22 showed no cell-based activity despite its marked biochemical
potency and selectivity against CDK2, possibly due to lack of
membrane permeability, compound efflux from the cell, or a
confounding off-target effect.
REFERENCES
■
(1) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman,
D. Global Cancer Statistics. CA Cancer J. Clin. 2011, 61, 69.
(2) Hurley, L. H. DNA and its associated processes as targets for
cancer therapy. Nat. Rev. Cancer 2002, 2, 188.
(3) Zhou, B.-B. S.; Elledge, S. J. The DNA damage response: Putting
checkpoints in perspective. Nature 2000, 408, 433.
(4) Kawabe, T. G2 checkpoint abrogators as anticancer drugs. Mol.
Cancer Ther. 2004, 3, 513.
(5) Xiao, Z.; Chen, Z.; Gunasekera, A. H.; Sowin, T. J.; Rosenberg, S.
H.; Fesik, S.; Zhang, H. Chk1 mediates S and G2 arrests through
Cdc25A degradation in response to DNA-damaging agents. J. Biol.
Chem. 2003, 278, 21767.
(6) Cho, S. H.; Toouli, C. D.; Fujii, G. H.; Crain, C.; Parry, D. Chk1
is essential for tumor cell viability following activation of the
replication checkpoint. Cell Cycle 2005, 4, 131.
(7) Arrington, K. L.; Dudkin, V. Y. Novel inhibitors of checkpoint
kinase 1. ChemMedChem 2007, 2, 1571.
(8) Ashwell, S.; Janetka, J. W.; Zabludoff, S. Keeping checkpoint
kinases in line: New selective inhibitors in clinical trials. Expert Opin.
Invest. Drugs 2008, 17, 1331.
(9) Blasina, A.; Hallin, J.; Chen, E.; Arango, M. E.; Kraynov, E.;
Register, J.; Grant, S.; Ninkovic, S.; Chen, P.; Nichols, T.; O'Connor,
P.; Anderes, K. Breaching the DNA damage checkpoint via PF-
00477736, a novel small-molecule inhibitor of checkpoint kinase 1.
Mol. Cancer Ther. 2008, 7, 2394.
(10) Zabludoff, S. D.; Deng, C.; Grondine, M. R.; Sheehy, A. M.;
Ashwell, S.; Caleb, B. L.; Green, S.; Haye, H. R.; Horn, C. L.; Janetka,
J. W.; Liu, D.; Mouchet, E.; Ready, S.; Rosenthal, J. L.; Queva, C.;
Schwartz, G. K.; Taylor, K. J.; Tse, A. N.; Walker, G. E.; White, A. M.
AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint
abrogation and potentiates DNA-targeted therapies. Mol. Cancer Ther.
2008, 7, 2955.
(11) Guzi, T. J.; Paruch, K.; Dwyer, M. P.; Labroli, M.; Shanahan, F.;
Davis, N.; Taricani, L.; Wiswell, D.; Seghezzi, W.; Penaflor, E.;
Bhagwat, B.; Wang, W.; Gu, D.; Hsieh, Y.; Lee, S.; Ming Liu, M.;
Parry, D. Targeting the replication checkpoint using SCH 900776, a
potent and functionally selective CHK1 inhibitor identified via high
content screening. Mol. Cancer Ther. 2011, 10, 591.
In summary, a new series of small molecules with distinctive
hinge binding modes, 2-aryl-N-(2-(piperazin-1-yl)phenyl)-
thiazole-4-carboxamide, were identified via ALIS screening of
mixture-based libraries and developed into potent inhibitors to
CHK1. Hit-to-lead chemistry efforts resulted in significant
improvements in biochemical potency and selectivity against
CDK2. Additional SAR development and improvement in cell-
based activity will be reported in due course.
(12) Annis, A.; Chuang, C.-C.; Nazef, N. ALIS: An Affinity Selection-
Mass Spectrometry System for the Discovery and Characterization of
Protein-Ligand Interactions. In Methods and Principles in Medicinal
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemistry; Wanner, K., Hofner, G., Eds.; Mass Spectrometry in
̈
Experimental procedures, spectroscopic characterization of key
compounds, and assay protocols. This material is available free
of charge via the Internet at http://pubs.acs.org.
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.
(13) Annis, D. A.; Athanasopoulos, J.; Curran, P. J.; Felsch, J. S.;
Kalghatgi, K.; Lee, W. H.; Nash, H. M.; Orminati, J.-P. A.; Rosner, K.
E.; Shipps, G. W. Jr.; Thaddupathy, G. R. A.; Tylera, A. N.; Vilenchik,
L.; Wagner, C. R.; Wintner, E. A. An affinity selection−mass
spectrometry method for the identification of small molecule ligands
from self-encoded combinatorial libraries Discovery of a novel
antagonist of E. coli dihydrofolate reductase. Int. J. Mass Spectrom.
2004, 238, 77.
(14) Cheng, C. C.; Shipps, G. W. Jr.; Yang, Z.; Sun, B.; Kawahata, N.;
Soucy, K. A.; Soriano, A.; Orth, P.; Xiao, L.; Mann, P.; Black, T.
Discovery and optimization of antibacterial AccC inhibitors. Bioorg.
Med. Chem. Lett. 2009, 19, 6507.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: xiaohua.huang@merck.com.
Present Addresses
†Merck Research Laboratories, 33 Avenue Louis Pasteur,
Boston, Massachusetts 02115.
‡Merck Research Laboratories, 2000 Galloping Hill Road,
Kenilworth, New Jersey 07033.
§Novartis Institutes for BioMedical Research, 100 Technology
Square, Cambridge, Massachusetts 02139.
(15) Cheng, C. C.; Huang, X.; Shipps, G. W. Jr.; Wang, Y.-S.; Wyss,
D. F.; Soucy, K. A.; Jiang, C.-k.; Agrawal, S.; Ferrari, E.; He, Z.; Huang,
H.-C. Pyridine Carboxamides: Potent Palm Site Inhibitors of HCV
NS5B Polymerase. ACS Med. Chem. Lett. 2010, 1, 466.
Notes
The authors declare no competing financial interest.
(16) Cheng, C. C.; Shipps, G. W. Jr.; Yang, Z.; Kawahata, N.;
Lesburg, C. A.; Duca, J. S.; Bandouveres, J.; Bracken, J. D.; Jiang, C.-k.;
Agrawal, S.; Ferrari, E.; Huang, H.-C. Inhibitors of hepatitis C virus
polymerase: Synthesis and characterization of novel 2-oxy-6-fluoro-N-
((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides. Bioorg. Med. Chem.
Lett. 2010, 20, 2119.
ACKNOWLEDGMENTS
■
Many thanks to Dr. Michael Ziebell for HRMS analysis,
Michael Starks, Jason Hill, and Mark Pietrafitta for analytical
support.
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Letter
(17) Shipps, G. W. Jr.; Deng, Y.; Wang, T.; Popovici-Muller, J.;
Curran, P. J.; Rosner, K. E.; Cooper, A. B.; Girijavallabhan, V.;
Butkiewicz, N.; Cable, M. Aminothiazole inhibitors of HCV RNA
polymerase. Bioorg. Med. Chem. Lett. 2005, 15, 115.
(34) Dwyer, M. P.; Paruch, K.; Labroli, M.; Alvarez, C.; Keertikar, K.
M.; Poker, C.; Rossman, R.; Fischmann, T. O.; Duca, J. S.; Madison,
V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J.
Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A
template-based approachPart 1. Bioorg. Med. Chem. Lett. 2011, 21,
467.
(35) Billingsley, K. L.; Anderson, K. W.; Buchwald, S. L. A highly
active catalyst for Suzuki−Miyaura cross-coupling reactions of
heteroaryl compounds. Angew. Chem., Int. Ed. 2006, 45, 3484.
(36) Gelman, D.; Buchwald, S. L. Efficient palladium-catalyzed
coupling of aryl chlorides and tosylates with terminal alkynes: Use of a
copper cocatalyst inhibits the reaction. Angew. Chem., Int. Ed. 2003, 42,
5993.
(18) Annis, D. A.; Shipps, G. W. Jr.; Deng, Y.; Popovici-Muller, J.;
̈
Siddiqui, M. A.; Curran, P. J.; Gowen, M.; Windsor, W. T. Method for
quantitative protein-ligand affinity measurements in compound
mixtures. Anal. Chem. 2007, 79, 4538.
(19) Lahue, B. R.; Ma, Y.; Shipps, G. W. Jr.; Seghezzi, W.; Herbst, R.
Substituted benzimidazoles: a novel chemotype for small molecule
hksp inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 3405.
(20) Lan, H.; Cheng, C. C.; Kowalski, T. J.; Pang, L.; Shan, L.;
Chuang, C.-C.; Jackson, J.; Rojas-Triana, A.; Bober, L.; Liu, L.; Voigt,
J.; Orth, P.; Yang, X.; Shipps, G. W. Jr.; Hedrick, J. A. Small-molecule
inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin
resistance in mice with diet-induced obesity. J. Lipid Res. 2011, 52, 646.
(21) Annis, D. A.; Cheng, C. C.; Chuang, C.-C.; McCarter, J. D.;
Nash, H. M.; Nazef, N.; Rowe, T.; Kurzeja, R. J. M.; Shipps, G. W. Jr.
Inhibitors of the lipid phosphatase SHIP2 discovered by high
throughput affinity selection-mass spectrometry screening of combi-
natorial libraries. Comb. Chem. High Throughput Screen. 2009, 12, 760.
(22) Huang, X.; Shipps, G. W. Jr.; Cheng, C. C.; Spacciapoli, P.;
Zhang, X.; McCoy, M. A.; Wyss, D. F.; Yang, X.; Achab, A.; Soucy, K.;
Montavon, D. K.; Murphy, D. M.; Whitehurst, C. E. Discovery and hit-
to-lead optimization of non-ATP competitive MK2 (MAPKAPK2)
inhibitors. ACS Med. Chem. Lett. 2011, 2, 632.
(23) Rosner, K. E.; Guo, Z.; Orth, P.; Shipps, G. W. Jr.; Belanger, D.
B.; Chan, T. Y.; Curran, P. J.; Dai, C.; Deng, Y.; Girijavallabhan, V. M.;
Hong, L.; Lavey, B. J.; Lee, J. F.; Li, D.; Liu, Z.; Popovici-Muller, J.;
Ting, P. C.; Vaccaro, H.; Wang, L.; Wang, T.; Yu, W.; Zhou, G.; Niu,
X.; Sun, J.; Kozlowski, J. A.; Lundell, D. J.; Madison, V.; McKittrick, B.;
Piwinski, J. J.; Shih, N.-Y.; Siddiqui, M. A.; Strickland, C. O. The
discovery of novel tartrate-based TNF-α converting enzyme (TACE)
inhibitors. Bioorg. Med. Chem. Lett. 2009, 20, 1189.
(24) Whitehurst, C. E.; Nazef, N.; Annis, D. A.; Hou, Y.; Murphy, D.
M.; Spacciapoli, P.; Yao, Z.; Ziebell, M. R.; Cheng, C. C.; Shipps, G.
W. Jr.; Felsch, J. S.; Lau, D.; Nash, H. M. Discovery and
characterization of orthosteric and allosteric muscarinic M2 acetylcho-
line receptor ligands by affinity selection−mass spectrometry. J.
Biomol. Screen. 2006, 11, 194.
(25) Meikrantz, W.; Schlegel, R. Suppression of apoptosis by
dominant negative mutants of cyclin-dependent protein kinases. J. Biol.
Chem. 1996, 271, 10205.
(26) Sampath, D.; Shi, Z.; Plunkett, W. Inhibition of cyclin-
dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase
checkpoint activated by fludarabine: dysregulation by 7-hydroxystaur-
osporine. Mol. Pharmacol. 2002, 62, 680.
(27) Annis, D. A.; Nazef, N.; Chuang, C.-C.; Scott, M. P.; Nash, H.
M. A general technique to rank protein-ligand binding affinities and
determine allosteric versus direct binding site competition in
compound mixtures. J. Am. Chem. Soc. 2004, 126, 15495.
(28) Zhao, L.; Curran, P. J.; Belanger, D. B.; Hamann, B.; Reddy, P.
A.; Paruch, K.; Guzi, T. J.; Dwyer, M. P.; Siddiqui, M. A.; Tadikonda,
P. K. Imidazopyrazines as protein kinase inhibitors. WO 2007/058942
A2.
(29) The atomic coordinates of compound 1 bound to CHK1 have
been deposited in the Protein Data Bank: PDB ID, 3U9N.
(30) For a previous example of such an interaction between a
thiazolo 5-CH and protein other than kinase, please see the crystal
structure in the Protein Data Bank: PDB ID, 2C5W.
(31) Pierce, A. C.; Sandretto, K. L.; Bemis, G. W. Kinase inhibitors
and the case for CH···O hydrogen bonds in protein−ligand binding.
Proteins: Struct. Funct. Genet 2002, 49, 567.
(32) Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding
in medicinal chemistry. J. Med. Chem. 2010, 53, 2601.
(33) Barillari, C.; Taylor, J.; Viner, R.; Essex, J. W. Classification of
water molecules in protein binding sites. J. Am. Chem. Soc. 2007, 129,
2577.
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