ACS Medicinal Chemistry Letters
Letter
(6) Adams, J. A. Activation Loop Phosphorylation and Catalysis in
Protein Kinases: Is There Functional Evidence for the Autoinhibitor
Model? Biochemistry 2002, 42, 601−607.
(7) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.;
Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan, J.
Inhibition of p38 MAP kinase by utilizing a novel allosteric binding
site. Nat. Struct. Biol. 2002, 9, 268−272.
(8) Iwata, H.; Imamura, S.; Hori, A.; Hixon, M. S.; Kimura, H.; Miki,
H. Biochemical Characterization of TAK-593, a Novel VEGFR/
PDGFR Inhibitor with a Two-Step Slow Binding Mechanism.
Biochemistry 2011, 50, 738−751.
(9) Liao, J. J.-L. Molecular Recognition of Protein Kinase Binding
Pockets for Design of Potent and Selective Kinase Inhibitors. J. Med.
Chem. 2007, 50, 409−424.
(10) Liu, Y.; Gray, N. S. Rational design of inhibitors that bind to
inactive kinase conformations. Nat. Chem. Biol. 2006, 2, 358−364.
(11) Rees, D. C.; Congreve, M.; Murray, C. W.; Carr, R. Fragment-
based lead discovery. Nat. Rev. Drug Dicovery 2004, 3, 660−672.
(12) Congreve, M.; Carr, R.; Murray, C.; Jhoti, H. A 'Rule of Three'
for fragment-based lead discovery? Drug Discovery Today 2003, 8,
876−877.
(13) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent
developments in fragment-based drug discovery. J. Med. Chem. 2008,
51, 3661−3680.
(14) Freire, E. Do enthalpy and entropy distinguish first in class from
best in class? Drug Discovery Today 2008, 13, 869−874.
(15) de Kloe, G. E.; Bailey, D.; Leurs, R.; de Esch, I. J. Transforming
fragments into candidates: Small becomes big in medicinal chemistry.
Drug Discovery Today 2009, 14, 630−646.
bins, the back pocket binders and the allosteric inhibitors, both
of which are novel hits for a lead generation effort. A follow up
assay of the hits in combination with A serves a rapid
confirmation that these hits engage the kinase remote from the
hinge binding region.
We chose to conduct full Yonetani−Theorell analysis as a
follow up for the hits in step one, a necessity since this study
was our proof of concept. When a greater number of hits are
produced, one can gain throughput at the cost of some accuracy
by screening the inhibitor pairs (A and fragment hit) at their
respective IC50 values. The null hypothesis is that the
compounds are mutually exclusive and the combination should
produce 67% inhibition (eq 1, α = infinity). Combinations with
A that produce greater than 67% inhibition are binned as
interesting. If the hits are solubility limited such that an IC50
cannot be determined, eq 224 affords a practical approximation
of the null hypothesis.
A FBDD methodology for the discovery of back pocket
inhibitors has been elusive as a result of both the fluidity of the
back pocket dynamics and the difficulty in executing an
appropriate screen against the unactivated kinase. Paradoxically,
weak binding of a fragment within this pocket will, when
optimally included in a larger molecule engaging the hinge
region, create a much more potent and selective inhibitor than
could be achieved in its absence. This study reports the first
robust, broadly applicable FBDD screening strategy capable of
discovering kinase back pocket-binding fragments.
(16) Baldwin, I.; Bamborough, P.; Haslam, C. G.; Hunjan, S. S.;
Longstaff, T.; Mooney, C. J.; Patel, S.; Quinn, J.; Somers, D. O. Kinase
array design, back to front: Biaryl amides. Bioorg. Med. Chem. Lett.
2008, 18, 5285−5289.
ASSOCIATED CONTENT
* Supporting Information
■
S
(17) Muller, G.; Sennhenn, P. C.; Woodcock, T.; Neumann, L. The
̈
Materials, characterization of all compounds, full experimental
procedures, supporting figures, and tables. This material is
'retro-design' concept for novel kinase inhibitors. IDrugs 2010, 13,
457−466.
(18) Simard, J. R.; Kluter, S.; Grutter, C.; Getlik, M.; Rabiller, M.;
Rode, H. B.; Rauh, D. A new screening assay for allosteric inhibitors of
cSrc. Nat. Chem. Biol. 2009, 5, 394−396.
AUTHOR INFORMATION
Corresponding Author
■
(19) Simard, J. R.; Getlik, M.; Grutter, C.; Pawar, V.; Wulfert, S.;
Rabiller, M.; Rauh, D. Development of a fluorescent-tagged kinase
assay system for the detection and characterization of allosteric kinase
inhibitors. J. Am. Chem. Soc. 2009, 131, 13286−13296.
(20) Iwata, H.; Imamura, S.; Hori, A.; Hixon, M. S.; Kimura, H.;
Miki, H. Biochemical characterization of a novel type-II VEGFR2
kinase inhibitor: Comparison of binding to non-phosphorylated and
phosphorylated VEGFR2. Bioorg. Med. Chem. 2011, 19, 5342−5351.
(21) Yonetani, T.; Theorell, H. Studies on liver alcohol dehydrogen-
ase complexes: III. Multiple inhibition kinetics in the presence of two
competitive inhibitors. Arch. Biochem. Biophys. 1964, 106, 243−251.
(22) Yonetani, T. The Yonetani-Theorell graphical method for
examining overlapping subsites of enzyme active centers. Methods
Enzymol. 1982, 87, 500−509.
(23) Oguro, Y.; Miyamoto, N.; Okada, K.; Takagi, T.; Iwata, H.;
Awazu, Y.; Miki, H.; Hori, A.; Kamiyama, K.; Imamura, S. Design,
synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-
d]pyrimidine derivatives: novel VEGFR2 kinase inhibitors binding to
inactive kinase conformation. Bioorg. Med. Chem. 2010, 18, 7260−
7273.
(24) Assumes that the hinge binder is present at its IC50 and that the
fragment was assayed alone under the same assay conditions
(especially [ATP]) as in the combination assay. β is the single point
estimated IC50 where V0 is the control kinase activity and v is the
kinase activity in the presence of the fragment.
*Tel: +81-466-32-2747. Fax: +81-466-29-4484. E-mail: Miki_
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Y. Nishikimi and K. Hamamura for compound
preparation and S. Tsuji and Y. Hayano for preparation of
recombinant human VEGFR2. We also thank T. Yoshida for
assistance with assays and I. Miyahisa and M. Lardy for
discussions, guidance, and editing. Finally, we thank J. Matsui
and N. Tarui for encouragement to undertake this study.
REFERENCES
■
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.;
Sudarsanam, S. The protein kinase complement of the human
genome. Science 2002, 298, 1912−1934.
(2) Hunter, T. The Croonian Lecture 1997. The phosphorylation of
proteins on tyrosine: its role in cell growth and disease. Philos. Trans.
R. Soc., B 1998, 353, 583−605.
(3) Schwartz, P. A.; Murray, B. W. Protein kinase biochemistry and
drug discovery. Bioorg. Chem. 2011, 39, 192−210.
(4) Blume-Jensen, P.; Hunter, T. Oncogenic kinase signalling. Nature
2001, 411, 355−365.
(5) Hubbard, S. R.; Mohammadi, M.; Schlessinger, J. Autoregulatory
mechanisms in protein-tyrosine kinases. J. Biol. Chem. 1998, 273,
11987−11990.
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