J. Am. Chem. Soc. 1999, 121, 627-631
627
Generation of Monospecific Nanomolar Tyrosine Kinase Inhibitors
via a Chemical Genetic Approach
Anthony C. Bishop,† Chi-yun Kung,† Kavita Shah,† Laurie Witucki,†
Kevan M. Shokat,*,†,‡ and Yi Liu†
Contribution from the Department of Chemistry and Department of Molecular Biology,
Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed September 14, 1998
Abstract: Selective protein kinase inhibitors are highly sought after as tools for studying cellular signal
transduction cascades, yet few have been discovered due to the highly conserved fold of kinase catalytic domains.
Through a combination of small molecule synthesis and protein mutagenesis, a highly potent (IC50 ) 1.5 nM)
and uniquely specific inhibitor (4-amino-1-tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine) of a rationally
engineered v-Src tyrosine kinase (Ile338Gly v-Src) has been identified. Both the potency and specificity of
this compound surpass those of any known Src family tyrosine kinase inhibitors. The molecule strongly inhibits
the engineered v-Src in whole cells but does not inhibit tyrosine phosphorylation in cells that express only
wild-type tyrosine kinases. In addition, the inhibitor selectively disrupts transformation in cells that express
the target v-Src. The structural degeneracy of kinase active sites should allow the same complementary inhibitor/
protein design strategy to be widely applicable across this entire enzyme superfamily.
Introduction
retinoid X receptor are sufficient to create two new classes of
receptors with novel ligand specificities.9 In a more medicinally
applicable system, Smith and co-workers engineered the pro-
tease, carboxypeptidase A1, to hydrolyze a prodrug of meth-
otrexate that is resistant to hydrolysis by wild-type proteases.10
Work in our laboratory has focused on engineering protein
kinases to uniquely recognize rationally designed small molecule
substrates and inhibitors.3,11-13 Protein kinase catalyzed phos-
phorylation of the hydroxyl moiety of serine, threonine, or
tyrosine is the central posttranslational control element in
eukaryotic signal transduction.14 The phosphorylation state of
a given protein can govern its enzyme activity, protein-protein
binding interactions, and cellular distribution. Phosphorylation
and dephosphorylation is thus a “chemical switch” that allows
the cell to transmit signals from the plasma membrane to the
nucleus to ultimately control gene expression in a highly
regulated manner.15 Highly selective, cell-permeable inhibitors
of individual kinases would allow for the systematic investiga-
tion of the cellular function of a kinase in real time and, thus
would provide invaluable tools for the deconvolution of
phosphorylation-dependent processes in signal transduction
cascades.16-21
The current explosion in the number of newly discovered
genes underscores the need for small molecule ligands that can
be used to elucidate and control gene function. Convergent
engineering of protein/small molecule interfaces has emerged
in recent years as a powerful method for generating novel ligand/
receptor pairs with high specificity.1-3 By introducing chemical
diversity into the target protein as well as the small molecule,
unique binding interactions can be designed and exploited more
efficiently than through traditional medicinal chemistry. Such
approaches have been used to chemically explore a number of
biological systems. FK506-binding protein has been engineered
to preferentially bind nonnatural FK506 analogues by Schreiber
and co-workers,4,5 as well as Clackson and co-workers.6,7 This
system has been used extensively to selectively dimerize
receptors and control gene expression in a cellular context.8
Nuclear hormone receptors have also been shown to be
amenable to chemical genetic design. Corey and co-workers
demonstrated that mutations at two amino acid residues in the
* To whom correspondence should be addressed. E-mail: shokat@
princeton.edu.
† Department of Chemistry.
‡ Department of Molecular Biology.
We recently described a combined chemical and genetic
approach which enables the rapid generation of highly selective
(1) Clackson, T. Curr. Opin. Struct. Biol. 1998, 8, 451-458.
(2) Cohen, P.; Goedert, M. Chem. Biol. 1998, 5, R161-R164.
(3) Bishop, A. C.; Shah, K.; Liu, Y.; Witucki, L.; Kung, C.; Shokat, K.
M. Curr. Biol. 1998, 8, 257-266.
(9) Peet, D. J.; Doyle, D. F.; Corey, D. R.; Mangelsdorf, D. J. Chem.
Biol. 1998, 5, 13-21.
(4) Belshaw, P. J.; Schoepfer, J. G.; Liu, K.-Q.; Morrison, K. L.;
Schreiber, S. L. Angew. Chem., Int. Ed. Engl. 1995, 34, 2129-2132.
(5) Belshaw, P. J.; Schreiber, S. L. J. Am. Chem. Soc. 1997, 119, 1805-
1806.
(6) Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M.; Amara, J. F.;
Rollins, C. T.; Stevenson, L. F.; Magari, S. R.; Wood, S. A.; Courage, N.
L.; Lu, X.; Cerasoli, F. J.; Gilman, M.; Holt, D. A. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 10437-10442.
(7) Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.; Keenan, T.;
Natesan, S.; Pollock, R.; Yang, W.; Courage, N. L.; Holt, D. A.; Gilman,
M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10618-10623.
(8) Klemm, J. D.; Schreiber, S. L.; Crabtree, G. R. Annu. ReV. Immunol.
1998, 16, 569-592.
(10) Smith, G. K.; Banks, S.; Blumenkopf, T. A.; Cory, M.; Humphreys,
J.; Laethem, R. M.; Miller, J.; Moxham, C. P.; Mullin, R.; Ray, P. H.;
Walton, L. M.; Wolfe, L. A., III. J. Biol. Chem. 1997, 272, 15804-15816.
(11) Shah, K.; Liu, Y.; Deirmengian, C.; Shokat, K. M. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 3565-3570.
(12) Liu, Y.; Shah, K.; Yang, F.; Witucki, L.; Shokat, K. Chem. Biol.
1998, 5, 91-101.
(13) Liu, Y.; Shah, K.; Yang, F.; Witucki, L.; Shokat, K. Bioorg. Med.
Chem. 1998, 6, 1219-1226.
(14) Hunter, T. Cell 1995, 80, 225-236.
(15) Shokat, K. M. Chem. Biol. 1995, 2, 509-514.
(16) Levitzki, A.; Gazit, A. Science 1995, 267, 1782-1787.
(17) Chang, C. J.; Geahlen, R. L. J. Nat. Prod. 1992, 55, 1529-1560.
10.1021/ja983267v CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/14/1999