4740 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Brief Articles
(3) Futterer, K.; Wong, J .; Grucza, R. A.; Chan, A. C.; Waksman,
G. Structural basis for syk tyrosine kinase ubiquity in signal
transduction pathways revealed by the crystal structure of its
regulatory SH2 domains bound to a dually phosphorylated ITAM
peptide. J . Mol. Biol. 1998, 281, 523-537.
(4) Kihara, H.; Siraganian, R. P. Src homology 2 domains of Syk
and Lyn bind to tyrosine-phosphorylated subunits of the high
affinity IgE receptor. J . Biol. Chem. 1994, 269, 22427-22432.
(5) Wan, Y.; Kurosaki, T.; Huang, X. Y. Tyrosine kinases in
activation of the MAP kinase cascade by G-protein-coupled
receptors. Nature 1996, 380, 541-544.
(6) Ottinger, E. A.; Botfield, M. C.; Shoelson, S. E. Tandem SH2
domains confer high specificity in tyrosine kinase signaling. J .
Biol. Chem. 1998, 273, 729-735.
(7) Schoepfer, J .; Fretz, H.; Gay, B.; Furet, P.; Garcia-Echeverria,
C.; End, N.; Caravatti, G. Highly potent inhibitors of the Grb2-
SH2 domain. Bioorg. Med. Chem. Lett. 1999, 9, 221-226.
(8) Yao, Z.-J .; King, C. R.; Cao, T.; Kelley, J .; Milne, G. W. A.; Voigt,
J . H.; Burke, T. R., J r. Potent inhibition of Grb2 SH2 domain
binding by non-phosphate-containing ligands. J . Med. Chem.
1999, 42, 25-35.
because of the small numbers of observed NOEs and
1
some H-15N cross-peaks from this region were more
broadened than those from other regions, which may
result from conformational exchange phenomena. Flex-
ibility of the pY pocket may be the reason that recogni-
tion in the pY pocket is allowed.
Crystallographic studies of the SykCSH2 indicated
that the methylene part of the glutamic acid of the
phosphopeptide at the pY+1 position interacted hydro-
phobically with LeuâD3, TyrâD5, and LeuBG5, whereas
the carboxylic group of the glutamic acid of the phos-
phopeptide at the pY+1 position was too far from the
amine group of LysâD1 to form a hydrogen bond (5.3
Å).3 Conversely, in our model, the carboxylic group of
12 is located within hydrogen-bonding distance of the
amine group of LysâD1 (Figure 1b) because 12 has a
methylene chain long enough to interact with the amine
group of LysâD1. In fact, compound 9, which has only
three methylene groups, did not show a binding affinity
for SykCSH2. The length of the methylene chain of 12
seems to be important for binding to SykCSH2.
(9) Gao, Y.; Luo, J .; Yao, Z. J .; Guo, R.; Zou, H.; Kelley, J .; Voigt, J .
H.; Yang, D.; Burke, T. R., J r. Inhibition of Grb2 SH2 domain
binding by non-phosphate-containing ligands. 2. 4-(2-Malonyl)-
phenylalanine as a potent phosphotyrosyl mimetic. J . Med.
Chem. 2000, 43, 911-920.
(10) Shakespeare, W. C.; Bohacek, R. S.; Azimioara, M. D.; Macek,
K. J .; Luke, G. P.; Dalgarno, D. C.; Hatada, M. H.; Lu, X.;
Violette, S. M.; Bartlett, C.; Sawyer, T. K. Structure-Based
Design of Novel Bicyclic Nonpeptide Inhibitors for the Src SH2
Domain. J . Med. Chem. 2000, 43, 3815-3819.
(11) Vu, C. B.; Corpuz, E. G.; Merry, T. J .; Pradeepan, S. G.; Bartlett,
C.; Bohacek, R. S.; Botfield, M. C.; Eyermann, C. J .; Lynch, B.
A.; MacNeil, I. A.; Ram, M. K.; van Schravendijk, M. R.; Violette,
S.; Sawyer T. K. Discovery of Potent and Selective SH2 Inhibi-
tors of the Tyrosine Kinase ZAP-70. J . Med. Chem. 1999, 42,
4088-4098.
(12) Burke, T. R., J r.; Smyth, M.; Nomizu, M.; Otaka, A.; Roller, P.
P. Preparation of fluoro- and hydroxy-4-phosphonomethyl-D,L-
phenylalanine suitably protected for solid-phase synthesis of
peptides containing hydrolytically stable analogues of O-phos-
photyrosine. J . Org. Chem. 1993, 58, 1336-1340.
The R1 moiety of 12 (5-methylhexyl) and the side
chain of Ile at the pY+3 position in the phosphopeptide
are well superimposed (Figure 1c). The isopropyl group
interacts hydrophobically with IleâE4, GlyEF3, GlyBG3,
and LeuBG4. Compounds 15 and 16, which have a
cyclohexane ring instead of an isopropyl group at the
pY+3 position, showed a 2-fold improvement of inhibi-
tory activity. Plummer et al. have also reported that
cyclohexane rings were effective pY+3 Ile side chain
replacements for the Src SH2 domain.17
Con clu sion
We applied a structure-based in-silico screening as
well as a docking simulation to the discovery of SykC-
SH2 inhibitors. In this trial, cyclohexylmalonic acid was
identified as a novel pTyr mimetic. It exhibited a higher
affinity (IC50 ) 5.9 mM) than pTyr itself (IC50 ) 14 mM)
and phenylmalonic acid (IC50 ) 12 mM). Cyclohexyl-
malonic acid was also effective in the linked compound.
The most active compound, 16, exhibited activity com-
parable to that of the monophosphorylated peptide
ligand (VpYTGLS). Further optimization of fragments
corresponding to each pocket and that of the linker
would lead to more potent inhibitors.
(13) Ye, B.; Akamatsu, M.; Shoelson, S. E.; Wolf, G.; Giorgetti-Peraldi,
S.; Yan, X. J .; Roller, P. P.; Burke, T. R. L-O-(2-malonyl)-
tyrosine: A new phosphotyrosyl mimetic for the preparation of
Src homology 2 domain inhibitory peptides. J . Med. Chem. 1995,
38, 4270-4275.
(14) Narula, S. S.; Yuan, R. W.; Adams, S. E.; Green, O. M.; Green,
J .; Philips, T. B.; Zydowsky, L. D.; Botfield, M. C.; Hatada, M.;
Laird, E. R.; Zoller, M. J .; Karas, J . L.; Dalgarno, D. C. Solution
structure of the C-terminal SH2 domain of the human tyrosine
kinase Syk complexed with a phosphotyrosine pentapeptide.
Structure 1995, 3, 1061-1073.
(15) Orita, M.; Yamamoto, S.; Katayama, N.; Aoki, M.; Takayama,
K.; Yamagiwa, Y.; Seki, N.; Suzuki, H.; Kurihara, H.; Sakashita,
H.; Takeuchi, M.; Fujita, S.; Yamada, T.; Tanaka, A. Coumarin
and chromen-4-one analogues as tautomerase inhibitors of
macrophage migration inhibitory factor: discovery and X-ray
crystallography. J . Med. Chem. 2001, 44, 540-547.
(16) (a) Kuntz, I. D. Structure-based strategies for drug design and
discovery. Science 1992, 257, 1078-1082. (b). Shoichet, B. K.;
Stroud, R. M.; Santi, D. V.; Kuntz, I. D.; Perry, K. M. Structure-
based discovery of inhibitors of thymidylate synthase. Science
1993, 259, 1445-1450.
Ack n ow led gm en t. This work was supported finan-
cially by Grant-in-Aid for Specially Promoted Research
from the Ministry of Education, Science, Sport and
Culture, J apan.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the protein structure analyses, measurements of
binding affinity, and the synthesis of 11-17 with analytical
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(17) Plummer, M. S.; Lunney, E. A.; Para, K. S.; Shahripour, A.;
Stankovic, C. J .; Humblet, C.; Fergus, J . H.; Marks, J . S.;
Herrera, R.; Hubbell, S.; Saltiel, A.; Sawyer, T. K. Design of
peptidomimetic ligands for the pp60src SH2 domain. Bioorg.
Med. Chem. 1997, 5, 41-47.
(18) Nicholls, A.; Sharp, K. A.; Honig, B. Protein folding and
association: insights from the interfacial and thermodynamic
properties of hydrocarbons. Proteins: Struct., Funct., Genet.
1991, 11, 281-296.
(19) Farrow, N. A.; Muhandiram, R.; Singer, A. U.; Pascal, S. M.;
Kay, C. M.; Gish, G.; Shoelson, S. E.; Pawson, T.; Forman-Kay,
J . D.; Kay, L. E. Backbone dynamics of a free and phosphopep-
tide-complexed Src homology 2 domain studied by 15N NMR
relaxation. Biochemistry 1994, 33, 5984-6003.
Refer en ces
(1) (a) Kurosaki, T. Molecular mechanisms in B cell antigen receptor
signaling. Curr. Opin. Immunol. 1997, 9, 309-318. (b) Turner,
M.; Schweighoffer, E.; Colucci, F.; Di Santo, J . P.; Tybulewicz,
V. L. Tyrosine kinase SYK: essential functions for immunore-
ceptor signalling. Immunol. Today 2000, 21,148-154.
(2) Bu, J . Y.; Shaw, A. S.; Chan, A. C. Analysis of the interaction of
ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen
receptor by plasmon resonance. Proc. Natl. Acad. Sci. U.S.A.
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