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Table 3 SH2 Specificity for Phosphotyrosine-Containing Peptides


SH2 Domain




Glu, Asp, Thr

Glu, Thr, Gln

Glu, Asn, Tyr

Glu, Asp

Ile, Met, Leu

Ile, Val, Met

Glu, Thr, Met

Gln, Thr, Glu


Gln, Tyr, Val

Asn, Glu, Asp

Glu, Gln, Thr



Pro, Val, Leu


Ile, Leu, Met

Tyr, Gln, Phe

Leu, Ile, Val

Val, Ile, Leu

Met, Ile, Val

Ile, Glu, Tyr

Glu, Asp

Ile, Leu

Leu, Ile, Val

Pro, Val, Ile


Ile, Leu, Met


Group 1A



Group 1Ba


Syk (N-SH2)

Syk (C-SH2)


Group 3b

PLCg (N-SH2)

PLCg (C-SH2)

p85 (N-SH2)



Group 1 contains Tyr or Phe at hD5.

Group 3 contains Ile, Cys, or Leu at hD5.

Sources: Refs. 6, 9.


electrostatic interactions, while the pY+3 pocket involves interactions

that are mostly hydrophobic.

Figure 4 represents the specific Src SH2 binding interactions with

pTyr-Glu-Glu-Ile sequences, as interpreted from x-ray structures [10,11].

The major intermolecular interactions in the pY pocket involve the

phosphate oxygens of the ligand pTyr side chain with the conserved basic

residues Arg158 and Arg178 of Src SH2. It is noted that Arg178 mutation

results in essentially a total loss of binding affinity [17]. Additional

intermolecular hydrogen-bonding interactions are also observed with

Ser180, Thr182, and the backbone NH of Glu181, whereas a hydrophobic

contact occurs between the alkyl side chain of Lys206 and the phenyl ring

of the ligand pTyr residue. The two adjacent glutamic acid residues

(pY+1 and pY+2) form relatively weak interactions (electrostatic and

hydrophobic) with the protein, albeit their extend side chain conformations (oriented away from each other) serve to align and rigidify the

peptide backbone. This is an important feature from a drug discovery

perspective and can be used in the design of rigid, nonpeptide templates

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Representation of the binding interactions involving the phosphopeptide motif pTyr-Glu-Glu-Ile with Src SH2 as interpreted from complexed x-ray

structures [10,11]. The binding regions of the protein, including the major pY and

pY+3 pockets, are represented by their key binding residues. Also included are

the observed structural waters and their interactions with the pY+1 Glu and pY+3

Ile phosphopeptide residues.

to advance Src SH2 inhibitors (see later: Sec. IV, Lead Discovery and

Combinatorial Chemistry).

The only direct ligand–protein hydrogen bond contact involves the

backbone NH of the pY+1 Glu with the carbonyl oxygen of the His 204

residue. In addition to the hydrophobic interactions involving the Ile

phosphopeptide residue and the pY+3 pocket, there exist potential

hydrogen-bonding possibilities from Tyr205, Ile217, and a buried

Tyr233 residue. Finally, two structural water molecules provide hydrogen-bonding networks between the pY+1 Glu (CO) and pY+3 Ile (NH)

phosphopeptide residues, and the Lys206 (NH) and Ile217 (CO) Src SH2

protein residues, respectively. Such structural waters act as drug design

elements to increase binding affinity (through favorable entropic contributions) and can be exploited by small molecules that bind to or displace

them (see later: Sec. VI, Structure-Based, Small-Molecule Libraries to

Explore Src SH2 Binding).

The importance of the pTyr group for SH2 binding is counterbalanced by the biological instability of the phosphate group to cellular

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 List of pTyr mimics containing nonhydrolyzable and reduced charge

functionality, which were explored in the context of a pTyr-Glu-Glu-Ile peptide.

(From Ref. 12.)

phosphatases as well as low cellular permeability posed by the highly

charged phosphate group [18]. These issues have prompted the pursuit of

pTyr mimics to discover cellulary active inhibitors. In a comparative

binding study involving pTyr mimics, in the context of a pTyr-Glu-GluIle sequence, researchers explored the ability of a variety of functional

groups to act as pTyr replacements (Fig. 5) [12]. The highest affinity,

nonhydrolyzable pTyr replacement was found to be the F2Pmp (difluorophosphonomethyl phenylalanine) group [19]. Although some of the

aforementioned pTyr replacements represent nonhydrolyzable moieties,

the design of a stable pTyr mimic providing both high affinity and adequate

cell permeability has remained challenging [20].



The integration of structural biology, drug design (molecular modeling

and ‘‘druglike’’ assessment), and synthetic chemistry to discover novel

small-molecule leads follows the general iterative process outlined in

Figure 6. Available structural knowledge is used to design pharmaceutically driven compounds that will bind a desired protein target; these

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 The iterative drug discovery process integrating structural biology,

drug design, synthetic chemistry, biological testing, and additional input from

other related research areas.

compounds are then synthesized and tested in the appropriate assays. The

biological data are analyzed in the context of available (x-ray or NMR)

structural information to impact the design of the next series of analogues. This process is repeated until a lead compound or series of compounds possessing the desired biological activities are obtained.

The database of available structural information during ARIAD’s

initial investigation into compounds targeting Src SH2 was limited; cases

involving ligand complexes utilized only peptide molecules [21]. Motivated

by an interest to develop orally active Src inhibitors (i.e., nonpeptides) we

adopted an exploratory approach to small-molecule lead discovery, using a

combinatorial chemistry strategy. Combinatorial libraries were biased

with a common phenyl phosphate group and systematically engineered

with diversity elements (selection guided by modeling) to probe the protein

surface for existing and new binding interactions (Fig. 7). Solid phase array

synthesis encompassing a novel phosphate ester linker strategy [22] was

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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