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in deconvoluting both enzyme-substrate and regulatory protein-protein interactions for a number of
signal-transduction pathways (for reviews see Reference 268). This emerging “superfamily” of proteins
includes SH2 and SH3 domains, the so-called “choreographers of multiple signalling pathways,” and
include very intriguing new therapeutic targets [85]. The SH2 domains have been determined to bind
cognate phosphotyrosine (pTyr) containing proteins in a sequence-dependent manner relative to the
amino acids contiguous to the C-terminal side of the pTyr residue (e.g., for Src SH2 a preferred
sequence is ~pTyr-Glu-Glu-Ile~ versus ~pTyr-Tyr-Asn-Tyr for Grb2 [87,269]. The SH3 domains have
been determined to specifically bind Pro-rich sequences of cognate proteins. Interestingly, as a result of
the pseudosymmetrical nature of the SH3 domains there is the possibility of binding both N rarrow.gif
C and C rarrow.gif N directions (e.g., for Src SH3 preferred sequences are ~Arg-Ala-Leu-Pro-Pro-Leu-
Pro-Arg-Tyr and Ala-Phe-Ala-Pro-Pro-Leu-Pro-Arg-Arg, wherein Arg binds to a site 3 pocket [249b]).
With respect to SH2 domain structure-based drug design, the first x-ray crystallographic structures of
pTyr-containing peptide ligands complexed with Src SH2 domain [239] have been utilized to design the
first peptidomimetic antagonists [89]. As illustrated in Figure 30, a molecular map of the tetrapeptide
sequence ~pTyr-Glu-Glu-Ile~ complexed with Src SH2 [239] shows the pTyr binding pocket and a
second binding site for the P +3 Ile residue. As previously described, a prototypic peptidomimetic AcpTyr-Glu-D-Hcy-NH
2 (66) was first discovered by a peptide scaffold design strategy (see Figure 16;
[77]) that took into account the x-ray crystallographic structure of Src SH2-phosphopeptide (Glu-Pro-
Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu, 127) complex. Further structure-based drug design
modifications have led to the discovery a series of potent peptidomimetics having novel C-terminal
functionalization (e.g., “transposed” side chain of the P +1 Glu or conformational constraint using a
pyrrolidine ring; see Figure 30) as represented by 128–130 [89,270]. Studies focused on Src SH2 [88]
have shown that the phosphate ester of pTyr is particularly critical for molecular recognition, and that
significant loss in binding occurs by replacement with sulfate, carboxylate, nitrosyl, hydroxy, and
amino. However, backbone modifications of pTyr which replace its acylated amino functionality with
aromatic rings designed to form π-cation type interactions with the Arg-αA2 were effective substitutions
[271].
Recently, high-resolution 3D structures have been described for the noncatalytic “adapter” protein Grb2
with respect to the apoprotein (SH3-SH2-SH3 [153]) as well as the individual SH2 and SH3 domains
[246 and 252, respectively]. In the case of the SH2 domain of Grb2 an x-ray crystallographic structure
of a phosphopeptide complex provided insight to the molecular basis of the specificity of Grb2 SH2
binding of ~pTyr-Xxx-Asn-Yyy~ sequences. As illustrated in Figure 31, the binding interactions of Lys-
Pro-Phe-pTyr-Val-Asn-
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