Development of Grb2 SH2 Domain Signaling Antagonists: A Potential New Class of Antiproliferative Agents

Aberrant signaling through protein-tyrosine kinase (PTK)-dependent pathways is associated with several proliferative diseases. Accordingly, PTK inhibitors are being developed as new approaches for the treatment of certain cancers. Growth factor receptor bound protein 2 (Grb2) is an important downstream mediator of PTK signaling that serves obligatory roles in many pathogenic processes. One of the primary functions of Grb2 is to bind to specific phosphotyrosyl (pTyr)-containing sequences through its Src homology 2 (SH2) domain. Agents that bind to the Grb2 SH2 domain and prevent its normal function could disrupt associated PTK signaling and serve as alternatives to kinase-directed inhibitors. Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these efforts. The presentation will be thematically arranged according to the region of peptide modified, proceeding from the N-terminus to the C-terminus, with a special section devoted to aspects of conformational constraint.


INTRODUCTION
Proliferative diseases often involve aberrations in protein-tyrosine kinase (PTK) dependent signaling (Blume-Jensen and Hunter, 2001), and for this reason PTK inhibitors are beginning to emerge as a new class of anticancer therapeutics (Shawver et al., 2002). However, blockade of oncogenic PTK signaling can be approached by means other than kinasedirected agents. Illustrative of this, the growth factor receptor bound protein 2 (Grb2) (Lowenstein et al., 1992) is a 25 kDa non-catalytic Src homology 2 (SH2) domain-containing protein providing connectivity downstream of PTKs that has been shown to be associated with a number of cancers (Million and Van Etten, 2000;Tari and Lopez-Berestein, 2001;Saucier et al., 2002), including breast cancer (Daly et al., 1994). Significant effort has been devoted to developing Grb2 SH2 domain-binding antagonists as potential therapeutics. Because the recognition and binding to phosphotyrosyl (pTyr) containing sequences by the Grb2 SH2 domain is critical for its proper function, most inhibitor development has focused on peptide and peptide mimetic-based analogues Vu, 2000;Garcia-Echeverria, 2001). Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these latter efforts. The reader is referred to an earlier review for a particularly fine presentation of work through the year 2000 .

RELATIONSHIP OF GRB2 SH2 DOMAIN-BINDING INTERACTIONS TO INHIBITOR DESIGN
SH2 domains comprise a family of proteins characterized by similar overall structures that can be described using a common nomenclature. This allows a systematic designation of individual protein residues based on consensus motifs (Eck et al., 1993;Waksman et al., 1993). Shared topographical features among SH2 domains include three principal antiparallel b-sheets (bB-bD) lying between C-proximal (aA) and N-proximal (aB) a-helixes, with shorter b-sheets (bE and bF) and several short loops (BC, EF and BG) connecting these elements (Songyang, 1999). The binding of pTyr-containing peptides to SH2 domains typically involves nestling of the critical pTyr residue into a positively-charged pocket lying under the aA helix next to the BC loop. Ligand residues C-proximal to the pTyr residue provide for differential affinity among various SH2 domain family members by extending in linear fashion through a 'specificity' region of the protein bounded by the EF and BG loops. The consensus sequence for peptides binding to the Grb2 SH2 is pTyr-(X+1)-Asn-(X+3), (where residue numbering starts at the pTyr residue and proceeds in the C-terminal direction) with the pTyr+2 Asn serving as the specificity determining residue (Songyang et al., 1994;Kessels et al., 2002). X-ray crystal structures of peptides complexed to Grb2 SH2 domains have shown that ligand cannot extend in the normal fashion between the EF and BG loops, because this region is blocked by the indole side chain of Trp EF1 (Rahuel et al., 1996Ettmayer et al., 1999;Furet et al., 1999;Schiering et al., 2000;Nioche et al., 2002). This blockade results in the preferential binding of peptides to Grb2 SH2 domains in type-I b-turn conformations (Rahuel et al., 1996). Figure 1 summarizes important Grb2 SH2 domain-binding interactions of the BCR-Abl 174-180 peptide Lys-Pro-Phe-pTyr-Val-Asn-Val, including its adaption of a b-turn conformation (Rahuel et al., 1996). Since it had been observed that the N-terminal Lys and Pro residues do not exhibit specific interactions with the protein, these were omitted from the Fig. 1. X-ray crystal structure of the BCR-Abl 174-180 sequence KPFpYVNV complexed to the Grb2 SH2 domain as reported in (Rahuel et al., 1996) with N-terminal Lys and Pro residues having been omitted for clarity. Important protein residues discussed in the text have been rendered in CPK style and labeled. Figure. However the carbonyl oxygen and phenyl ring of the pTyr-1 Phe residue do engage in hydrogen bonding and van der Waals interactions, respectively, with the Arg aA2 guanidinium group and these were retained in the Figure. The critical pTyr phosphoryl group forms hydrogen bonds with the Arg aA2 and Arg bB5 side chains, while the pTyr phenyl ring undergoes hydrophobic interactions with side chain methylenes of Lys bD6 in addition to p-cation interactions with the Arg aA2 guanidinium group. The pTyr+1 Val residue exhibits hydrophobic interactions with the Phe bD5 and Gln bD3 side chains, while the carboxamido side chain group of the critical pTyr+2 Asn residue forms three hydrogen bonds with the Lys bD6 and Leu bE4 residues in the 'specificity determining pocket.' Finally, the pTyr+3 Val residue rests on a hydrophobic patch formed by the side chains of the Lys bD6 and Leu bD1 and bounded by Phe bE3. In the remainder of this review each of the above regional interactions will be used as the basis for summarizing studies directed toward the development of Grb2 SH2 domain-binding antagonists.

AMINO-TERMINAL MODIFICATIONS
Amino-terminal modifications have most often been directed at enhancing interactions with the Arg aA2 guanidinium group. Furet et al. found that an approximate 7-fold increase in binding affinity resulted relative to H-pTyr-Ile-Asn-amide (1a), by introducing an N-terminal carboxamido moiety provided either by an acetyl group (1b) or by a Glu residue (1c) (Fig. 2) (Furet et al., 1997). Surprisingly, acylation of the Glu residue with a 2-aminobenzoyl group (Abz) increased affinity more than 300-fold (peptide 1d). X-ray crystallographic structure determination of 1d complexed to the Grb2 SH2 domain showed that the 2-amino group formed a salt bridge with the pTyr phosphoryl group, thereby positioning the Abz phenyl ring for efficient p-cation stacking with the Arg aA2 guanidinium group . A similar, though slightly less potent effect could be achieved by acylating the pTyr residue directly with a 3-aminobenzyloxycarbonyl ((3-amino)Z) group (peptide 1e).  (Furet et al., 1997). phosphoryl group was shown by the nearly 100-fold loss of affinity following removal of the 3-amino group (peptide 1f).
Using as a display platform, a tripeptide disclosed by Furet et al. (2, Fig. 3) , Burke et al. examined a series of N-terminal amides containing carboxyl and tetrazolyl groups intended to undergo ionic interactions with the Arg aA2 guanidinium group (peptides 2a-2e, Fig. 3) . The N-oxalyl moiety (2b) provided the best affinity enhancement. Although affinity was only approximately 3-fold greater than the N-Acetyl containing analogue (2a) in extracellular binding assays, potency in whole cells was increased in spite of the fact that the N-oxalyl group would have been expected to adversely affect cellular bioavailability .

pTyr MIMETICS
Interactions within the SH2 domain pTyr-binding pocket are central to overall ligand affinity, with ionic bonding between the phosphoryl group and the Arg bB5 residue being particularly important (Bradshaw et al., 1999). However, the pTyr phosphoryl group (3a, Fig. 4) presents physiochemical properties that are unsuitable for therapeutically-relevant SH2 domainbinding antagonists. These properties include poor bioavailability due to the di-anionic nature of the phosphoryl group at pH 7 and hydrolytic lability of the phosphoryl ester to phosphatases. Accordingly, significant effort has been devoted to developing pTyr mimetics that address these drawbacks while retaining recognition within the pTyr-binding pocket Burke and Lee, 2003). Among the di-acidic pTyr mimetics that have been successfully used in high affinity Grb2 SH2 domain-binding antagonists are phosphorus-containing (phosphonomethyl)phenylalanine (Pmp, 3b) and (difluorophosphonomethyl)phenylalanine (F 2 Pmp, 3c) . Non-phosphorus-containing analogues include the malonyl-containing O-malonyltyrosine (OMT, 3d) (Ye et al., 1995), O-fluoromalonyltyrosine (FOMT, 3e) and p-malonylphenylalanine (3f) (Gao et al., 2000). Modification of OMT by translocation of one carboxyl from the malonyl group to the 3-position of the tyrosyl ring provided analogue 3g .

MODIFICATIONS TO THE pTyr+1 POSITION
Exploiting X-ray crystallographic data of Grb2 SH2 domain complexed with a peptide ligand showing that the pTyr+1 residue adapts a 3 10 helical structure (Rahuel et al., 1996), Garcia-Echeverria et al. substituted the pTyr+1 residue of the peptide Ac-pTyr-Val-Asn-amide (5b, Fig. 6) with a series of bend-inducing cycloalkyl amino acids . The intent was to promote a local 3 10 helical structure and to afford van der Waals interactions with the side chains of Phe bD5 and Gln bD3. Starting with a 3-membered ring (Ac 3 c, 5c), binding affinity increased progressively with ring size, reaching a maximum with 1-aminocyclohexane carboxylic acid (Ac 6 c, 5f) ( Fig. 6). Binding affinity fell off with the larger, 7-membered ring (Ac 7 c, 5g).
pTyr+3 REPLACEMENTS X-ray crystal structures of ligand-complexed Grb2 SH2 domain reveal the presence of a lipophilic region formed by the side chains of the Lys bD6 and Leu bD1 and bounded by Phe bE3, which is situated proximal to the binding region of the pTyr+3 residue (Rahuel et al., 1996;Furet et al., 1998). Through a systematic study, Furet et al. showed that replacement of the pTyr+3 residue with a 3-naphthalen-propyl amide significantly enhanced binding affinity, particularly when used in combination with a pTyr+1 Ac 6 c residue (8a, Fig. 9) . Subsequently using a convergent synthetic approach, Schoepfer et al. prepared a series of hydroxy and methoxy-substituted congeners, including 8b and 8c, that exhibited higher binding affinity (Schoepfer et al., 2001). In further work, Schoepfer replaced the naphthyl ring with a variety of substituted indolyl rings   Fig. 7. Examination of bend-inducing pTyr mimetics at the i+1 position as reported in (Liu et al., 1999).
of propyl amide attachment (for example, 8d as compared to 8e) and substituents appended to the indolyl ring, highly potent binding inhibitors could be obtained. These included compounds such as 8f that were the first analogues reported to exhibit subnanomolar Grb2 SH2 domain-binding affinities. Higher affinity of indolyl vs. naphthyl-containing compounds was attributed to enhanced CH/p interactions of the former with segments of the Lys bD6 and Leu bD1 side chains that formed the lipophilic binding surface.

USE OF CONFORMATIONAL CONSTRAINT
The net Grb2 SH2 domain-binding affinity of flexible analogues is adversely affected by free energy terms derived from the ordering of ligands in solution to conformations needed for binding. Theoretically, affinity enhancement can be achieved through reduction of these entropy penalties by induction of conformational constraint. This has already been shown above by the beneficial binding effects of pTyr+1 bend-inducing residues such as Ac 6 c (5f). In the following, further examples will be provided where induction of conformational constraint has been applied to the design of Grb2 SH2 domainbinding ligands. The approaches presented can be categorized generally into local conformational constraint and global conformational constraint.

Monomeric Constrained pTyr Mimetics
Introduction of ring structures into amino acid analogues can be used to achieve conformational constraint. The bicyclic indanyl-containing analogue 10 and the tricyclic methanobenzazocino compound 11 were designed to contain within their frameworks, pTyr residues having v 2 (for 10) and /, v 1 and v 2 (for 11) angles constrained to values approximating those needed for binding as determined by X-ray crystal structures of SH2 domain-bound pTyr residues (9) (Fig. 10)  . Although this approach is conceptually appealing, little if any affinity enhancement is achieved, and the binding constants of pTyr itself and the constrained analogues 10 and 11 are in the millimolar range Wang et al., 2003).

Constrained pTyr Mimetics Within Peptide Platforms
Conformationally constrained pTyr mimetics have also been examined within peptide platforms. Analogues 12 (Plake et al., 2003) and 13   (Fig. 11) utilized cyclopropyl and cyclohexyl rings, respectively, to lock both / and v angles. While peptide 12 maintained a binding on-rate similar to the parent pTyr-containing congener, analogue 13 exhibited a greater than two orders of magnitude loss of binding affinity relative to the un-constrained parent. A similarly large loss of binding affinity was observed for compound 14, which made an alternate use of a cyclohexyl moiety to constrain v 1 and v 2 angles (Oishi et al., 2004). To date, local conformational constraint of the pTyr mimicking residue has not proven to be a useful approach toward affinity enhancement of Grb2 SH2 domain-binding ligands.  Fig. 8. Replacement of the pTyr+2 Asn residue as reported in    [Schoepferet al., (2001)]  [Schoepferet al., (2001)] ] ]

Global Conformational Constraint Through Head-to-Tail Cyclization pTyr-Containing Peptides
While local conformational constraint of pTyr mimetics has not proven beneficial to date, the preferential binding of Grb2 SH2 domain peptides in b-bend conformations has made induction of global conformational constraint through macrocyclization an important field of investigation. Ettmeyer et al. examined a series of cyclic peptides (15) based on the sequence pTyr-Val-Asn-Val-Pro) (Fig. 12) (Ettmayer et al., 1999). These included analogues 15a (IC 50 = 0.06 lM) and 15b (IC 50 =0.07 lM), which exhibited approximately 10-fold higher affinity than the open chain congeners such as 15c (IC 50 =1.63 lM). This is in contrast to the findings of Dekker et al. that ring closing metathesis (RCM) of open-chain 16 (K d =0.44 lM) to unsaturated 17 (K d =0.60 lM) and saturated 18 (K d =0.48 lM) failed to enhance binding affinity ( Fig. 13) (Dekker et al., 2003).

Cyclic Peptides Based a Non-pTyr-Containing Phage Library Lead
High affinity binding to the SH2 domains normally requires a pTyr or pTyr-mimicking residue that can form ionic interactions with positively-charged residues within the pTyr binding pocket, particularly with the Arg bB5 residue (Bradshaw et al., 1999). In this light, a noteworthy body of work has appeared on several Grb2 SH2 domain-binding peptides whose designs are predicated on a non-pTyr-containing disulfide-cyclized phage library-derived lead termed 'G1' (19a, IC 50 =10-25 lM) and its thioether homologue 'G1TE' (19b, IC 50 =20 lM) (Fig. 14) (Oligino et al., 1997). Through the preparation of an extensive series of analogues, binding affinity was enhanced to low nanomolar values (Li et al., 2003a). The use of a Tyr instead of a pTyr in the original G1TE (19b) with maintenance of binding affinity, was shown to be due to the compensatory action of a Glu residue in the Tyr-2 position that supplied anionic interactions with the critical protein Arg bB5 residue (Li et al., 2003c). Further modifications including addition of a second carboxyl group to the Glu residue, replacement of the Tyr residue with a 3-amino-Tyr residue and conversion of the ring-closing thioether to an (R)-sulfoxide, lead to cyclic peptide 20, which exhibited an IC 50 value of 50 nM (Song et al., 2004). High affinity variants of these peptides have been prepared that utilize pTyr mimetics, such as 21, IC 50 =2.4 nM.   Fig. 13. RCM-derived peptides described in (Dekker et al., 2003).   [Shi et al., 2003[Shi et al., , 2004Kang et al., (2005) Kang et al., (2005)] [Kang et al., (2005)] [Kang et al., (2005)] [Kang et al., (2005)] [Kang et al., (2005)] However, these require that the compensatory acidic residue at the Tyr-2 position also be replaced by a non-acidic residue, such as Ala (Li et al., 2003b).

Macrocycles Derived From Ring Closure at the pTyr Mimetic b-Methylene
Molecular modeling studies of open-chain peptides such as 8a (Fig. 9) bound to the Grb2 SH2 domain indicated that the b-methylene of the N-terminal pTyr mimicking residue was in close proximity to C2 of the C-terminal naphthylpropylamido group . Ring closure from these two positions using RCM chemistries yielded the new family of macrocycles shown in Fig. 15 Wei et al., 2003). Macrocycle 24, which lacked any functionality at the pTyr mimetic a-position, exhibited an IC 50 value of 20 nM in extracellular ELISA binding assays, yet was unable to block the intracellular association of Grb2 with cytoplasmic erbB-2 PTK in whole cells following extracellular administration . However, introduction of an a-carboxymethyl group onto 24 to yield 25 not only resulted in an order of magnitude enhancement of binding affinity in extracellular assays, but also imparted the ability to block the association of Grb2 with cytoplasmic erbB-2 PTK in whole cells following extracellular administration (Wei et al., 2003). By analogy to SAR studies conducted on open-chain peptides (Fig. 9), the naphthyl ring of 25 was replaced with a 5-hydroxynaphthyl group (compound 26) (Shi et al., 2005b) or a 5methylindolyl group (compound 27) (Shi et al., 2003(Shi et al., , 2004; Kang et al., 2005). This resulted in potency enhancement, such that 27 was able to block the association of Grb2 with cytoplasmic erbB-2 PTK in whole cells at low nanomolar concentrations when given in extracellular medium. Interestingly, the absolute configuration at the C2 ring juncture has little effect on binding affinity (Shi et al., 2005a). A variety of phosphoryl-mimicking moities were also examined (compounds 28-34), including several mono-acidic groups that provided low nanomolar binding affinities (Kang et al., 2005).
A modification of this approach has recently been reported, whereby allylglycine residues were used to form the C-terminal ring-closing segments ( (Fig. 16). Similar to above, the absolute configuration at the C-terminal ring junction had little effect on binding affinity.

CELLULAR STUDIES
The highly polar nature of pTyr and pTyr mimicking residues limits cell membrane transport of many Grb2 SH2 domain-binding antagonists. However, whole cell studies conducted following prodrug derivatization of the inhibitor phosphoryl or phosphonic acid moieties have been reported. Work done by Gay et al. using CGP78850 (compound 41 ,  Fig. 18), a selective Grb2 SH2 domain-binding inhibitor having a mixed phosphoramidite/phenyl ester -protected phosphonic acid group, showed that this compound could block the association of Grb2 with activated epidermal growth factor receptor (EGFR) in living cells and inhibit the growth of cells driven by PTK signaling through Ras (Gay et al., 1999a). Further studies showed that CGP78850 could inhibit hepatocyte growth factor (HGF)-induced cell motility (Gay et al., 1999b). Using a monobenzyl phosphoryl protection protocol Garbay and co-workers showed that compound 42 could block soft agar colony formation of HER2 transfected cells with a sub-micromolar IC 50 value . In work that did not rely on prodrug protection, Bottaro and co-workers showed that compound 43, which employs a malonyl-based phosphoryl mimetic, was able to block HGF-induced cell motility, matrix invasion and morphogenesis with IC 50 values of 300 nM or less (Atabey et al., 2001;Soriano et al., 2004).

CONCLUSIONS
This review has presented an overview of Grb2 SH2 domain-directed inhibitor development. Although several high affinity inhibitors have been reported, including agents that exhibit potent effects in cellular systems, studies in whole animal cancer models have yet to appear. This may be due in part to the success of PTK inhibitors that has muted enthusiasm within the pharmaceutical industry for SH2 domain-directed inhibitors. It may also reflect scale up and bioavailability issues presented by some of the most promising inhibitors. Only time will tell whether these latter issues will be overcome to a sufficient extent to allow clinical trials of this class of agents.