N-terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding.

High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that N(alpha)-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-NH(2) motifs, significant potency enhancement can be incurred by N(alpha)-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-NH(2). Using ligands based on the high affinity pY-Ac(6)c-Asn-(naphthylpropylamide) motif, (where Ac(6)c=1-aminocyclohexanecarboxylic acid), additional reports have shown moderate potentiating effects of N(alpha)-oxalyl derivatization. The current study examined variations of the N(alpha)-oxalyl theme in the context of a Xxx-Ac(6)c-Asn-(naphthylpropylamide) platform, where Xxx=the hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of N(alpha)-(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH(2) could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was found for the Pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality with a tetrazole isostere, resulted in binding potency greater than the parent N(alpha)-acetyl-containing compound, with enhancement approximating that observed for the N(alpha)-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC(50) values were observed for the series. Examination of the N(alpha)-(3-amino)Z derivatized Pmp-Ac(6)c-Asn-(naphthylpropylamide), showed that binding affinity was reduced relative to the parent N(alpha)-acetyl analogue, in contrast to the reported significant enhancement of affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the N(alpha)-oxalyl and N(alpha)-malonyl-containing compounds exhibiting IC(50) values (4.3 and 4.6 microM, respectively) approximately five-fold lower than the parent N(alpha)-acetyl-containing compound. Tetrazole and N(alpha)-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays.     

Design of tetrapeptide ligands as inhibitors of the Src SH2 domain

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide pTyr-Glu-Glu-Ile (pYEEI) binds to Src SH2 domain with high affinity (K(d)=100 nM). The development of five classes of tetrapeptides as inhibitors for the Src SH2 domain is described. Peptides were prepared via solid-phase peptide synthesis and tested for affinity to Src SH2 domain using a fluorescence polarization based assay. All of the N-terminal substituted pYEEI derivatives (class II) presented binding affinity (IC(50)=of 2.7-8.6 microM) comparable to pYEEI (IC(50)=6.5 microM) in this assay. C-Terminal substituted pYEEI derivatives (class III) showed a lower binding affinity with IC(50) values of 34-41 microM. Amino-substituted phenylalanine derivatives (class IV) showed weak binding affinities (IC(50)=16-153 microM). Other substitutions on phenyl ring (class I) or the replacement of the phenyl ring with other cyclic groups (class V) dramatically decreased the binding of tetrapeptides to Src SH2 (IC(50)>100 microM). The ability of pYEEI and several of the tetrapeptides to inhibit the growth of cancer cells were assessed in a cell-based proliferation assay in human embryonic kidney (HEK) 293 tumor cells. The binding affinity of several of tested compounds against Src SH2 domain correlates with antiproliferative activity in 293T cells. None of the compounds showed any significant antifungal activity against Candida albicans ATCC 14053 at the maximum tested concentration of 10 microM. Overall, these results provided the structure-activity relationships for some FEEI and YEEI derivatives designed as Src SH2 domain inhibitors.     

Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).     

Development of a phosphatase-stable phosphotyrosyl mimetic suitably protected for the synthesis of high-affinity Grb2 SH2 domain-binding ligands

Synthesis of (2R)-2-carboxymethyl-3-(4-(phosphonomethyl)phenyl) proprionic acid (5) in tert-butyl-protected form (6) and its use for the preparation of a Grb2 SH2 domain-directed tripeptide (8a) is reported. In extracellular ELISA-based assays, 8a exhibits potent Grb2 SH2 domain binding affinity (IC(50)=8 nM). Against cultures of MDA-MB-453 breast cancer cells, which over-express erbB-2 tyrosine kinase, 8a is also antimitogenic at concentrations equivalent to those required to inhibit intracellular association of Grb2 protein with phosphorylated p185(erbB-2) protein (IC(50)=8 microM). Analogue 6 may be useful for the preparation of a variety of phosphatase-stable SH2 domain-directed ligands.     

A novel macrocyclic tetrapeptide mimetic that exhibits low-picomolar Grb2 SH2 domain-binding affinity

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic (5) that binds to Grb2 SH2 domain protein with K(d)=75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC(50)<10nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations (IC(50)=0.6 microM). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents.     

Novel peptide inhibitors for Grb2 SH2 domain and their detection by surface plasmon resonance.

One of the critical intracellular signal transduction pathways involves the binding of the Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factor receptors, such as epidermal growth factor receptor (EGFR) and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for the development of potential anticancer agents. As a continuation of our earlier work, herein we report the design and synthesis of new peptide analogs, and their inhibitory effect on the Grb2 SH2 domain using surface plasmon resonance (SPR) technology. These novel agents do not contain phosphotyrosine or phosphotyrosine mimics. Binding interactions between these peptides and the Grb2 SH2 domain were measured and analyzed using a BIAcore X instrument, which provides detailed information on the real-time detection of the binding interaction. The results of this study should provide important information for the further development of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain.     

Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide.

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.     

Structure-based design of thioether-bridged cyclic phosphopeptides binding to Grb2-SH2 domain

A series of phosphotyrosine containing cyclic peptides was designed and synthesized based upon the phage library derived cyclopeptide, G1TE. Considering the type-I beta-turn feature of peptidic ligand binding to Grb2 SH2 domain, we introduce alpha,alpha-disubstituted cyclic amino acid, Ach, into the 4th position of the cyclic peptide to induce a local right handed 3(10) helical conformation. In order to stabilize the favorable binding conformation, the bulky and hydrophobic amino acids, neopentylglycine (NPG) and phenylalanine, were introduced into the 8th and 2nd positions of the peptide ligand, respectively. To facilitate the sidechain of pTyr3 reaching into the phosphotyrosine binding pocket, a less bulky alanine was preferred in position 1. Based upon these global modifications, a highly potent peptide ligand 12 was discovered with an IC(50)=1.68 nM, evaluated by ELISA binding essay. Ligand 12 is at least 10(5) more potent than the lead peptide, termed G1TE.     

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGo<1.4 kcal/mol) reduction in affinity, indicating that Arg betaB5 is the single most important determinant of pTyr recognition. However, mutation of Cys betaC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg betaB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.     

Characterization of the beta-dystroglycan-growth factor receptor 2 (Grb2) interaction

The beta-dystroglycan/Grb2 interaction was investigated and a proline-rich region within beta-dystroglycan that binds Grb2-src homology 3 domains identified. We used surface plasmon resonance (SPR), fluorescence analysis, and solid-phase binding assay to measure the affinity constants between Grb2 and the beta-dystroglycan cytoplasmic tail. Analysis of the data obtained from SPR reveals a high-affinity interaction (K(D) approximately 240 nM) between Grb2 and the last 20 amino acids of the beta-dystroglycan carboxyl-terminus, which also contains a dystrophin-binding site. A similar K(D) value (K(D) approximately 280 nM) was obtained by solid-phase binding assay and in solution by fluorescence. Both Grb2-SH3 domains bind beta-dystroglycan but the N-terminal SH3 domain binds with an affinity approximately fourfold higher than that of the C-terminal SH3 domain. The Grb2-beta-dystroglycan interaction was inhibited by dystrophin in a range of concentration of 160-400 nM. These data suggest a highly regulated and dynamic dystrophin/dystroglycan complex formation and that this complex is involved in cell signaling.     

Structure-based design of novel nonpeptide inhibitors of the Src SH2 domain:phosphotyrosine mimetics exploiting multifunctional group replacement chemistry

A series of novel nonpeptide inhibitors of the pp60(c-Src) (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x-ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono- or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4' and 3' substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA, 2000, Vol. 97, pp. 9373-9378). These pTyr mimetics included 4',3'-diphosphono-Phe (Dpp), 4',3'-dicarboxymethyloxy-Phe (Dcp), and 4'-phosphono-3'-carboxymethyloxy-Phe (Cpp). Noteworthy were nonpeptide inhibitors 8-11 that were 5- to 10-fold more potent than the cognate tetrapeptide ligand Ac-pTyr-Glu-Glu-Ile-NH(2) in binding to the Src SH2 domain.     

A phosphotyrosine displacement mechanism for activation of Src by PTPalpha

Protein tyrosine phosphatase alpha (PTPalpha) is believed to dephosphorylate physiologically the Src proto-oncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPalpha overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPalpha is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPalpha activity. We show here that this phosphorylation also specifically enables PTPalpha to dephosphorylate pTyr527. Tyr789-->Phe mutation abrogates PTPalpha-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPalpha in vivo. We suggest that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, we find that excess SH2 domains can disrupt PTPalpha-Src binding and can block PTPalpha-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, we show that, as predicted by the model, catalytically defective PTPalpha has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways.     

Development of l-3-aminotyrosine suitably protected for the synthesis of a novel nonphosphorylated hexapeptide with low-nanomolar Grb2-SH2 domain-binding affinity

Synthesis of orthogonally protected (2S)-2-amino-3-(3-amino-4-hydroxy-phenyl)-propionic acid (10) suitable for solid phase peptide synthesis and its first use for the preparation of nonphosphorylated Grb2-SH2 domain antagonists (4a-c) are reported. The 3-aminotyrosine containing sulfoxide-cyclized hexapeptide (4b) exhibited potent Grb2-SH2 domain binding affinity with IC50 = 50 nM, which represents the highest affinity yet reported for a peptide inhibitor against Grb2-SH2 domain with only 6 residues free of phosphotyrosine or phosphotyrosine mimics. This potent small peptidomimetic 4b may be representative of a new class of therapeutically relevant Grb2-SH2 domain-directed agents, and acts as a chemotherapeutic lead for the treatment of erbB2-related cancers.     

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.     

Macrocyclization in the design of a conformationally constrained Grb2 SH2 domain inhibitor.

Grubbs' olefin metathesis reaction was utilized to prepare a macrocyclic variant of a linear Grb2 SH2 domain antagonist in an attempt to induce a beta-bend conformation known to be required for high affinity binding. In extracellular Grb2 SH2 domain binding assays, the macrocyclic analogue exhibited an approximate 100-fold enhancement in binding potency relative to its linear counterpart. The macrocycle was not as effective in whole cell binding assays as would be expected based on its extracellular binding potency.     

Natural and synthetic analogues of actinomycin D as Grb2-SH2 domain blockers

Natural analogues (D, C2, and VII) of actinomycin inhibit Grb2 SH2 domain binding with phosphopeptide-derived from Shc in vitro and in intracellular system. To study structure-activity relationships, 13 actinomycin analogues were synthesized and we found that the inhibition activity depended on the substituents of cyclic peptide groups in actinomycin and two analogues with Tyr residue are the most potent inhibitors with IC50 value of 0.5 and 0.8 microM, respectively.     

Structural basis for specificity switching of the Src SH2 domain

The Src SH2 domain binds pYEEI-containing phosphopeptides in an extended conformation with a hydrophobic pocket, which includes ThrEF1, binding Ile(pY +3). Mutating ThrEF1 to tryptophan switches specificity to an Asn(pY +2) requirement, yielding a biological mimic of the Grb2 SH2 domain. Here we show that the Src ThrEF1Trp SH2 domain mutant binds pYVNV phosphopeptides in a beta turn conformation, which, despite differing conformations of the interacting tryptophan, closely resembles the native Grb2/pYVNV cognate peptide binding mode. The ThrEF1Trp substitution therefore switches specificity by physically occluding the pTyr +3 binding pocket and by providing additional interaction surface area for Asn(pY +2). This demonstrates structurally how novel SH2 domain specificities may rapidly evolve through single amino acid substitutions and suggests how new signaling pathways may develop.     

Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.     

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity

Src‐homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7‐18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7‐18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7‐18NATE is specific for the Grb7‐SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7‐18NATE binds with micromolar binding affinity to Grb7‐SH2 domain (K_D = 4–6 μm ) compared with 50–200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2‐(N‐Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7‐18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7‐18NATE binding to the Grb7‐SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition. Copyright © 2011 John Wiley & Sons, Ltd.     

The difluoromethylenesulfonic acid group as a monoanionic phosphate surrogate for obtaining PTP1B inhibitors

Three peptides, 7-9, bearing sulfono(difluoromethyl)phenylalanine (F(2)Smp, 2), a nonhydrolyzable, monoanionic phosphotyrosine mimetic, were prepared and evaluated as PTP1B inhibitors. The most effective inhibitor was the nonapeptide, ELEF(F(2)Smp)MDYE-NH(2), (9) which exhibited a K(i) of 360 nM. A comparison of F(2)Smp-bearing peptides 7 [DADE(F(2)Smp)LNH(2), K(i)=3.4 microM] and 8 [EEDE(F(2)Smp)LNH(2), K(i)=0.74 microM] with their phosphono(difluoromethyl)phenylalanine (F(2)Pmp)-bearing analogues indicated that F(2)Smp is not as effective a pTyr mimetic as F(2)Pmp by 100- to 130-fold. Although F(2)Smp is not as effective as F(2)Pmp, a comparison of peptide 7 with analagous peptides bearing other monoanionic pTyr mimetics recently reported in the literature indicates that F(2)Smp is about 65-fold more effective than any other non-hydrolyzable, monanionic pTyr mimetic reported to date. To further assess the difluoromethylenesulfonic acid (DFMS) group as a monoanionic phosphate mimetic, a series of 24 nonpeptidyl biaryl compounds bearing the DFMS group were prepared using polymer-supported methodologies and screened for PTP1B inhibition. Several of these compounds were selected for further study and their IC(50)'s compared to their difluoromethylenephosphonic (DFMP) analogues. The differences in IC(50)'s between the DFMS and DFMP non-peptidyl compounds was not as great as with the F(2)Smp- and F(2)Pmp-bearing peptides. Possible reasons for this and its implication to the design of small molecule PTP1B inhibitors is discussed.