Examination of novel non-phosphorus-containing phosphotyrosyl mimetics against protein-tyrosine phosphatase-1B and demonstration of differential affinities toward Grb2 SH2 domains

Inhibitory potencies were compared of several mono- and dicarboxy-based pTyr mimetics in Grb2 SH2 domain versus PTP1B assays. Although in both systems pTyr residues provide critical binding elements, significant differences in the manner of recognition exist between the two. This is reflected in the current study, where marked variation in relative potencies was observed between the two systems. Of particular note was the poor potency of all monocarboxy-based pTyr mimetics against PTP1B when incorporated into a hexapeptide platform. The recently reported high PTP1B inhibitory potency of similar phenylphosphate mimicking moieties displayed in small molecule, non-peptide structures, raises questions on the limitations of using peptides as platforms for pTyr mimetics in the discovery of small molecule inhibitors.     

Phosphoryltyrosyl mimetics in the design of peptide-based signal transduction inhibitors

The central roles played by protein-tyrosine kinase (PTK)-dependent signal transduction in normal cellular regulation and homeostasis have made inappropriate or aberrant functions of certain of these pathways contributing factors to a variety of diseases, including several cancers. For this reason, development of PTK signaling inhibitors has evolved into an important approach toward new therapeutics. Since in these pathways phosphotyrosyl (pTyr) residues provide unique and defining functions either by their creation under the catalysis of PTKs, their recognition and binding by protein modules such as SH2 and phosphotyrosyl binding (PTB) domains, or their destruction by protein-tyrosine phosphatases, pTyr mimetics provide useful general starting points for inhibitor design. Important considerations in the development of such pTyr mimetics include enzymatic stability (particularly toward PTPs), high affinity recognition by target pTyr binding proteins, and good cellular bioavailability. Although small molecule, nonpeptide inhibitors may be ultimate objectives of inhibitor development, peptides frequently serve as display platforms for pTyr mimetics, which afford useful and conceptually straightforward starting points in the development process. Reported herein is a limited overview of pTyr mimetic development as it relates to peptide-based agents. Of particular interest are recent findings that highlight potential limitations of peptides as display platforms for the identification of small molecule leads. One conclusion that results from this work is that while peptide-based approaches toward small molecule inhibitor design are often intellectually satisfying from a structure-based perspective, extrapolation of negative findings to small molecule, nonpeptide contexts should be undertaken with extreme caution.     

Tripeptide inhibitors of Yersinia protein-tyrosine phosphatase

The protein-tyrosine phosphatase (PTP) 'YopH' is a virulence factor of Yersinia pestis, the causative agent of plague. Potential use of Yersinia as a bioterrorism agent renders YopH inhibitors of therapeutic importance. Previously, we had examined the inhibitory potencies of a variety of phosphotyrosyl (pTyr) mimetics against the human PTP1B enzyme by displaying them in the EGFR-derived hexapeptide sequence, 'Ac-Asp-Ala-Asp-Glu-Xxx-Leu-amide', where Xxx=pTyr mimetic. The poor inhibitory potencies of certain of these pTyr mimetics were attributed to restricted orientation within the PTP1B catalytic pocket incurred by extensive peripheral interaction of the hexapeptide platform. Utilizing the smaller tripeptide platform, 'Fmoc-Glu-Xxx-Leu-amide' we demonstrate herein that several of the low affinity hexapeptide-expressed pTyr mimetics exhibit high PTP1B affinity within the context of the tripeptide platform. Of particular note, the mono-anionic 4-(carboxydifluoromethyl)Phe residue exhibits affinity equivalent to the di-anionic F(2)Pmp residue, which had previously been among the most potent PTP-binding motifs. Against YopH, it was found that all tripeptides having Glu residues with an unprotected side chain carboxyl were inactive. Alternatively, in their Glu-OBn ester forms, several of the tripeptides exhibited good YopH affinity with the mono-anionic peptide, Fmoc-Glu(OBn)-Xxx-Leu-amide, where Xxx=4-(carboxymethyloxy)Phe providing an IC(50) value of 2.8 microM. One concern with such inhibitors is that they may potentially function by non-specific mechanisms. Studies with representative inhibitors, while failing to provide evidence of a non-specific promiscuous mode of inhibition, did indicate that non-classical inhibition may be involved.     

New syntheses of tetrazolylmethylphenylalanine and O-malonyltyrosine as pTyr mimetics for the design of STAT3 dimerization inhibitors

Investigation within the pTyr-binding pocket of the STAT3 SH2 domain led us to develop a novel synthesis of two pTyr mimetics, l-tetrazolylmethylphenylalanine (l-Tmp) and l-O-malonyltyrosine (l-OMT), that were next incorporated in a high affinity ligand of STAT3 SH2 domain. Biological evaluation of peptidomimetics on STAT3 dimerization identified l-OMT as the first non-phosphorus pTyr mimetic so far reported against STAT3 SH2 domain, harboring an activity similar to that of the Pmp-containing reference peptidomimetic.     

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.     

Enantioselective synthesis of (S)-3-carboxy-4-((carboxy)difluoromethyl)phenylalanine in protected form and its incorporation into a PTP-1B-directed tripeptide

Recent findings have shown that, when expressed in the tripeptide platform, 'Fmoc-Glu-Xxx-Leu-amide', the phosphotyrosyl mimetic (pTyr), Xxx=(S)-3-carboxy-4-(carboxymethyl)-Phe, provides higher PTP-1B affinity than that obtained with Xxx=(S)-difluorophosphonomethyl-Phe (F(2)Pmp). This was of note, since difluorophosphonomethyl-containing pTyr mimetics have typically exhibited higher PTP-inhibitory potencies than carboxy-based mimetics, indicating the potential value of 3-carboxy-4-(carboxymethyl)-Phe as a starting point for further analogue development. Therefore, relying on precedence that alpha-fluorination often enhances PTP-1B affinity, (S)-3-carboxy-4-((carboxy)difluoromethyl)-Phe was designed as a PTP-1B-directed pTyr mimetic. Reported herein is the synthesis of this new amino acid analogue through application of commercially available Williams chiral auxiliary. The target was prepared in an orthogonally protected form suitable for peptide synthesis according to Fmoc chemistries and utilization for the synthesis of a PTP-1B-directed tripeptide bearing the sequence indicated above. Biological evaluation with in vitro PTP-1B assays indicated nearly total loss of affinity for this peptide. Evidence is provided that the unexpected deleterious effect of fluorination is probably not related to pK(a) effects.     

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.     

Monocarboxylic-based phosphotyrosyl mimetics in the design of GRB2 SH2 domain inhibitors

Three monocarboxylic-containing analogues, O-carboxymethyltyrosine (cmT, 5), 4-(carboxymethyl)phenylalanine (cmF, 6), and 4-(carboxydifluoromethyl)phenylalanine (F2cmF, 7) were utilized as phosphotyrosyl (pTyr) replacements in a high affinity B-bend mimicking platform, where they exhibited IC50 values of 2.5 microM, 65 microM and 28 microM, respectively, in a Grb2 SH2 domain Biacore binding assay. When a terminal N(alpha)-oxalyl axillary was utilized to enhance ligand interactions with a critical SH2 domain Arg67 residue (alphaA-helix), binding potencies increased from 4- to 10-fold, resulting in submicromolar affinity for cmF (IC50 = 0.6 microM) and low micromolar affinity for F2cmF (IC50 = 2 microM). Cell lysate binding studies also showed inhibition of cognate Grb2 binding to the p185erbB-2 phosphoprotein in the same rank order of potency as observed in the Biacore assay. These results indicate the potential value of cmF and F2cmF residues as pTyr mimetics for the study of Grb2 SH2 domains and suggest new strategies for improvements in inhibitor design.     

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.     

Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.     

Synthesis of fluorine-containing bioisosteres corresponding to phosphoamino acids and dipeptide units

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine-containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF(2)) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF(2)-substituted pThr mimetics and development of a two-step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that gamma,gamma-difluoro-alpha,beta-enoates were reduced to gamma-fluoro-beta,gamma-enoates by organocopper reagents and then applied to the synthesis of (Z)-fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper-mediated reduction led us to development of a SmI(2)-mediated approach toward the synthesis of the fluoroalkene isosteres.     

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.     

Synthesis and biological evaluation of (3′-amino-[1,1′-biphenyl]-4-yl) sulfamic acid derivatives as novel HPTPβ inhibitors

A series of novel (3′-amino-[1,1′-biphenyl]-4-yl) sulfamic acid derivatives were designed as nonphosphonate-based phosphotyrosy (pTyr) mimetics, synthesized and screened for use as HPTPβ inhibitors. Compounds C22 and C2 showed favorable HPTPβ inhibitory activity and better selectivity for HPTPβ than for PTP1B and SHP2. Docking results suggested that compounds C2 and C22 could not only efficiently fit into the catalytic site of the HPTPβ enzyme but also interact with the Lys1807, Arg1809 and Lys1811 residues of the secondary binding site, which was next to the catalytic center of the enzyme. The mode of interaction of the synthesized compound with the protein was different from the one found in a complex crystal of small molecules with HPTPβ (2I4H), in which the inhibitory molecule formed hydrogen bonds with the Gln1948 and Asn1735 residues of the secondary binding site.     

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.     

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.     

Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Global impact of oncogenic Src on a phosphotyrosine proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.