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Biomedical subjects

Z J Yao

Publications and source records attributed to Z J Yao.

At least 19 recordsLinked to original sources
PubMed2001

Phospho-Azatyrosine, a less effective protein-tyrosine phosphatase substrate than phosphotyrosine.

Azatyrosine (AzaTyr, 4) is a natural product isolated from Streptomyces chibanesis, whose structure is characterized by a nitrogen atom in the aryl ring of a tyrosyl residue. This seemingly minor modification to the tyrosyl residue results in profound physiological effects, as AzaTyr has been shown to promote permanent reversion of ras-dependent transformed cells to the normal phenotype in culture and to inhibit chemical induction of carcinogenesis in transgenic mice bearing oncogenic human ras. The mechanisms underlying these effects are not known, however ras-pathways involve an intricate balance between both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). The present study was undertaken to examine the general utility of AzaTyr as a structural motif for PTP inhibitor design by examining the phospho-azatyrosine (pAzaTyr)-containing peptide Ac-Asp-Ala-Asp-Glu-pAzaTyr-Leu-amide (8) in a PTP1 enzyme system. Kinetic analysis indicated that 8 binds with a Km value of 210 microM and a catalytic turnover rate, kcat of 52 s(-1). This represents a greater than 50-fold reduction in binding affinity relative to the parent phosphotyrosine-containing peptide, indicating that the aryl nitrogen adversely affects binding affinity. The much lower PTP affinity of the pAzaTyr-containing peptide reduces the potential utility of the AzaTyr pharmacophore for PTP inhibitor design. These results are discussed from the point of view that incorporation of AzaTyr residues into proteins could result in perturbation of protein-tyrosine phosphorylation,dephosphorylation cascades that control signal transduction processes, including ras-dependent pathways.

Alanine↗
PubMed2001

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Evaluation of a recombinant Listeria monocytogenes expressing an HIV protein that protects mice against viral challenge.

Vaccine strategies that utilize cell mediated immunity to control infection will be a necessary component of human immunodeficiency virus (HIV) vaccines. In previous studies we have shown that a Listeria monocytogenes recombinant expressing HIV-Gag elicits strong CD8+ and CD4+ T cell responses against HIV Gag in addition to its own secreted proteins. Here, we show that Lm-Gag can protect mice against a viral challenge with a recombinant vaccinia virus expressing Gag, in an antigen specific manner, and that this protection is T cell mediated. These results further support the use of L. monocytogenes as a vaccine approach for HIV through the induction of T cell immunity.

AIDS Vaccines↗
PubMed2001

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.

Binding Sites↗
PubMed2001

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.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Peptide mimicking antigenic and immunogenic epitope of double-stranded DNA in systemic lupus erythematosus.

Autoantibodies to double-stranded (ds) DNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Identifying dsDNA mimotopes is a way to discover diagnostic and therapeutic candidates for SLE. 'Mono-specific' SLE anti-dsDNA antibodies were obtained by affinity purification using dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide library, we were able to identify a mimotope that has a motif peptide sequence of RLTSSLRYNP. This chemically synthesized peptide could be recognized by 88% (37 out of 42) of anti-dsDNA antibody-positive SLE sera with a cut-off point at mean + 3 SD of the negative control sera at OD(492). The reaction of the peptide with SLE sera in ELISA was highly correlated with that of dsDNA (r = 0.809, P < 0.0001). Of particular interest, not only dsDNA but also single-stranded (ss) DNA and native RNA could inhibit the binding of the peptide with SLE sera, suggesting that the mimotope is shared by ds and ssDNAs as well as native RNA, whereas denatured RNA was not observed to inhibit the binding. The peptide was also able to elicit an immune response in rabbits and the anti-peptide rabbit serum was observed to cross-react with the peptide, ss and dsDNAs, and ss and dsDNAs could inhibit the binding of the anti-peptide serum and the peptide. However, the inhibition was not obtained with RNA. Our findings demonstrate the potential of the peptide mimic in diagnostic tests of SLE, and in the investigation of anti-DNA antibody origin and of DNA-anti-DNA antibody interaction.

Adolescent↗
PubMed2001

[Capillary array electrophoresis and high-throughput DNA sequencing].

Depending on the outputs of 10,080 DNA sequencing samples from the human genome project, technical features of capillary array electrophoresis are discussed. The results show that 88% of the total readlength were higher than 50 kbp/run. It means that most of the readlength could reach 500 bp or more with reasonable quality. The results were much better than expected when the technology of capillary array appeared about one year ago. A key parameter affecting the quality of DNA sequencing is the purity of sequencing template. Therefore, a batch agarose slab electrophoresis was applied to check the purity before loading. Other conditions, such as concentration of template and loading amount, are discussed as well. Finally, comparisons based on the electrophoresis timing, the pre-treatment of samples, the loading procedures and the results analysis between capillary array electrophoresis and slab gel electrophoresis have been made. From the point of view of high-throughput DNA sequencing, especially in the field of genome research, the capillary array electrophoresis should be the better choice as a more efficient technical platform.

Base Sequence↗
PubMed2000

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants.

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.

Amino Acid Sequence↗
PubMed2000

Polyether mimics of naturally occurring cytotoxic annonaceous acetogenins.

On the basis of the ionophore model, polyether analogues 4 and 6 were designed and synthesized to mimic the naturally occurring annonaceous acetogenins corossolin (2) and bullatin (5), which were discovered as members of a large family of novel polyketides with cytotoxicity, antitumoral, and other biological activities since 1982. The preliminary screening shows that they have compatible cytotoxicity with the corresponding natural annonaceous acetogenins. These results open a potential way to find more active antitumor agents with simplified structures based on natural annonaceous acetogenins.

Antineoplastic Agents↗
PubMed2000

Inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. 2. 4-(2-Malonyl)phenylalanine as a potent phosphotyrosyl mimetic.

Nonhydrolyzable phosphotyrosyl (pTyr) mimetics serve as important components of many competitive Grb2 SH2 domain inhibitors. To date, the most potent of these inhibitors have relied on phosphonate-based structures to replace the 4-phosphoryl group of the parent pTyr residue. Reported herein is the design and evaluation of a new pTyr mimetic, p-malonylphenylalanine (Pmf), which does not contain phosphorus yet, in Grb2 SH2 domain binding systems, approaches the potency of phosphonate-based pTyr mimetics. When incorporated into high affinity Grb2 SH2 domain-directed platforms, Pmf is 15-20 times more potent than the closely related previously reported pTyr mimetic, O-malonyltyrosine (OMT). Pmf-containing inhibitors show inhibition constants as low as 8 nM in extracellular Grb2 binding assays and in whole cell systems, effective blockade of both endogenous Grb2 binding to cognate erbB-2, and downstream MAP kinase activation. Evidence is provided that use of an N(alpha)()-oxalyl auxiliary enhances effectiveness of Pmf and other inhibitors in both extracellular and intracellular contexts. As one of the most potent Grb2 SH2 domain-directed pTyr mimetics yet disclosed, Pmf may potentially have utility in the design of new chemotherapeutics for the treatment of various proliferative diseases, including breast cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Potent inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands.

Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has important implications for treatment of a variety of diseases, including several cancers. In cellular studies, inhibitors of Grb2 SH2 domain binding have to date been large, highly charged peptides which relied on special transport devices for cell membrane penetration. Work presented in the current study examines a variety of pTyr mimetics in the context of a high-affinity Grb2 binding platform. Among the analogues studied are new non-phosphorus-containing pTyr mimetics 23a and 23b which, when incorporated into tripeptide structures 18f and 20f, are able to inhibit Grb2 SH2 domain binding with affinities among the best yet reported for non-phosphorus-containing SH2 domain inhibitors (IC50 values of 6.7 and 1.3 microM, respectively). The present study has also demonstrated the usefulness of the Nalpha-oxalyl group as an auxiliary which enhances the binding potency of both phosphorus- and non-phosphorus-containing pTyr mimetics. When combined with the (phosphonomethyl)phenylalanine (Pmp) residue to give analogues such as L-20d, potent inhibition of Grb2 SH2 domain binding can be achieved both in extracellular assays using isolated Grb2 SH2 domain protein and in intracellular systems measuring the association of endogenous Grb2 with its cognate p185erbB-2 ligand. These latter effects can be achieved at micromolar to submicromolar concentrations without prodrug derivatization. The oxalyl-containing pTyr mimetics presented in this study should be of general usefulness for the development of other Grb2 SH2 domain antagonists, independent of the beta-bend-mimicking platform utilized for their display.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A phosphotyrosyl mimetic peptide reverses impairment of insulin-stimulated translocation of GLUT4 caused by overexpression of PTP1B in rat adipose cells.

Biological actions of insulin are initiated by activation of the insulin receptor tyrosine kinase. Protein tyrosine phosphatases (PTPases) PTP1B and PTPalpha are known to dephosphorylate the insulin receptor and may contribute to insulin resistance in diseases such as diabetes. We previously reported that overexpression of PTP1B in rat adipose cells significantly impairs insulin-stimulated translocation of GLUT4 [Chen, H., et al. (1997) J. Biol. Chem. 272, 8026]. In the present study, we treated adipose cells with a PTPase inhibitor containing the phosphotyrosyl mimetic difluorophosphonomethyl phenylalanine (F2Pmp) to determine whether we could improve the insulin resistance caused by overexpression of PTP1B or PTPalpha. Rat adipose cells transfected by electroporation with either PTP1B or PTPalpha were treated without or with the inhibitor, and effects on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. The IC50 of the F2Pmp-containing inhibitor is 180 nM for PTP1B and 10 mM for PTPalpha in vitro. As expected, in the absence of the inhibitor, overexpression of either PTP1B or PTPalpha caused a significant decrease in the amount of GLUT4 at the cell surface both in the absence and in the presence of insulin when compared with control cells transfected with epitope-tagged GLUT4 alone. Interestingly, the insulin resistance caused by overexpression of PTP1B (but not PTPalpha) was reversed by treating the transfected cells with the F2Pmp-containing inhibitor. Furthermore, the inhibitor blocked the insulin-stimulated association of PTP1B with the insulin receptor. We conclude that the F2Pmp-containing compound is a potent and specific inhibitor of overexpressed PTP1B that may be useful for designing rational therapies for treating insulin resistant diseases such as diabetes.

Adipocytes↗
PubMed1998

Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics.

Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of phosphotyrosine mimetics, the (difluoronaphthylmethyl)phosphonic acids and the fluoromalonyl tyrosines, have been determined to resolutions greater than 2.3 A. The fluoromalonyl tyrosine residue was incorporated within a cyclic hexapeptide modeled on an autophosphorylation site of the epidermal growth factor receptor. The structure of this inhibitor bound to PTP1B represents the first crystal structure of a non-phosphonate-containing inhibitor and reveals the mechanism of phosphotyrosine mimicry by the fluoromalonyl tyrosine residue and the nature of its interactions within the catalytic site of PTP1B. In contrast to complexes of PTP1B with phosphotyrosine-containing peptides, binding of the fluoromalonyl tyrosine residue to the catalytic site of PTP1B is not accompanied by closure of the catalytic site WPD loop. Structures of PTP1B in complex with the (difluoronaphthylmethyl)phosphonic acid derivatives reveal that substitutions of the naphthalene ring modulate the mode of inhibitor binding to the catalytic site and provide the potential for enhanced inhibitor affinity and the generation of PTP-specific inhibitors. These results provide a framework for the rational design of higher affinity and more specific phosphotyrosine mimetic inhibitors of not only protein tyrosine phosphatases but also SH2 and PTB domains.

Binding Sites↗
PubMed1998

An immunogenic epitope of Chlamydia pneumoniae from a random phage display peptide library is reactive with both monoclonal antibody and patients sera.

Random 15-mer peptides displayed on filamentous phages were screened in binding studies using a Chlamydia pneumoniae-specific monoclonal antibody (RR-402) and affinity-purified, polyclonal sera from patients seropositive for C. pneumoniae infections by the microimmunofluorescence (MIF) test. One 15-mer epitope, epitope Cpnl5A (LASLCNPKPSDAPVT) was identified in both the monoclonal and polyclonal screenings, and showed higher ELISA reactivity with C. pneumoniae MIF-positive sera compared to patients with other chlamydial infections, non-chlamydial respiratory infections and normal healthy sera (MIF-negative). Interestingly, epitope Cpnl5A also showed significant (52%) amino acid sequence homology to the 56 kDa type-specific antigen of Rickettsia tsutsugamushi, a protein implicated in the virulence of this organism.

Amino Acid Sequence↗
PubMed1998

Structure-based design and synthesis of small molecule protein-tyrosine phosphatase 1B inhibitors.

Protein-tyrosine phosphatase (PTP) inhibitors are attractive as potential signal transduction-directed therapeutics which may be useful in the treatment of a variety of diseases. We have previously reported the X-ray structure of 1,1-difluoro-1-(2-naphthalenyl)methyl] phosphonic acid (4) complexed with the human the protein-tyrosine phosphatase 1B (PTP1B) and its use in the design of an analogue which binds with higher affinity within the catalytic site (Burke, T. R., Jr. et al. Biochemistry 1996, 35, 15989). In the current study, new naphthyldifluoromethyl phosphonic acids were designed bearing acidic functionality intended to interact with the PTP1B Arg47, which is situated just outside the catalytic pocket. This residue has been shown previously to provide key interactions with acidic residues of phosphotyrosyl-containing peptide substrates. Consistent with trends predicted by molecular dynamics calculations, the new analogues bound with 7- to 14-fold higher affinity than the parent 4, in principal validating the design rationale.

Drug Design↗