[Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.
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ABSTRACT:
SUBMITTER: Desmarais S
PROVIDER: S-EPMC1219955 | biostudies-other | 1999 Jan
REPOSITORIES: biostudies-other
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Project description:Proper control of the phosphotyrosine content in signal transduction proteins is essential for normal cell behavior and is lost in many pathologies. Attempts to normalize aberrant tyrosine phosphorylation levels in disease states currently involve either the application of small compounds that inhibit tyrosine kinases (TKs) or the addition of growth factors or their mimetics to boost receptor-type TK activity. Therapies that target the TK enzymatic counterparts, the multi-enzyme family of protein tyrosine phosphatases (PTPs), are still lacking despite their undisputed involvement in human diseases. Efforts to pharmacologically modulate PTP activity have been frustrated by the conserved structure of the PTP catalytic core, providing a daunting problem with respect to target specificity. Over the years, however, many different protein interaction-based regulatory mechanisms that control PTP activity have been uncovered, providing alternative possibilities to control PTPs individually. Here, we review these regulatory principles, discuss existing biologics and proteinaceous compounds that affect PTP activity, and mention future opportunities to drug PTPs via these regulatory concepts.
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Project description:A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes.
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Project description:Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upon subsequent treatment with chymotrypsin. Analysis by HPLC showed that chymotrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. Thus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed between PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding (32)P-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic substrate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme preparations. The lower detection limit of our assay (1 microM substrate in 200 microliter of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was estimated to be about 1 pmol in the assay that used (32)P-labelled peptide (specific radioactivity of approx. 1000 c.p.m. /pmol). Our assay is simple, specific, highly sensitive and non-radioisotopic, and hence would contribute greatly to the development of PTP biology.
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Project description:In all, 15 aryl-containing phosphonates have been synthesized and tested for their effect on protein-tyrosine phosphatase (PTPase) activity. Two compounds, (naphth-2-yl) difluoromethylphosphonic acid (12) and (napthy-1-yl) difluoromethylphosphonic acid (13) have been found to inhibit dephosphorylation of [32P]insulin receptors by PTP-1B, a protein tyrosine phosphatase (PTPase), with IC50 values of 40-50 microM. Compound 12 competitively inhibited insulin-receptor dephosphorylation by PTP-1B. Compound 12 also inhibited PTP-1B-catalysed dephosphorylation of a synthetic tyrosine phosphorylated substrate poly(Glu80-Tyr20) at the same potency, indicating that 12 acted via interaction with the PTPase. Additionally, 12 inhibited insulin-receptor PTPase(s) and epridermal-growth-factor-receptor PTPase(s) present in solubilized membranes from CHO (Chinese-hamster ovary)/HIRc and A431 cells respectively. IC50 values of 40-50 microM were obtained in all cases with compound 12. Of note is the fact that these compounds did not have any effect on insulin-receptor autophosphorylation. Nine out of the 15 compounds potently inhibited serine/threonine phosphatase PP-2A activity without any effect on serine/threonine phosphatase PP-1 when tested at a concentration as high as 675 microM. The most potent compounds acting toward PP-2A had IC50 values of 45-50 microM. These PP-2A inhibitors could be useful tools for studying serine/threonine-phosphatase-mediated signal transduction. Two compounds, 12 and 13, inhibited both tyrosine phosphatase PTP-1B and serine/threonine phosphatase PP-2A with similar potency; IC50 values being 40-50 microM in both cases. Details of the synthesis of compounds 10, 11 and 13 are given in Supplementary Publication SUP 50177 (6 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1995) 305, 9.
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Project description:Several protein tyrosine phosphatases (PTPs), particularly PTPN1, PTPN2, PTPN6, PTPN9, PTPN11, PTPRS, and DUSP9, are involved in insulin resistance. Therefore, these PTPs could be promising targets for the treatment of type 2 diabetes. Our previous studies revealed that PTPN2 and PTPN6 are potential antidiabetic targets. Therefore, the identification of dual-targeting inhibitors of PTPN2 and PTPN6 could be a potential therapeutic strategy for the treatment or prevention of type 2 diabetes. In this study, we demonstrate that methyl syringate inhibits the catalytic activity of PTPN2 and PTPN6 in vitro, indicating that methyl syringate acts as a dual-targeting inhibitor of PTPN2 and PTPN6. Furthermore, methyl syringate treatment significantly increased glucose uptake in mature 3T3-L1 adipocytes. Additionally, methyl syringate markedly enhanced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in 3T3L1 adipocytes. Taken together, our results suggest that methyl syringate, a dual-targeting inhibitor of PTPN2 and PTPN6, is a promising therapeutic candidate for the treatment or prevention of type 2 diabetes.
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Project description:Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases (PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a useful tyrosine surrogate during peptide library screening for optimal PTP substrates.
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Project description:Protein tyrosine phosphatases (PTPs) are a large family of 107 signaling enzymes that catalyze the hydrolytic removal of phosphate groups from tyrosine residues in a target protein. The phosphorylation status of tyrosine residues on proteins serve as a ubiquitous mechanism for cellular signal transduction. Aberrant function of PTPs can lead to many human diseases, such as diabetes, obesity, cancer, and autoimmune diseases. As the number of disease relevant PTPs increases, there is urgency in developing highly potent inhibitors that are selective towards specific PTPs. Most current efforts have been devoted to the development of active site-directed and reversible inhibitors for PTPs. This review summarizes recent progress made in the field of covalent inhibitors to target PTPs. Here, we discuss the in vivo and in vitro inactivation of various PTPs by small molecule-containing electrophiles, such as Michael acceptors, α-halo ketones, epoxides, and isothiocyanates, etc. as well as oxidizing agents. We also suggest potential strategies to transform these electrophiles into isozyme selective covalent PTP inhibitors.
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Project description:Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN-triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN-triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (kcat/KM) by more than 102-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN-triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.
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Project description:Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival. Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome. Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for functional characterization of these enzymes. To facilitate the global analysis of PTPs, we designed and synthesized two activity-based probes that consist of alpha-bromobenzylphosphonate as a PTP-specific trapping device and a linker that connects the trapping device with a biotin tag for visualization and purification. We showed that these probes are active site-directed irreversible inactivators of PTPs and form a covalent adduct with PTPs involving the active site Cys residue. Additionally, we demonstrated that the probes are extremely specific toward PTPs while remaining inert to other proteins, including the whole proteome from Escherichia coli. Consequently, these activity-based PTP probes can be used to profile PTP activity in complex proteomes. The ability to interrogate the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets.
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Project description:The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs). Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs). In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs). Although PTPs have already been well-characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.
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