Identifying Potent, Selective Protein Tyrosine Phosphatase Inhibitors from a Library of Au(I) Complexes

Karver, Mark R.; Krishnamurthy, D.; Kulkarni, Rhushikesh A.; Bottini, Nunzio; Barrios, Amy

doi:10.1021/jm901220m

Cited by 73 publications

(75 citation statements)

References 45 publications

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“…S6 in the supplemental material). SVZ explants were also treated with a synthetic molecule that blocks the enzymatic activities of PTP-PEST (51). Pharmacologic inhibition of PTP-PEST resulted in the blockade of cell migration from SVZ explants (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Lee

Cheerathodi

Chaki

et al. 2015

Molecular and Cellular Biology

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f Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor ␤8 integrin that plays essential roles in directional cell motility. ␤8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.

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Section: Resultsmentioning

confidence: 99%

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Lee

Cheerathodi

Chaki

et al. 2015

Molecular and Cellular Biology

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“…Replacement of phosphotyrosine with pCAP in known PTP peptide substrates results in efficient fluorogenic probes of in vitro PTP activity. Peptides containing the pCAP moiety provide a sensitive, fluorogenic assay that has been useful in inhibitor screens (12) and combinatorial peptide substrate libraries (13).…”

Section: Resultsmentioning

confidence: 99%

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Stanford

Panchal

Walker

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.single-cell assay | peptide substrate P rotein tyrosine phosphatases (PTPs) are important regulators of signal transduction and are emerging as drug targets for common and debilitating human diseases, including cancer, diabetes, arthritis, and infectious diseases (1). The transmembrane PTP CD45, a critical regulator of signaling through the T-cell receptor (TCR), was one of the first PTPs to be considered as a drug target for treatment of human autoimmune diseases (2). In addition, inhibitors of PTP-1B and SHP-2 currently are being sought for therapy of metabolic diseases and cancer, respectively (3-5). Unfortunately, development of small-molecule inhibitors of PTPs has been a challenging task. Poor cell permeability of PTP inhibitors has been a limitation difficult to overcome in later stages of drug development (5). Several methods to assess intracellular PTP activity are available (6-8), yet no assay has been able to capture intracellular PTP activity at the single-cell level. Cell-based PTP assays that work at the single-cell level and are suited to high-throughput screening might accelerate PTP inhibitor development.In this report we describe the development and optimization of a method to monitor PTP activity at the single-cell level by using cell-permeable fluorogenic peptides that, once delivered into cells, are dephosphorylated by PTPs. The dephosphorylation of these peptides by PTPs generates a signal that can be monitored by flow cytometry and h...

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“…All these results demonstrate that the structures of copper complexes influence the potency and selectivity of PTP inhibition. Compared with other metal complexes, the PTPs inhibition ability of copper complexes is commonly comparable to vanadium complexes Gao et al 2009;Lu et al 2010), stronger than gold complexes which have IC 50 values at micromolar level (Karver et al 2009: Krishnamurthy et al 2008, and much stronger than mononuclear dicitrate iron which displays strong PTPs inhibition at 500 lM (Gomez et al 2010). Our results also show that, although the five copper complexes have obviously different structures, they display little difference in the inhibition of each PTP except PTP-MEG2.…”

Section: Ptp Inhibition Studiesmentioning

confidence: 96%

“…The structures of vanadium complexes influence the inhibition over different PTPs Gao et al 2009;Lu et al 2010). Recent researches reveal other metal complexes such as Au, Cu, Fe complexes are also potent PTP inhibitors Gomez et al 2010;Karver et al 2009: Krishnamurthy et al 2008). Barrios' researches show gold complex auranofin potently and selectively inhibit PTP-PEST with IC 50 of 9 lM over HePTP, CD45, TCPTP, YopH, PTP1B and LYP, while complexes [(p-MeBzMeIm)Au I Cl] and [(BzMeIm)Au I Cl] potently inhibit PTP-PEST, HePTP, PTP1B and LYP with IC 50 from 7 to 40 lM.…”

Section: Introductionmentioning

confidence: 99%

Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives

et al. 2011

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A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV-Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC(50) at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 10(6) and 1.87 × 10(6) M(-1) at 310 K for 2 and 4, respectively.

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Identifying Potent, Selective Protein Tyrosine Phosphatase Inhibitors from a Library of Au(I) Complexes

Cited by 73 publications

References 45 publications

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives

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