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

Abstract

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...

CD45-mediated control of TCR tuning in naïve and memory CD8+ T cells

“…5f), caused a prominent increase in p-ERK induction following CD3 ligation (Fig. 6a); this applied to three additional CD45-specific inhibitors (NSC95397, R164259 and S349631)35 with a similar and even more marked enhancing effect relative to CD45i (Fig. 6b,c and Supplementary Fig.…”

“…LCK inhibitor PP2 and various PTP inhibitors, LYPi II, PTPi IV, PTPi XVIII, HePTPi, SHP1/2i, CD45i, NSC95397 and vanadate (Na 3 VO 4 ) were all purchased from Calbiochem and dissolved in DMSO (Sigma-Aldrich). Two CD45-specific inhibitors, R164259 and S34963135, were generously provided by N. Bottini (La Jolla Institute for Allergy and Immunology).…”

pCAP-based peptide substrates: The new tool in the box of tyrosine phosphatase assays

“…Peptide 19 could be introduced into cells via microinjection (sea urchin oocytes [17]) or electroporation (Jurkat T cells [17]). The addition of a basic and hydrophobic tag, either in a linear peptide ( peptide 20 ) or a cyclic peptide ( peptide 21 ), facilitated spontaneous uptake primarily to the cytosol in a variety of cell types, including Jurkat T cells, RAW 264.7 macrophages and MCF-7 cells [17, 18]. In all cases, the internalized pCAP peptide was rapidly dephosphorylated by intracellular phosphatases to produce fluorescent cells.…”

“…Importantly, the pCAP peptides can be hydrolyzed only by PTP activity. Serine/threonine phosphatases are not able to dephosphorylate the pCAP peptides, and kinases are unable to appreciably rephosphorylate the resulting CAP peptides [17, 18]. …”

Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

“…We recently reported the development of a single-cell pCAP peptide-based assay for PTP activity, which is amenable to either high-content microscopy or flow cytometry (108) (Fig. 5).…”