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Updated: May 19, 2026
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of
Stephanie M Stanford1, Rekha G Panchal, Logan M Walker
1Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
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.
Insights
Researchers developed a novel assay to track protein tyrosine phosphatase activity within single cells. This method identified cell-permeable inhibitors for CD45, a key target for autoimmune and infectious diseases.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Tyrosine phosphorylation regulates cellular signaling via protein tyrosine kinases and phosphatases.
- Protein tyrosine phosphatases are promising drug targets, but cell permeability issues hinder inhibitor development.
- Monitoring phosphatase activity at the single-cell level is crucial for drug discovery.
Purpose of the Study:
- To develop a method for monitoring protein tyrosine phosphatase activity at the single-cell level.
- To identify cell-permeable inhibitors targeting protein tyrosine phosphatases, specifically CD45.
- To validate a new assay for high-throughput screening of phosphatase inhibitors.
Main Methods:
- Utilized fluorogenic phosphorylated coumaryl amino propionic acid (pCAP) analogs incorporated into peptides.
- Delivered pCAP peptides into cells to monitor dephosphorylation by protein tyrosine phosphatases.
- Employed flow cytometry and high-content imaging to measure cell fluorescence and validate assay sensitivity.
- Screened for CD45 inhibitors using the developed assay.
Main Results:
- Successfully monitored single-cell protein tyrosine phosphatase activity using pCAP peptides.
- Validated assay robustness and sensitivity with CD45 and T-cell tyrosine phosphatase.
- Identified four novel cell-permeable CD45 inhibitors with activity in T cells and macrophages.
- Demonstrated the assay's applicability for primary screening of protein tyrosine phosphatase inhibitors.
Conclusions:
- The developed assay enables single-cell monitoring of protein tyrosine phosphatase activity.
- This method facilitates the discovery of cell-permeable inhibitors for therapeutic targets like CD45.
- The assay has broad potential for screening inhibitors of various protein tyrosine phosphatases, improving drug development yields.
