High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45.
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.
Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
Stephanie M
SM
Stanford
Rekha G
RG
Panchal
Logan M
LM
Walker
Dennis J
DJ
Wu
Matthew D
MD
Falk
Sayantan
S
Mitra
Sagar S
SS
Damle
David
D
Ruble
Teodora
T
Kaltcheva
Sheng
S
Zhang
Zhong-Yin
ZY
Zhang
Sina
S
Bavari
Amy M
AM
Barrios
Nunzio
N
Bottini
D000881:Anthrax / Q000188:drug therapy / Q000378:metabolism; D001408:Bacillus anthracis; D019610:Cytoprotection / Q000187:drug effects; D055808:Drug Discovery; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology
D004791:Enzyme Inhibitors; D009842:Oligopeptides; C047135:protein C activator peptide; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D017027:Protein Tyrosine Phosphatases