An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

Sheng Zhang, Lan Chen, Sanjai Kumar, Li Wu, David S. Lawrence, Zhong-Yin Zhang

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.

Original languageEnglish
Pages (from-to)261-267
Number of pages7
JournalMethods
Volume42
Issue number3
DOIs
StatePublished - Jul 2007

Fingerprint

Protein Tyrosine Phosphatases
Fluorescence Polarization
Assays
Fluorescence
Polarization
Catalytic Domain
Mutant Proteins
Alkylating Agents
Alkylation
Medical problems
Phosphoric Monoester Hydrolases
Oxidants
Libraries
Screening
Substitution reactions
Adhesion
Throughput
Apoptosis
Ligands
Oxidation

Keywords

  • Assay development
  • Fluorescence polarization (FP)
  • High throughput screening (HTS)
  • Protein tyrosine phosphatases (PTPs)
  • PTP inhibitor

ASJC Scopus subject areas

  • Molecular Biology

Cite this

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases. / Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin.

In: Methods, Vol. 42, No. 3, 07.2007, p. 261-267.

Research output: Contribution to journalArticle

Zhang, Sheng ; Chen, Lan ; Kumar, Sanjai ; Wu, Li ; Lawrence, David S. ; Zhang, Zhong-Yin. / An affinity-based fluorescence polarization assay for protein tyrosine phosphatases. In: Methods. 2007 ; Vol. 42, No. 3. pp. 261-267.
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