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Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically
K I Skorey1, B P Kennedy, R W Friesen
1Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec H9R 4P8, Canada. skorey@merck.com
Abstract:
Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.
Insights
A new assay method was developed to screen for inhibitors of Protein Tyrosine Phosphatase 1B (PTP1B), an enzyme linked to Type II diabetes. This method uses a catalytically inactive PTP1B mutant to overcome interference and accurately identify potential drug compounds.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Protein tyrosine phosphatases (PTPs) regulate cellular functions by modulating protein tyrosine phosphorylation.
- PTP1B is a key negative regulator of insulin signaling, making it a therapeutic target for Type II diabetes.
- Developing effective PTP1B inhibitors requires overcoming assay interference.
Purpose of the Study:
- To develop a robust high-throughput screening (HTS) assay for identifying PTP1B inhibitors.
- To address and overcome interference issues in enzymatic assays targeting cysteine active-site enzymes.
- To demonstrate the utility of a catalytically inactive mutant in differentiating enzyme activity.
Main Methods:
- A scintillation proximity assay (SPA) was employed using a radiolabeled inhibitor and recombinant PTP1B.
- A catalytically inactive mutant, GST-PTP1B(C215S), was utilized to prevent interference from oxidizing/alkylating agents.
- The assay was validated by simultaneously performing binding and kinetic assays for PTP1B(C215S) and active FLAG-CD45 in a single 96-well plate.
Main Results:
- The developed SPA effectively screened for PTP1B inhibitors by measuring competition for radiolabeled inhibitor binding.
- Using the catalytically inactive GST-PTP1B(C215S) mutant successfully minimized interference in the binding assay.
- The method allowed for simultaneous assessment of inhibition on both PTP1B (binding) and CD45 (activity) under identical conditions.
Conclusions:
- A novel and reliable HTS assay for PTP1B inhibitors has been established, overcoming common interference issues.
- The use of catalytically inactive mutants is a viable strategy for developing specific binding assays for cysteine active-site enzymes.
- This approach facilitates the differentiation of inhibitors between related enzymes, aiding in drug development for metabolic diseases like Type II diabetes.