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Updated: Jun 30, 2026
PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions
Published on: July 27, 2017
Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay
Glenn S Van Aller1, Jeff D Carson, Christine Fernandes
1Department of Enzymology and Mechanistic Pharmacology, Oncology CEDD, GlaxoSmithKline, Collegeville, PA 19426, USA. glenn.s.van.aller@gsk.com
Abstract:
Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110alpha, p110beta, and p110delta, each coexpressed with the regulatory subunit p85alpha or splice variant p55alpha. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.
Insights
A new scintillation proximity assay (SPA) enables efficient study of phosphoinositide 3-kinase (PI3K) enzymes. This method aids in discovering novel PI3K inhibitors for cancer treatment.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently dysregulated in various cancers due to gene amplification and mutations.
- Specific mutations in PIK3CA, encoding the p110alpha catalytic subunit, are common in tumors like breast, colon, and ovarian cancers.
- Targeting PI3K kinase activity presents a potential therapeutic strategy for multiple human cancers.
Purpose of the Study:
- To develop and validate a scintillation proximity assay (SPA) for detecting phosphatidylinositol 3-kinase catalytic activity.
- To compare steady-state kinetic parameters of PI3K class IA enzymes (p110alpha, p110beta, p110delta) using the developed SPA.
- To assess the utility of the SPA for evaluating PI3K inhibitors.
Main Methods:
- A scintillation proximity assay (SPA) was established to measure PI3K catalytic activity.
- Steady-state kinetic parameters were determined for PI3K class IA enzymes (p110alpha, p110beta, p110delta) with regulatory subunits p85alpha or p55alpha.
- Inhibition of PI3K activity by known inhibitors (wortmannin, LY294002) was assessed using the SPA.
Main Results:
- The SPA format successfully detected PI3K catalytic activity.
- Kinetic parameters were compared across different PI3K isoforms and regulatory subunits.
- The SPA demonstrated sensitivity in detecting inhibition by wortmannin and LY294002, consistent with other assay formats.
- The assay is simple, inexpensive, sensitive, and high-throughput.
Conclusions:
- The developed SPA is a valuable tool for studying PI3K enzymology.
- This assay facilitates isoform-specific biochemical characterization of PI3K.
- The SPA platform will accelerate the discovery and development of novel PI3K inhibitors for cancer therapy.
