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Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy
Mahesh Yanamandra1, Labanyamoy Kole2, Archana Giri3
1a Department of In-vitro Biology , GVK Biosciences Private Limited , Hyderabad.
Abstract:
Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.
Insights
A new cell-based ELISA assay quantifies phosphorylated and total AKT kinase, essential for assessing phosphatidylinositol-3 kinase (PI3K) inhibitors. This tool aids drug discovery by evaluating PI3K inhibitor efficacy and mechanism of action.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The Phosphatidylinositol-3 kinase (PI3K) pathway regulates critical cellular processes like survival, growth, and metabolism.
- Dysregulation of the PI3K pathway is implicated in diseases including cancer, atherosclerosis, and inflammation.
- PI3K/AKT pathway inhibitors are significant therapeutic targets in drug discovery.
Purpose of the Study:
- To develop a sensitive, in-vitro cell-based assay for evaluating PI3K inhibitors.
- To quantify phosphorylated AKT kinase (pAKT) and total AKT kinase (tAKT) in cells treated with compounds.
- To establish a reliable screening system for assessing the mechanism of action and target engagement of novel PI3K inhibitors.
Main Methods:
- Development of a cell-based kinase ELISA to measure pAKT and tAKT.
- Utilized NIH3T3 cells overexpressing PI3Kβ, stimulated with lysophosphatidic acid for pathway activation.
- Assessed assay reliability using percentage coefficient of variation (%CV) and Z factor, adhering to established guidelines.
- Employed a 96-well plate format for medium-throughput screening.
Main Results:
- The developed cell-based kinase ELISA demonstrated high sensitivity and reliability.
- Assay performance metrics (%CV and Z factor) confirmed its robustness and suitability for screening.
- The assay successfully quantified both phosphorylated and total AKT kinase levels within cells post-treatment.
- The system proved effective in screening novel molecules targeting the PI3K/AKT pathway.
Conclusions:
- A robust and sensitive cell-based kinase ELISA has been developed for evaluating PI3K inhibitors.
- This assay serves as a valuable tool for drug discovery, enabling assessment of target engagement and mechanism of action.
- The methodology supports medium-throughput screening of novel compounds targeting the PI3K/AKT pathway.
