Abstract 1666: A novel sox-based continuous and homogeneous assay for the discovery of inhibitors of inactive and active AKT

Abstract

Abstract Introduction: The activity of protein kinases plays a critical role in the aberrant activation of oncogenic signaling pathways which can drive tumorigenesis and malignant transformation in cancer. AKT has been shown to be both upregulated and mutated in cancer cells allowing it to serve as a driver of cancer cell growth and progression. Inhibition of AKT activation and activity are both attractive targets for effective cancer drug discovery. Experimental Procedures: We developed continuous, homogeneous assays for unactive AKTs. A peptide substrate, modified with a sulfonamido-oxine fluorophore (Sox), utilizes chelation-enhanced fluorescence to enable a real-time readout of AKT-driven phosphorylation. First, a subset of 30,000 Sox-containing sequences were evaluated for AKT substrate candidates, selecting for assay robustness and specificity. A physiologically relevant peptide substrate was identified and used to develop a kinetic assay to monitor AKT1 activation and activity. Unactive AKT1 was incubated with DOPS/DOPC and PIP3, which mimics the plasma membrane, allowing the PH domain of AKT to bind, leading to a conformational change that enables full activation of AKT by PDK1 and MK2. Upon assay initiation, active AKT phosphorylates the sensor peptide, and the resulting signal is read in kinetic mode using a fluorescence intensity readout. First derivative plots for each progress curve were generated; the slope of the linear regions of each plot, representing acceleration towards a steady-state, were used to determine relative rates of activation for each AKT. Results: We developed a novel assay for AKT activation and substrate phosphorylation utilizing AQT0076, DOPS/DOPC, PIP3, PDK1, and MK2. With a mix of classical AKT inhibitors and allosteric inhibitors that rely on an inactive “PH-in” conformation, we demonstrated inhibition and quantified inhibitor potency of both active “PH-out” AKT activity and inactive “PH-in” AKT activation via dose-response measurements of steady-state rate and rate acceleration, respectively. Conclusions: A robust, homogeneous assay was developed to simultaneously monitor AKT activation and substrate phosphorylation kinetically over time. Through a continuous assay, we captured both steady-state rates and rate acceleration as a function of inhibitor concentration, allowing for accurate quantitation of both classes of AKT inhibitors in a single experimental format. With some inhibitors, we observed potencies that matched reported literature values, while with others we observed marked differences that may reflect a more physiological context that could translate through to higher efficacy in the clinic. This novel assay format provides a new tool that can be used in drug discovery to generate more effective next generation inhibitors of both AKT activation and subsequent substrate phosphorylation to prevent cancer cell growth and progression. Citation Format: Susan Cornell-Kennon, Daniel Urul, Matthew Hakar, Hayley McMahon, Khanh Huynh, Earl May, Erik Schaefer. A novel sox-based continuous and homogeneous assay for the discovery of inhibitors of inactive and active AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1666.