Activity-based Sensor Peptides for Direct, High-throughput, and Kinetic Quantitation of Oncogenic Protein Kinases in Unfractionated Cell and Tissue Samples

PROJECT SUMMARY 30 lines Protein kinases catalyze selective and timely protein phosphorylation and are essential for the normal function of all cells. Unregulated kinase activity is directly associated with hundreds of human diseases, including cancer; an estimated 30% of all new drug development focuses on this target class. Unfortunately,

although protein kinases are validated drug targets and kinase inhibitors represent promising treatments for diseases, the challenge for small molecule drug discovery and development is that the current kinase assay methods are inefficient and commonly only work under limited conditions. A significant barrier to progress

centers on the tools applied to assay protein kinases throughout the drug discovery pipeline. Although many assay platforms exist, severe limitations in the various sensing mechanisms lead to substantial disconnects in the discovery process, resulting in less well-validated clinical candidates and often limited efficacy in

treating disease. The proposed research exploits AQT’s PhosphoSens® platform, a kinase assay for purified enzymes that uses sensor peptides featuring a kinase-recognition sequence and a sulfonamido-oxine fluorophore conjugated to cysteine (CSox) proximal to the (Ser/Thr/Tyr) phosphorylation site—the synthetic sensor

peptides report on phosphorylation via chelation-enhanced fluorescence (ChEF) in a time-dependent manner. The PhosphoSens format addresses the limitations of the other kinase assays in a single platform. This Direct to Phase II proposal will leverage AQT’s PhosphoSens and KinSightTM (a high throughput kinase

profiling platform based on PhosphoSens) for establishing commercial PhosphoSens® Lysate products. For broad adoption, the new assays must address a range of physiologically and therapeutically critical kinases, be compatible with complex cell and tissue lysate samples, and be straightforward to apply,

quantitative, rapid, reproducible, and readily run on standard laboratory microplate readers. Aim 1 focuses on mitogen-activated protein kinases (MAPKs), which play critical roles in cell signaling, including responses to proliferation and inflammatory signals that drive tumor phenotypes. AQT will commercialize lysate-compatible sensor peptides for ERK1/2 based on Phase 1 product AQT0491. Sensor

peptides for eight other MAPKs will be developed and validated following Milestone steps 1-3, biochemical validation, and deployment of sensor peptides to beta test sites for testing with complex samples (e.g., cell or tissue lysates). AQT will refine sensor peptides as needed before commercial product release.

Aim 2 focuses on additional oncology-centric kinases. Lysate-compatible sensor peptides for PIM1/3 and GSK3α/ will be commercialized based on Phase 1 products AQT0473 and AQT0157, respectively. AQT will develop data-driven approaches to prioritize at least six additional protein kinase targets for sensor

peptide development and commercialization following approaches outlined for Aim 1.