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Introduction: The aberrant activation of oncogenic signaling pathways can drive tumorigenesis and malignant transformation, where deregulation of the activity of protein kinases or phosphatases can play a critical role. Several protein tyrosine phosphatases (PTPs) that are overexpressed in human cancers have recently been shown to activate signaling pathways and promote tumor development and progression (Sivaganesh et al., 2021. Protein Tyrosine Phosphatases: Mechanisms in Cancer. Int J Mol Sci. 22(23):12865). This reinforces the need for protein phosphatase inhibitors for cancer therapy, which requires robust assays to monitor PTP activity. Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on known physiological substrates. PTP enzyme activity was monitored using the PhosphoSens® platform in kinetic mode (a progress curve in every well) using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode with Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: Using commercially available phosphatases, we demonstrated the ability to rapidly identify novel Sox-based phosphopeptide substrates for each phosphatase. Performance measures included initial reaction rates, Km's, signal/background, linear reaction kinetics, absence of initial lags, assay sensitivity and specificity. Final optimized assay conditions were established for 28 different phosphatases. Each phosphatase was titrated to determine the lowest optimal assay concentration, demonstrating linearity and detection of low nM enzyme. Potency assessments using a set of compounds revealed sub nM inhibitor IC50 values and this included using sodium orthovanadate as a positive control. Finally, we demonstrated the ability to profile a range of novel phosphatase inhibitors to determine selectivity. Conclusions: The generation of robust activity-based Sox-sensor phosphopeptide substrates opens new areas for effective drug discovery with protein phosphatases as an emerging target class. The PhosphoSens-Kinetic assay format is ideal for assessing enzyme regulation and elucidating drug mechanism of action, potency, and profiling. The PhosphoSens-Red endpoint format is ideal for HTS and high-throughput SAR. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a dramatic improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Susan Cornell-Kennon, Erik Schaefer, Earl May, Matthew Hakar. Sox-based sensor phosphopeptides for continuous, homogeneous and quantitative monitoring of protein phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1426. |
