Introduction: Protein kinases are a diverse group of over 500 enzymes whose dysregulation lies at the center of many human diseases, spanning all therapeutic areas. Oncology is the most active area, where 30% of all drug development efforts are focused on protein kinases. Although 30 drugs have been approved by the FDA, and another 120 are in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been a surge in the generation of kinase inhibitors with different modes of action, where new tools are needed to effectively and efficiently characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of sensors that can be used for the continuous, quantitative and homogenous detection of kinase activity to enable target discovery and drug development.

Experimental Procedures: We have harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to create degenerate peptide libraries containing the consensus sequence for Ser, Thr or Tyr kinases that are implicated in cancer. Kinase reactions included 10 μM substrate, 10 mM MgCl2 and 1 mM ATP and fluorescence intensity was monitored in kinetic mode using a Biotek Synergy Neo2 microplate reader (excitation 360 nm, emission wavelength of 485-505 nm).

Results: By exploiting the continuous, quantitative and homogeneous nature of Sox-based detection, we demonstrate the ability to rapidly identify novel optimum substrates simply from observing and analyzing the resulting progress curve (fluorescence intensity over time). Performance measures included higher reaction rates, lower Km's, lower and flatter backgrounds, improved signal/background, increased sensitivity (down to low pM levels), and increased specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We highlight recently developed novel substrates to monitor activity of high-profile tyrosine kinases, including the EGFR and multiple clinically-relevant mutants and Tec-family kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including MAP4K's, MAPKs, CDKs and DRAK1/2.

Conclusions: The generation of robust activity-based assays, especially where previously only binding assay formats were available, opens up new areas of the kinome for effective drug discovery. The Sox-based assay technology is ideal for elucidating drug mechanism of action, potency, kinase activation and profiling, and therefore can be applied across the entire target discovery and drug development workflow. These developments provide a quantum improvement in performance and productivity that is needed to address the challenges and opportunities of next generation protein kinase inhibitors. These assays run on commonly available microplate instruments, providing access across the cancer research and drug development community.

Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Eric Lamsa, Erik McCauley, Eric Berg, Jordan Fishman, Barbara Imperiali. Optimized Sox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-024. doi:10.1158/1538-7445.AM2017-LB-024