Abstract 2393: Next-generation CSox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity
Authors: Schaefer Erik M. , Cornell-Kennon Susan , Imperiali Barbara 
Published: August 2018

Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases across therapeutic areas and especially oncology. Currently, 30% of all drug development efforts are focused on protein kinases. Although many drugs are approved or in clinical trials, these are predominantly 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 characterize inhibitor mechanism of action, predict drug potency, and 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 with recombinant enzymes and crude lysates to enable target discovery and drug development.

Experimental Procedures: We harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to identify optimized sequences based on physiologic substrates for Ser, Thr or Tyr kinases. Kinase reactions were monitored using fluorescence intensity in kinetic mode (excitation 360 nm, emission wavelength of 485-505 nm).

Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Kms, higher 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 clinically relevant mutants, DDR1/2, JAK kinases (JAK1-3, Tyk2), MET, SYK, Tec-kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including Aurora, CDKs, MAPK pathway (MAP4Ks, Raf, MEK1/2, ERK1/2, RSK), PKR/EIF2AKs and PIM1.

Conclusions: The generation of robust activity-based assays opens 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 and phosphatase 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, Barbara Imperiali. Next-generation CSox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2393.