(1393-B) Using Mass Spectrometry detection to mitigate fluorescence interference and potency detection limits in a plate-based kinase assay panel.
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: The development of highly selective inhibitors for kinase drug targets remains a considerable challenge within the pharmaceutical industry. Often, compound testing cascades will include biochemical assays for both the desired kinase target of the project as well as other kinases to provide relative selectivity data which informs SAR optimization. For one such program, we needed an assay platform to monitor the selectivity of project compounds with all three isoforms of a kinase target. For that work, we established an assay panel using the AssayQuant PhosphoSens technology. During SAR optimization of our series, we encountered two limitations: (1) we experienced an unanticipated fluorescence interference with the readout by the pharmacophore of the lead series, and (2) emerging high-potency leads had bottomed out the pharmacological dynamic range of the fluorescent assay- i.e., we were not able to quantify further improvements to potency using the established assay method. We were challenged to identify a solution to both problems of assay interference and limits of potency characterization in a quick and resource-sparing manner to both sustain momentum as well as stay within the budget for the project. In this poster we highlight a series of innovative solutions delivered by a cross-departmental collaboration to implement Mass Spec detection of the phosphorylated peptides that addressed these issues. We describe our multiplexed detection panel that allowed for the simultaneous detection of phospho-peptide products from all three isoforms and reduced our Mass Spec run time 3-fold such that the assay panel could be deployed as the primary biochemical assay for the project.