Extracellular signal-regulated kinases (ERKs) regulate downstream signaling of RAS-RAF-MEK in the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is prevalent in numerous human cancers and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. The rapid rise of resistance to clinical RAF and MEK inhibitors have led to new interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. However, current methods for measuring activity of inhibitors against ERK isoforms are based primarily on use of recombinant proteins. Strategies to directly and independently probe native ERK1 and ERK2 activity would greatly complement current cell biology tools used to study and target ERK function. Here, we present a chemical biology and quantitative mass spectrometry strategy that uses active-site directed probes to directly quantify native ERK activity in an isoform-specific manner. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across diverse biological samples. We used our chemoproteomic strategy to determine potency and selectivity of ERK inhibitors used in research labs as well as those currently undergoing clinical trials. Using our quantitative mass spectrometry strategy, we determined that &gt;90% inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of ERK inhibitors and drugs. Thus, we introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to enable drug discovery of oncogenic ERK isoforms.<br/>

Using mass spectrometry to exploit a single isoleucine/leucine difference in ERK substrate binding sites for activity-based profiling of MAPK signaling

256th National Meeting (2018)

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