Immunoassay for Measuring Mutation Activated Kinases Across Multiple Cell Signaling Pathways - ABSTRACT
Due to the vast number of potential drug combinations, “testing them all” in clinical trials is impossible, putting
pressure on preclinical research to find the most promising compounds to advance to human studies. While
certain drug combination effects can now be modeled in silico, this approach is generally limited to the best-
studied biological pathways and may not work for analyzing complex disease mechanisms that involve multiple
interconnected pathways.
Protein immunoassays detect activated biological pathways by analyzing site-specific phosphorylation of kinases
and/or their substrates, normalized against the abundance of unmodified proteins. Recently, an interest in
detecting and quantifying protein mutations has emerged, due to the development of novel therapeutic options
based on covalent binding of a drug to a mutant protein or targeted protein degradation. Currently, there are no
immunoassays for measuring multiple protein targets (total and phospho-) across multiple major cancer
pathways, which can resolve differentially phosphorylated kinases and kinase substrates.
While several platforms for building multiplexed protein-based assays exist they either a) have a limited ability
to detect phosphorylation (immunoassay-based technologies); or b) are low-throughput, expensive and often
difficult to standardize (LC-MS), making them suitable for discovery proteomics but not for routine screening of
tens, hundreds or thousands of samples. Importantly, none of these analytical technologies are ideal for
quantifying protein targets containing multiple phosphorylated sites within a specific protein region. Therefore,
the existing assays cannot accurately measure kinases that are activated by sequential phosphorylation events.
We propose to initiate development of Path10™ - a multiplexed, protein-based, multi-pathway profiling assay for
the Bead-Assisted Mass Spectrometry (BAMS™) platform - which will simultaneously measure protein targets
across 10 major cell signaling pathways: RAF-MEK-ERK, cell cycle, Hippo, Myc, Notch, Nrf2, PI3K/Akt, TGFβ,
p53 and β-catenin/Wnt. It will achieve broad, multi-pathway coverage by including targets unique to each
pathway and also common targets shared by different pathways, e.g. protein substrates of multiple kinases. By
selecting relevant proteins and modification sites, future assays can be configured to extend coverage within
selected pathways or to profile disease pathways other than cancer, such as diabetes.
In the proposed Phase I we will start the product development, focusing initially on the RAF-MEK-ERK pathway,
which has the highest frequency of genomic alterations. In the follow-up Phase II, we will rapidly expand assay
coverage to the remaining 9 pathways using the methods developed in this Phase I. Post-Phase II, we will focus
on assay automation and applying Path10™ to human tissue and animal models, e.g., patient-derived xenografts
(PDXs).
