Project Details
Description
Kinases are a central hub for signaling in multiple disease settings, including cancer, autoimmune disease, heart
disease, and beyond. There is significant need for technologies that could streamline multiplexed measurement
of kinase activities in live cells using high throughput screening and clinical lab-compatible read-outs for future
translation. We develop these kinds of cell-based assay approaches, designing novel substrates tuned to
particular read-out technologies and characterize them in vitro and in cell-based assays. Through prior work, we
prototyped an in silico pipeline called KINATEST-ID, in which kinase substrate preferences are identified, cross-
checked against other “off-target” kinases, then novel peptides are designed based on predicted compatibility
with a read-out technique and tested empirically. Our initial iterations of this pipeline have focused on tyrosine
kinases and in vitro lanthanide fluorescence assay read-outs, which have limited multiplexability. Mass
spectrometry detection of cell-deliverable kinase substrates that report kinase activity in live cells would provide
far higher multiplexability, but in prior work we ran up against a fundamental physiochemical limitation of our
design pipeline: selecting sequences for Tb3+ chelation produces substrates biased towards acidic amino acids
(e.g. D, E) that ionize very poorly in standard MS analyses. We also found that while we could predict biochemical
efficiency for kinases that had high-quality preference data available, prediction of selectivity is still inadequate
because most kinases lack such data for cross-referencing against each other. These barriers have limited further
progress on developing substrates for cell-based kinase profiling assays, particularly with multiplexed MS
detection. Further, in order to be more robust for higher throughput analyses, the workflows for the assay,
sample processing, and MS detection need to be simplified. In Aim 1, we will expand the KINATEST-ID platform
functionality with a focus on MS detection. In Aim 2, we will develop multiplexed cell-based deliverable substrate
kinase assays using targeted parallel reaction monitoring (PRM) MS methods in collaboration with colleagues at
Cedars Sinai who are developing cutting edge, high throughput proteomics methods that are clinical lab-
compatible. This work will produce an optimized platform for developing and implementing MS-compatible,
cell-based assays enabling kinase activity profiling in live cells, as well as a path to new tools for understudied
kinases. Overall, these tools will have high potential to impact both basic research for rapid profiling of signaling
activities, and kinase inhibitor drug discovery with eventual translation to the clinic.
Status | Finished |
---|---|
Effective start/end date | 5/1/22 → 4/30/24 |
Funding
- National Institute of General Medical Sciences: $10,075.00
- National Institute of General Medical Sciences: $313,840.00
- National Institute of General Medical Sciences: $112,963.00
- National Institute of General Medical Sciences: $327,590.00
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