# A new multi-pathway kinase activity assay applied to compound library screening in cancer biology

> **NIH NIH K99** · HARVARD MEDICAL SCHOOL · 2022 · $117,363

## Abstract

A new multi-pathway kinase activity assay applied to compound library screening in cancer biology
Project Abstract
Dysregulation of protein kinase signaling pathways is a hallmark of most human cancers. Although targeting
kinases has been an effective therapeutic strategy in diverse cancer types, limited druggable targets, tumor
heterogeneity and drug resistance remain problems in kinase-targeted cancer therapies. Finding new modulators
of kinase activity in complex cancer models is therefore an urgent need, but current kinase activity measurement
methods suffer from being low throughput and only provide surrogates for kinase activity.
This proposal aims to apply the latest sample multiplexing technologies using Tandem Mass Tag (TMT) reagents
and mass spectrometry to create a novel, multi-pathway, high-throughput, direct kinase activity readout assay
and apply it to profile kinase activity for drug screening. An in vitro peptide phosphorylation assay provides a
way to directly measure kinase activities for many pathways, while TMT increases throughput by sample
multiplexing. First, I will select peptide substrates to represent specific kinase pathways (e.g., AKT, SRC, MAPK,
CDK, etc.) and create a high-throughput in vitro kinase assay based on the TMT reagents. This assay will then
be adapted to a 96-well plate format (Aim1). In Aim2, I will expand the assay to a three-dimensional (3D) cell
culture model, profile and compare the kinase activities in response to 137 known kinase inhibitors in both
standard two-dimensional (2D) and 3D cell culture models. In Aim3, I will perform high-throughput profiling of
kinase activities across a library of 3200 compounds in both 2D and 3D ovarian cancer models using the 96-well
plate platform for drug screening. Upon completion of these Aims, I will have developed and applied a highly
optimized method for direct kinase activity measurement. This novel assay will result in the ability to quantitatively
measure the activation state of dozens of signaling pathways directly from cellular lysates and, therefore, identify
kinase modulators for future drug development.
This proposal draws on my training in mass spectrometry-based method development and kinase and cancer
biology. However, to ensure completion of the goals and successful transition to independence, I have a detailed
training plan, under the guidance of my mentors Dr. Steven Gygi and Dr. Joan Brugge, as well as my advisory
team, Dr. Peter Sicinski, Dr. Jennifer Smith and Dr. Joao Paulo. During the mentored phase, I plan to (1) deepen
my understanding of mass spectrometry instrumentation, (2) develop new expertise in drug screening, (3)
strengthen my knowledge base in cancer biology, especially in 3D cell culture and cellular signaling, and (4)
enhance my scientific writing and communication skills while gaining experience in lab management and
leadership. The environment at Harvard Medical School presents an excellent opportunity for training in drug
sc...

## Key facts

- **NIH application ID:** 10505710
- **Project number:** 1K99CA273170-01
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Tian Zhang
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $117,363
- **Award type:** 1
- **Project period:** 2022-08-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10505710

## Citation

> US National Institutes of Health, RePORTER application 10505710, A new multi-pathway kinase activity assay applied to compound library screening in cancer biology (1K99CA273170-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10505710. Licensed CC0.

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