Over the past 30 years, the field of protein kinase inhibitors development has rapidly expanded. As a result,
several dozens of small-molecule kinase inhibitors have been approved by FDA, that led to improved outcomes
in cancer treatment for thousands of patients. Moreover, kinase inhibition was recently extended towards non-
oncology applications such as autoimmune and inflammatory diseases.
The ultimate success of a drug development process relies on the availability of prompt, reliable, and cost-
efficient analytical methodologies for potential inhibitor profiling. In the case of kinase inhibition, the screening
is often performed against purified recombinant kinase domains in vitro. While this approach has led to
successful identification of kinase inhibitors, it suffers from some major limitations that hamper drug development
process. A critical limitation is the poor correlation between in vitro and cellular behavior of inhibitor candidates.
This limitation is a reason for late-in-the-process failures of some drug candidates, that overall contribute to high
cost of the drug development process. This limitation can be overcome via development and use of highly
efficient cellular assays for inhibitor profiling. However, currently there are no efficient cellular assays for kinase
inhibitor profiling in native environments.
In the proposed work, we will design and develop a new cellular assay for profiling inhibitors of a model kinase,
EGFR tyrosine kinase. The assay will enable screening inhibitor activity in native conditions, inside the living
cells. To develop the cellular assay, we will accomplish three specific aims: (1) rational design, synthesis, and
in vitro characterization of the new cell-permeable fluorescent sensors that selectively respond on low-nanomolar
concentrations of EGFR; (2) development and characterization of intracellular delivery, localization, and EGFR
binding of these new sensors; (3) development of a competitive cellular assay for inhibitors profiling and
quantitative assessment of the inhibitor binding.
Upon successful completion of this work, the new assay will not only provide a foundation for the high-throughput
profiling of EGFR inhibitors, but also will establish general development principles applicable for other protein
In addition, this project will enhance research environment and infrastructure at Northern Illinois University.
Thus, generations of undergraduate students will get valuable experience in design and synthesis of the new
sensors, characterizing biomolecule/sensor interaction, working on developing in vitro and cellular assays.
Eventually, participation in this project will prepare and empower the undergraduate students for future careers
in biomedical field.