# Assessing the polypharmacology of kinase inhibitors by transcription factor activity profiling

> **NIH NIH R44** · ATTAGENE, INC. · 2020 · $999,998

## Abstract

ABSTRACT
Evaluating the biological activity of protein kinase inhibitors (PKI) is one of the most challenging problems of
drug development. Here, we propose a new evaluation approach (the ATTAKIN™) in which we infer PKI
bioactivities from responses of cellular signaling. As a readout, we assess the activity of transcription factors
that connect the signaling pathways to regulated genes. In preliminary studies, we found specific TF activity
profiles (TFAPs) for inhibitors of AKT, mTOR, MEK, ERK, CDK, and Aurora kinases. Remarkably, in each
case, most inhibitors of a given kinase produced a common TFAP signature, regardless of their structural
dissimilarities and MOAs. Therefore, these invariant PKI TFAP signatures may represent specific markers for
the on-target PKI activity. Individual PKIs exhibited the on-target TFAPs within certain concentration ranges
(the “specificity windows”); beyond those, we observed distinctly different signatures, reflecting off-target
effects. Querying the off-target PKI signatures against reference signatures of chemicals with known
bioactivities afforded straightforward identification of the off-target effects on unintended kinases and non-
kinase effectors. Therefore, PKI TFAPs provide clear quantitative metrics for the evaluation of PKI
polypharmacology. Under this proposal, we will identify the on-target PKI TFAP signatures for a focused set of
40 to 50 therapeutically relevant kinases (Specific Aim 1). Using these signatures, we will assess the specificity
windows and the off-target effects of the evaluated PKIs (Specific Aim 2). Under Specific Aim 3, we will test the
utility of the ATTAKIN™ approach for discovery of novel inhibitors. For that, we will query our database,
containing over 30,000 TFAP entries for drugs and environmental chemicals, and will validate the retrieved
chemicals by a proteomics-based kinase profiling technique. This project will (1) validate the new, unbiased
approach to evaluation of PKI polypharmacology; (2) produce specific markers of the on-target activity for
many therapeutically relevant kinases; and (3) assess the polypharmacology of a large number of PKIs. In
summary, this project will firmly validate the ATTAKIN™ approach as a tool for biological evaluation of kinase
inhibitors, which is orthogonal and complementary to existing methodologies. This study will establish the
new, signature-guided PKI assessment approach as a mainstream drug evaluation technology. The
ATTAKIN™ will complement and expand the existing Attagene line of drug evaluation services.

## Key facts

- **NIH application ID:** 9909795
- **Project number:** 1R44GM136083-01
- **Recipient organization:** ATTAGENE, INC.
- **Principal Investigator:** SERGEI S MAKAROV
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $999,998
- **Award type:** 1
- **Project period:** 2020-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9909795

## Citation

> US National Institutes of Health, RePORTER application 9909795, Assessing the polypharmacology of kinase inhibitors by transcription factor activity profiling (1R44GM136083-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9909795. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*