Assessing the polypharmacology of kinase inhibitors by transcription factor activity profiling

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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.