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Abstract

Signal transducer and activator of transcription 3 (STAT3) is widely recognized as a prominent anticancer drug target, yet its complex signaling and limited “druggability” present major challenges for developing potent and selective inhibitors that directly target STAT3. Recent advances in thermal shift assays like differential scanning fluorimetry (DSF) and Cellular thermal shift assay (CETSA) have expanded the toolkit for STAT3 drug discovery by enabling the identification and validation of inhibitors that directly bind STAT3 in biochemical and cellular conditions, respectively. My doctoral work proposed the execution of an integrated drug discovery funnel using thermal shift assays alongside phenotypic STAT3 assays, aiming to enable the discovery and optimization of direct STAT3 inhibitors. To this end, the protein thermal shift assay (PTSA) was adapted as a supportive screening and validation tool linking DSF and CETSA, facilitating the assessment of small molecules for their interaction with STAT3 in biochemical settings. I introduce a target engagement-focused screening and optimization pipeline combining these chemical biology techniques with other STAT3 inhibition assays. From a STAT3 DSF high-throughput screen, to complementary and orthogonal assays, I identified a series of compounds that appeared to stabilize STAT3 towards thermal aggregation and moderately inhibited cellular STAT3 activity. However, thoroughly evaluating these compounds in auxiliary and orthogonal assays illuminated their high-affinity for tropomyosin receptor kinase A (TrkA) and other kinase targets, disputing them as direct STAT3 antagonists. Structural activity relationship studies were then employed to further refine inhibitor binding for TrkA, a prominent target in rare NTRK1 fusion-positive cancers. Applying a similar target engagement-inspired approach, I optimized this inhibitor series to identify compounds with improved binding affinity and selectivity towards TrkA. Cellular activity in TrkA cancer models was evaluated with top compounds, including PI-15, which demonstrated successful target-engagement in cell lysate CETSA and potently inhibited TPM3-TrkA phosphorylation in TrkA fusion-positive colon cancer cells. Further lead modifications yielded compound PI-15R, a metabolically stable, cell-permeable and potent Trk kinase inhibitor with single-digit nanomolar IC50 values. Overall, identifying a promising kinase inhibitor through comprehensive validation screening approaches, this study highlights the value of rigorous target engagement validation early in the drug discovery pipeline.