Development of N546K and K656E Mutant FGFR Small Molecule Inhibitors

National Center For Advancing Translational Sciences

Investigators

Abstract

During this reporting period, the biology team focused on assay optimization for three key biochemical assays: FGFR1 N546K mutant, FGFR1 K656E mutant, and wild-type (WT) FGFR1. Optimization efforts included refining assay conditions (e.g., substrate concentration, enzyme stability, and buffer composition) and evaluating assay robustness metrics such as signal-to-background ratio, Z′-factor, and reproducibility to ensure suitability for medium-throughput screening and reliable comparison of mutant versus WT activity. In parallel, the informatics team assembled a focused compound library of ~120 FGFR inhibitors, encompassing both pan-FGFR and selective FGFR inhibitors, curated from literature reports, commercial sources, and clinical-stage compounds. This collection is being prepared for pilot screening to benchmark assay performance and to establish reference activity profiles across WT and mutant FGFR1 targets. On the computational front, homology models of FGFR1 N546K and K656E mutants were constructed using high-resolution FGFR1 and FGFR2 crystal structures as templates. To probe conformational dynamics and mutation-induced structural perturbations, molecular dynamics (MD) simulations are currently underway. These simulations aim to characterize differences in activation loop flexibility, ATP-binding pocket geometry, and potential allosteric sites between mutant and WT enzymes. Insights from these analyses are expected to guide the design and prioritization of mutant-selective inhibitors, enabling strategies to overcome limitations of pan-FGFR inhibition.