Abstract:
Dysregulation and mutation of fibroblast growth factor (FGF) signalling pathways can promote the development of human malignancies, including non-small cell lung cancer (NSCLC) associated with FGFR1 aberrations, FGFR2 related intrahepatic cholangiocarcinoma (ICC), FGFR3 linked bladder urothelial carcinoma, and FGFR4 amplified hepatocellular carcinoma. Therefore, fibroblast growth factor receptors (FGFRs) are attractive molecular targets for drug development. Structural knowledge of how novel small molecule inhibitors (SMIs) interact with FGFR binding sites provides opportunities to rationally design new generations of drugs. The majority of SMIs targeting FGFRs that have been tested in clinical trials belong to the type I1/2 class of inhibitor and compete with ATP binding in a reversible manner, forming hydrogen bonds with the hinge region connecting to the two lobes of the receptor kinase domain. Over the past decade, research and clinical focus has turned increasingly to inhibitors that covalently modify the protein target after an initial reversible binding event.
This study characterised the structure-activity relationships of a series of novel irreversible FGFR inhibitors developed by our collaborators based on the structure of another potent FGFR inhibitor, FIIN-1. FIIN-1 was also studied in this project. FIIN-1 was never advanced to clinical evaluation, but it was the first reported successful FGFR-selective irreversible inhibitor designed using structure-guided lead optimisation. The pan-FGFR inhibitor TAS-120 was also studied in this project as an exemplar of a highly FGFR-reactive molecule with a novel structure. TAS-120 is currently under phase II/III clinical trials in patients with ICC and advanced solid tumours.
The covalent binding of the selected series of compounds and their relative reactivity (selectivity) toward the FGFR family members (FGFR1-4) were examined using liquid chromatography-mass spectrometry (LC-MS) and these results compared to the profiles of FIIN-1 and TAS-120. All the inhibitors studied target a specific cysteine residue located in the P-loop, a conserved ATP-binding element in the target proteins. The novel series of compounds feature a dimethylaminocrotonamide moiety, which modules the reactivity of their acrylamide functionalities towards cysteine modification, potentially minimising undesirable off-target reactivity in a cellular setting. The control compounds FIIN-1 and TAS-120 by comparison, display much faster cysteine adduct formation that might be explained by their unprotected “naked” acrylamide functionality.
The high-resolution X-ray structures of TAS-120 as a free ligand, in reversible binding mode, and as the first reported irreversible FGFR1-bound structure, were determined in this project and highlighted the importance of acrylamide placement in determining covalent reactivity. Computational analysis of TAS-120 and selected novel inhibitors using molecular dynamics further emphasized that structural flexibility in both inhibitor and the target P-loop is a critical molecular mechanism in determining both reactivity and selectivity in FGFR targeting.