Unravelling Gatekeeper Resistance In Fibroblast Growth Factor Receptor 4

Abstract

Hepatocellular carcinoma is the third leading cause of cancer death globally. This primary liver cancer develops from dysregulation of cellular control-factors, leading to increased cell division and tumour formation. Fibroblast growth factor receptors 1-4 (FGFR1-4) are signalling proteins that interact with growth-factors to regulate cellular proliferation. In particular, aberrant signalling of the FGFR4 protein family is associated with acquired “gatekeeper” mutations that lead to drug-resistance. As a result, much research has been invested in developing small molecule inhibitors that target the active site of the protein for inhibition. However, the presence of “gatekeeper” mutations severely limits the potency and selectivity of such drugs. BLU9931 and BLU554 were the first reported irreversible small molecule kinase inhibitors that were able to effectively target FGFR4 proteins. Based on the structure of these compounds, our collaborators developed several new inhibitors with varying degrees of structural flexibility. This project aimed to investigate the selectivity and potency of the new FGFR4 inhibitors towards gatekeeper FGFR4 protein, and to make comparisons with the activity of BLU554 and BLU9931. To understand the selectivity profiles of the inhibitors, differential scanning fluorimetry (DSF), liquid chromatography-mass spectrometry (LC-MS) and computational docking techniques were employed. The DSF experiments provided several unexpected results between FGFR4 and inhibitors, while LC-MS confirmed the covalent modification of the inhibitors to the FGFR4 proteins. Computational docking offered a visual representation of protein and inhibitor binding to further complement and explain the results from DSF and LC-MS. Clinical drug Erdafitinib and LY2874455, a known gatekeeper active FGFR inhibitor, were included in experiments to complement the known literature around these compounds. This project reports that DSF and mass spectrometry are suitable assays to determine gatekeeper targeting ability in the FGFR system. However, none of the novel compounds, nor BLU554 and BLU9931, showed significant gatekeeper activity compared to their ability to inhibit wild type protein.