Probing conformational changes in c-Met activation loop using single molecule fluorescence spectroscopy

Abstract

The catalytic activity of protein kinases is governed by the conformation of the activation loop, thought to exist in dynamic equilibrium between active and inactive states. This equilibrium position can be influenced by post-translational modification, ligand binding or mutation, and may impact the efficacy and design of small molecule kinase inhibitors. c-Met is a receptor tyrosine kinase, and several small molecule inhibitors of c-Met are approved for clinical use for the treatment of non-small cell lung cancer (NSCLC) and papillary renal cell carcinoma (PRCC) patients. The emergence of acquired resistance mutations in c-Met poses a challenge for the efficacy of these targeted therapies, in particular acquired resistance to crizotinib, a first line therapy for NSCLC patients. Currently, all approved c-Met inhibitors are classified as either type I or type II inhibitors, which interact with the ATP-binding site via different binding mechanisms, and type I inhibitors, including crizotinib, are more susceptible to acquired resistance mutations D1228N/V/H and Y1230C/H.

Using a single molecule fluorescence spectroscopy assay, this thesis explores the activation loop conformational equilibrium of c-Met, and of clinically arising mutants D1228V and Y1230H in apo and ligand-bound states. Following successful expression optimisation of recombinant c-Met kinase domain in E. coli through coexpression with GroES-EL chaperone plasmid, samples were labelled with two self-quenching TMR fluorescent dye molecules for TIRF microscopy.

Intensity frequency histograms reveal that unphosphorylated c-Met kinase exists in equilibrium between both active and inactive conformations, with the majority of wildtype (83%), D1228V mutant (84%) and Y1230H mutant (84%) molecules occupying the inactive conformation. Type I c-Met inhibitors (crizotinib and savolitinib) drove the activation loop equilibrium of unphosphorylated wildtype c-Met towards almost 100% inactivation and type II inhibitors, (foretinib and BMS-777607) shifted the wildtype c-Met equilibrium to 87% and 91% inactive, respectively. Our experiments show that small molecule c-Met inhibitors bind both activation loop conformations, maintaining an equilibrium between active and inactive conformations. In addition, both type I inhibitors display greater conformational discrimination towards binding the inactive activation loop conformation of unphosphorylated wildtype c-Met kinase domain.

The conformational equilibrium of c-MetY1230H was unaltered in the presence of both type I inhibitors (84%) and the c-MetD1228V equilibrium was unchanged by crizotinib (83%) but shifted towards the inactive population with savolitinib (91%). We suggest two distinct mechanisms behind type I inhibitor resistance, including reduced inhibitor binding affinity and altered conformational discrimination between activation loop states, likely due to structural interactions involving Asp1228 and Tyr1230 in the inactive activation loop conformation. The conformational equilibrium of c-MetD1228V was shifted towards the inactive state, consistent with wildtype (foretinib = 89% and BMS-777607 = 91% inactive). However, the conformational equilibrium of c-MetY1230H in the presence of BMS-777607 remained unchanged (86% inactive), suggesting that Y1230H reduces BMS-777607 conformational discrimination towards the inactive state, an impact previously unrecognised in prior structural investigations.
Bioinformatic analysis has elucidated highly conserved residues and structures among receptor tyrosine kinases regulated by double phosphorylation, suggesting the applicability of our single molecule spectroscopy and thesis conclusions to related kinases such as INSR, FGFR1/4, ALK, ROR1/2, TKRA/B/C, and RON. This thesis is the first research to use single molecule fluorescence spectroscopy to elucidate the complex interplay between kinase conformational dynamics and resistance mutations, with implications for understanding kinase structure-function relationships and structure-based drug design approaches.

Date of Award	24 Jul 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Ian Eggleston (Supervisor) & Charlotte Dodson (Supervisor)