Validation of ion mobility spectrometry ‐ mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase

Abstract

The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required.Methods: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods.Electrospray ionisation -ion mobility spectrometry -mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II.Results: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method.Conclusions: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

Exploring the Conformational Landscape and Stability of Aurora A Using Ion-Mobility Mass Spectrometry and Molecular Modeling

“…At the outset of this study, we hypothesized that the activation status of Aur A and the binding of different classes of small molecule inhibitor would alter the conformational landscape adopted by this protein, as has been established previously, 85,86 for other protein kinases such as PKA, 56,87 c-Abl, 88 and FGFR1 89 as well as intrinsically disordered proteins such as p53 12,90 and Aβ40. 91 While we do observe some differences, the effects are subtler than might be anticipated, being broadly consistent with the findings of others.…”

Meta-learning-based Inductive Logistic Matrix Completion for Prediction of Kinase Inhibitors

“…However, this method cannot be used to determine the inhibition ability of compounds to the whole Kinome. With the development of technology, it is possible to screen new high-throughput kinase profiles [12][13][14][15][16][17]. However, the long experimental cycle, high equipment requirements, and high cost make it difficult to use it as an early screen approach for drug discovery [18].…”

Native mass spectrometry reveals binding interactions of SARS-CoV-2 PLpro with inhibitors and ISG15

“…46,47 Additionally, changes in CCS can be monitored during low energy collisional activation of protein, a process known as collision induced unfolding (CIU), thus allowing discernment of the impact of ligands on stabilization of proteins. [48][49][50] vT-ESI allows determination of melting temperatures of proteins and protein complexes 42 and measurement of thermodynamic parameters associated with ligand binding. 51,52 An alternative ion activation method, UVPD has been used to localize inhibitor binding regions of proteins 53 and reveal unfolded/extended vs folded protein regions, 54 thus providing deeper insight into protein tertiary structure.…”