Conformational Plasticity in Tyrosine Kinase Inhibitor–Kinase Interactions Revealed with Fluorescence Spectroscopy and Theoretical Calculations

Abstract

To understand drug-protein dynamics, it is necessary to account for drug molecular flexibility and binding site plasticity. Herein, we exploit fluorescence from a tyrosine kinase inhibitor, AG1478, as a reporter of its conformation and binding site environment when complexed with its cognate kinase. Water-soluble kinases, aminoglycoside phosphotransferase APH(3')-Ia and mitogen-activated protein kinase 14 (MAPK14), were chosen for this study. On the basis of our prior work, the AG1478 conformation (planar or twisted) was inferred from the fluorescence excitation spectrum and the polarity of the AG1478-binding site was deduced from the fluorescence emission spectrum, while red-edge excitation shift (REES) probed the heterogeneity of the binding site (protein conformation and hydration) distributions in the protein conformational ensemble. In the AG1478-APH(3')-Ia complex, both twisted (or partially twisted) and planar AG1478 conformations were evidenced from emission wavelength-dependent excitation spectra. The binding site environment provided by APH(3')-Ia was moderately polar (λ = 480 nm) with evidence for considerable heterogeneity (REES = 34 nm). In contrast, in the AG1478-MAPK14 complex, AG1478 was in a predominantly planar conformation with a lower degree of conformational heterogeneity. The binding site environment provided by the MAPK14 protein was of relatively low polarity (λ = 430 nm) with a smaller degree of heterogeneity (REES = 11 nm). The results are compared with the available X-ray data and discussed in the context of our current understanding of tyrosine kinase inhibitor conformation and protein conformational ensembles.

Future of computational molecular spectroscopy—from supporting interpretation to leading the innovation

“…There are three major challenges in drug development summarised by Habgood et al 79 That is, (a) development of a good method to generate ensembles of a molecule's bioactive conformation; (b) rational analysis and modification of a pre-established bioactive conformation; and (c) approximation of real solution phase conformational ensembles in tandem with spectroscopic data such as IR, NMR and UV-Vis spectra. Further CMS studies of anilinoquinazoline TKIs with high potency (PD150335) 80 reveal that optically reportable conformation of the TKI is often more potent than the planar global minimum structure of the TKI. It seems that a correlation with flexibility and potency of a TKI exists, as flexible TKI conformers are able to fit and dock in the TK domain of EGFR.…”

Optical spectra of EGFR inhibitor AG-1478 for benchmarking DFT functionals

“…This approach is usually a good approximation for absorption (e.g. UV-vis) calculations [4,16,[33][34][35]. The GS properties include geometry, dipole moment, and electronic transitions (absorption) [36].…”

Intrinsically Fluorescent Anti-Cancer Drugs

“…The plasticity of structural elements of the ATP-binding site is critical for determining the inhibitor conformation and binding mode. For instance, Khattab et al [ 63 ] showed that the binding interaction of AG1478 with either a kinase important for bacterial drug resistance (APH(3′)-Ia) or a kinase important for pancreatic cancer (MAPK14) can result in a discrete change in AG1478 conformation on a rugged matrix of protein backbone. In this study the authors inferred conformational heterogeneity of the drug AG1478 from excitation spectra, polarity of the AG1478 binding pocket from the emission spectrum and restricted molecular dynamics in the AG1478–kinase complex from excitation-dependent emission spectra.…”