Binding-induced, turn-on fluorescence of the EGFR/ERBB kinase inhibitor, lapatinib

Abstract

We report the photophysical properties, binding-induced turn-on emission, and fluorescence imaging of the cellular uptake and distribution of lapatinib, an EGFR/ERBB inhibitor. Lapatinib, a type II, i.e. inactive state, inhibitor that targets the ATP binding pocket of the EGFR family of receptor tyrosine kinases. DFT calculations predict that the 6-furanylquinazoline core of lapatinib should exhibit an excited state with charge transfer character and an S0 to S1 transition energy of 3.4 eV. Absorption confirms an optical transition in the near UV to violet, while fluorescence spectroscopy shows that photoemission is highly sensitive to solvent polarity. The hydrophobicity of lapatinib leads to fluorescent aggregates in solution, however, binding to the lipid-carrier protein, BSA or to the kinase domain of ERBB2, produces spectroscopically distinct photoemission. Confocal fluorescence microscopy imaging of lapatinib uptake in ERBB2-overexpressing MCF7 and BT474 cells reveals pools of intracellular inhibitor with emission profiles consistent with aggregated lapatinib.

Fig. 1

Chemical structures of EGFR/ERBB-targeted small molecule inhibitors. While gefitinib and erlotinb target active state kinases and compete directly with ATP, lapatinib is a so-called type 2 inhibitor that targets the inactive state. The pendant furan ring of lapatinib can rotate modulating the electronic interaction with the quinazoline core.

Fig. 2

Frontier molecular orbitals of lapatinib calculated at the 6-31G^* level.

Fig. 3

Calculated (dashed lines) versus experimentally measured solution (solid lines) spectra of lapatinib. The solution spectra are slightly red-shifted when compared to the TD-DFT predicted spectra and extend into the visible spectrum enabling excitation with near-UV or violet sources; the typical window for a DAPI filter set and the 405 nm laser line are show. While lapatinib is emissive in a relatively non-polar solvent like THF (orange line), fluorescence is quenched in methanol (black line at bottom). 10 μM and 1 μM solutions were used for absorption and emission spectroscopy, respectively.

Fig. 4

Lapatinib (3 μM) forms aggregates immediately upon introduction of stock DMSO solutions into PBS; between t = 0 to t = 60 min the aggregates increase in size from 150 nm to approximately 1500 nm. In the presence of BSA, aggregation is eliminated (open circles). Data points are the average of three independent measurements; error bars show S.D. (too small to be seen for lapatinib in the presence of BSA).

Fig. 5

Excitation (solid lines) and emission spectra (dashed lines) of lapatinib bound to BSA (violet), ERBB2 (blue) and as aggregates in PBS (green). Solution concentrations are noted in the text.

Fig. 6

A comparison of ERBB2-overexpressing MCF7 cells without lapatinib (A), after 15 min of 3 μM lapatinib treatment (B) and 24 h of lapatinib treatment (C) reveals the turn-on fluorescence of intracellular lapatinib. All images were captured under identical conditions (e.g. laser intensity, detector gain, exposure time). The emission spectrum (D) of lapatinib at 15 min (orange line) closely matches that of lapatinib aggregates in PBS solutions (green dashed line). At longer time points, the emission is red-shifted (red line); emission of ERBB2-bound lapatinib (blue dashed line) is not observed either at the cell membrane or intracellularly.