Switching from ultrafast electron transfer to proton transfer in excited drug-protein complexes upon biotransformation

Abstract

Photosensitization by drugs is directly related with the excited species and the photoinduced processes arising from interaction with UVA light. In this context, the ability of gefitinib (GFT), a tyrosine kinase inhibitor (TKI) used for the treatment of a variety of cancers, to induce phototoxicity and photooxidation of proteins has recently been demonstrated. In principle, photodamage can be generated not only by a given drug but also by its photoactive metabolites that maintain the relevant chromophore. In the present work, a complete study of O-desmorpholinopropyl gefitinib (GFT-MB) has been performed by means of fluorescence and ultrafast transient absorption spectroscopies, in addition to molecular dynamics (MD) simulations. The photobehavior of the GFT-MB metabolite in solution is similar to that of GFT. However, when the drug or its metabolite are in a constrained environment, i.e. within a protein, their behavior and the photoinduced processes that arise from their interaction with UVA light are completely different. For GFT in complex with human serum albumin (HSA), locally excited (LE) singlet states are mainly formed; these species undergo photoinduced electron transfer with Tyr and Trp. By contrast, since GFT-MB is a phenol, excited state proton transfer (ESPT) to form phenolate-like excited species might become an alternative deactivation pathway. As a matter of fact, the protein-bound metabolite exhibits higher fluorescence yields and longer emission wavelengths and lifetimes than GFT@HSA. Ultrafast transient absorption measurements support direct ESPT deprotonation of LE states (rather than ICT), to form phenolate-like species. This is explained by MD simulations, which reveal a close interaction between the phenolic OH group of GFT-MB and Val116 within site 3 (subdomain IB) of HSA. The reported findings are relevant to understand the photosensitizing properties of TKIs and the role of biotransformation in this type of adverse side effects.

Fig. 1. Chemical structure of gefitinib (GFT) and its O-desmorpholinopropyl metabolite GFT-MB.

Fig. 2. Normalized fluorescence spectra (A) and decay traces (B) for GFT-MB in acetonitrile (black), 1,4-dioxane (red), toluene (blue) and cyclohexane (green) after excitation at 340 nm.

Fig. 3. (A) Femtosecond transient absorption spectra from 0.5 ps (black) to 2 ns (blue) for GFT-MB. (B) Kinetic traces monitored at 480 (black) and 435 nm (dark gray). Measurements were performed in acetonitrile after excitation at 330 nm.

Fig. 4. LFP spectra (from 0.2 to 3 μs) and decay traces at 600 nm for GFT-MB in MeCN (A) and toluene (B) under deaerated conditions after excitation at 355 nm.

Fig. 5. (A) LFP spectra monitored 0.08 μs after the laser pulse for GFT (black) and a mixture of GFT/NAc-TyrMe in a molar ratio of 1 : 200 (blue); the concentration of GFT was 120 μM. (B) Kinetic trace at 400 nm. Measurements were performed in deaerated MeCN after excitation at 355 nm.

Fig. 6. Fluorescence spectra (A) and normalized spectra (B) for GFT (gray line), GFT-MB (dashed gray line), GFT@HSA (black line) and GFT-MB@HSA (black dashed line) after excitation at 340 nm in aqueous PBS under air. For the protein complexes, mixtures were at 1 : 1 ligand@HSA molar ratio, using isoabsorptive solutions at the excitation wavelength. The inset in (A) shows a zoom of the weakly emitting species, while in (B) shows the normalized fluorescence spectra for GFT-MB@HSA and GFT-MB in PBS.

Fig. 7. (A) Absorption spectra for GFT-MB (black line), HSA (gray line) and GFT-MB@HSA (dashed black line). (B) Absorption spectra for GFT (black line), HSA (gray line) and GFT@HSA (dashed black line). All solutions were prepared at 10 μM in aqueous PBS. For the protein complexes, mixtures were at 1 : 1 ligand@HSA molar ratio.

Fig. 8. (A) Femtosecond transient absorption spectra from 1 ps (black) to 0.5 ns (blue) for GFT-MB@HSA; the absorption spectra of GFT@HSA from 1 ps (light gray) to 1 ns (light blue) is also shown for comparison. (B) Kinetic traces for GFT-MB@HSA at 500 nm (black) and for GFT@HSA at 460 nm (light gray) after excitation at 330 nm of a 1 : 1 molar ratio ligand@protein complexes in aerated aqueous PBS solution.

Fig. 9. Binding mode of GFT and GFT-MB with subdomain IB (site 3) of HSA obtained by MD simulation studies. (A) Overall and detailed views of GFT (yellow) binding mode. Snapshot after 100 ns is shown. Protein subdomains and main binding sites 1–3 of HSA are labelled and highlighted in the overall view. (B) Detailed view of GFT-MB (violet) binding mode. Snapshot after 90 ns is shown. Note how GFT-MB is anchored in the pocket thanks to a strong hydrogen bonding interaction with Val116 (yellow shadow) and a double π–π stacking interaction with two tyrosine residues (green spheres), which are located on both sides of the aromatic ring. (C) Superposition of the arrangements of GFT and GFT-MB with subdomain IB of HSA. Note the different arrangements of GFT and GFT-MB, which is more buried in the pocket. Hydrogen bonding interactions between the ligands and the protein are shown as red dashed lines. Relevant side chain residues are shown and labelled. The tyrosine residues Tyr161 and Tyr138 are highlighted in green color.

Fig. 10. (A) Comparison of several snapshots of GFT-MB@HSA during 100 ns of MD simulation. GFT-MB and Val116 are shown as sticks. Note that during the simulation GFT-MB was displaced towards the bottom part of the pocket to locate its hydroxyl group pointing towards the main carbonyl group of Val116, remaining in this arrangement after ∼15 ns of simulation. (B) Variation of the relative distance between the oxygen atom (OH group) of the quinazoline moiety in GFT-MB and the oxygen atom of the main carbonyl group of Val116 in the GFT-MB@HSA protein complex during whole simulation. (C) Variation of the relative distance between the mass center of the phenol groups in Tyr138 and Tyr161 and GFT-MB in the GFT-MB@HSA protein complex during whole simulation. Note how, after stabilization, both residues remain in close contact with the ligand during the simulation.