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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., Financial support from the Spanish Ministry of Science and Innovation (RYC-2015-17737, BEAGAL 18/00211, PID2020-115010RB-I00/AEI/10.13039/501100011033, FPU19/00048, PID2019-105512RB-I00/AEI/10.13039/501100011033), the Xunta de Galicia (ED431C 2021/29), the Centro singular de investigación de Galicia accreditation 2019–2022 (ED431G 2019/03), and the European Regional Development Fund (ERDF) is gratefully acknowledged. All authors are grateful to the Centro de Supercomputación de Galicia (CESGA) for use of the Finis Terrae computer.

[EN] Gefitinib (GFT) is a tyrosine kinase inhibitor currently used for the treatment of metastatic non-small cell lung cancer. Although it has been suggested that GFT can be phototoxic, there are no systematic studies on this issue. Here, the photosensitizing potential of GFT has been assessed by means of NRU assays and protein photooxidation. In addition, a thorough photophysical study is presented based on ultrafast transient absorption spectroscopy, fluorescence and laser flash photolysis. Transient species generated after excitation of GFT have been characterized in solution and in biological environments (i.e. HSA and HaCaT cells) to gain insight into the mechanisms involved in photodamage. The photobehavior of GFT was strongly medium-dependent. Excitation of the drug resulted in the formation of locally excited (LE) singlet states ((1)GFT*), which were found to be the main emissive species in non-polar solvents and also within HSA and HaCaT cells. By contrast, in polar solvents, LE states rapidly evolved (similar to 1 ps) towards the formation of longer-lived intramolecular charge transfer (ICT) states. The triplet excited state of GFT ((3)GFT*) can be formed through intersystem crossing from (1)GFT* in non-polar solvents and from ICT states in the polar ones, or in the particular case of ethanol, by photosensitization using 2-methoxyacetophenone as an energy donor. In the HSA environment, (3)GFT* was hardly detected due to quenching of its LE (1)GFT* precursor by Trp through an electron transfer process. Accordingly, HSA photooxidation by GFT was demonstrated using the protein carbonylation method. In summary, a good correlation is established between the photophysical behavior and the photobiological properties of GFT, which provides a mechanistic basis for the observed phototoxicity., Financial support from the Spanish Government (RYC-2015-17737, CTQ2017-89416-R, BEAGAL 18/00211, FPU19/00048 and PID2020-115010RB-I00; the Carlos III Institute (ISCIII) of Health, grants: PI16/01877, and CPII16/00052) and Conselleria d'Educacio, Cultura i Esport (PROMETEO/2017/075) is gratefully acknowledged.