Joint Research Unit in in Molecular mechanisms of Drug adverse reactions

Research lines

Photoactive species as probes for proteins. This line aims to develop a new metrology, based on the behavior of transient species generated from photoactive probes in the presence of porteins. In this way, we intend to produce relevant information about the interactions that take place in the active sites of the biomolecules, focusing attention on the transducing proteins (serum albumin and acidic alpha-glycoprotein). The probes are selected from among the drugs and their metabolites, due to their pharmacological and toxicological interest. An essential requirement is that these probes can generate transient species with properties that depend on the medium, detectable by photophysical techniques in steady state and in resolved time. At a later stage it is intended to apply this methodology to less common proteins of biological importance, such as pharmacological receptors and metabolic enzymes.

Photochemical mechanisms of DNA damage and its repair. Photosensitization versus protection. This line focuses on studying the carcinogenic and mutagenic effects of solar radiation. In this context, the knowledge of DNA photolesions is a central issue since it has been unequivocally demonstrated that exposure to ultraviolet solar radiation is involved in the pathologies of carcinomas and melanomas. To protect themselves from these harmful effects, living organisms have enzymes that repair the lesions to their original form, thus maintaining genetic integrity. In mammalian cells, damage is usually eliminated through repair by excision of nucleotides or bases. However, the repair of some injuries represents a challenge for the organism. In this context, multiple injuries such as double-strand breaks or cluster-type damage are of special relevance. The study of these lesions is of great complexity due to its difficult analytical characterization and the determination of its role in the induction of harmful effects to the cells. Investigating the photochemical mechanisms is essential to understand most of the key processes involved in both damage and DNA repair.

Functionalization of gold nanoparticles with biological targets and singlet oxygen photosensitisers for their use in biomedicine. The design of gold nanoparticles (AuNPs) and exploration of their physical and chemical properties is a topic of high interest due to their extensive use in biomedicine and new technologies. Besides, they exhibit exceptional electronic and optical properties making them suitable for imaging applications. The biological function of AuNPs depends on a number of parameters such as size, shape, surface charge and functionalization, properties of the environment, etc. Moreover, their reactivity in solution and within a biological media can be modified due to the spontaneous coating of their surface by biomolecules such as proteins, lipids, nucleic acids, etc. The functionalization of AuNPs using suitable ligands such as drugs, polyethylene glycol, amino acids (or peptides), folic acid, etc., may help to decrease or even eliminate undesired side effects, but also to enhance the biological function, delivery and targeting of the nanomaterial. Therefore, the main goal of this research line is the photophysical design and caracterization of funcionalized AuNPs with drugs and biological probes in order to deeply discover the parameters which affect their formation and stablity, and in consequence improving their biological function and decrease their toxicity.

Photoactive metabolites. This line addreesses the chemical mechanisms of phototoxicity, photoallergy and photocarcinogenesis mediated by interaction of sunlight with metabolites of widely used xenobiotics. While the photosensitizing potential of the parent xenobiotics can be screened following relatively straightforward methodologies, identification of metabolites with phototoxic or adduct forming capability still remains a major challenge. Hence, a well-planned effort is essential for improving the knowledge in this field. Accordingly, the ultimate goal of the project is to develop a methodology for early assessment of the photobiological risk linked to formation of photoactive metabolites before introduction of the parent xenobiotics in the market. This would contribute to a rational design and should result in economic advantage for the chemical-pharmaceutical industry. Thorough photochemical studies of a number of selected xenobiotic metabolites will be performed, centered on identification of excited states and reaction intermediates as well as on their interaction with biological substrates; the studies will be complemented by collaborative work on the cellular and translational aspects of the problem. The specific steps include: i) characterization of the electronically excited states of the selected metabolites in biologically relevant media, ii) direct detection of the photogenerated short-lived intermediates (radicals, radical ions, reactive oxygen species, etc.), iii) determination of the photochemical reactivity and identification of the stable photoproducts, iv) analysis of the interactions between the excited metabolites and biological targets (lipids, proteins, DNA and their building blocks), v) in vitro studies based on the bioactivation of xenobiotics by human cells expressing the relevant biotransformation activities, vi) evaluation of the possibility for xenobiotics lacking photosensitizing properties per se to undergo effective conversion into photoactive metabolites, vii) assessment of whether xenobiotic metabolism is a likely mechanism for the generation of phototoxins, and viii) establishment of qualitative structure-activity relationships and definition of alert rules to identify potential photosensitizers acting through their reactive metabolites.

Photodiagnostic: clinical evaluation, biological assays and mechanistic studies. The goal of the present project is to investigate photosensitivity phenomenon by xenobiotics and their metabolites, including drugs widely employed in the treatment of severe diseases and several relevant sunscreens used in photoprotection. For this purpose a photodiagnostic multidisciplinary approach will be carried out encompassing clinical assessment and chemical and biological mechanistic studies. Thus, it is intended to identify potential photosensitizing agents and to clarify the mechanistic aspects involved in photo(geno)toxic, photoallergic and photocarcinogenic underlying effects. To achieve the proposed objectives, it will be combined several techniques: photodiagnostic (clinical history, phototest, photopatch), organic synthesis in order to prepare (photo)metabolites not commercially available, biological (3T3 NRU phototoxicity test, lipid peroxidation assay, COMET assay, mutagenicity test) in vitro from commercial cell lines or primary cell cultures of fibroblasts or keratinocytes isolated from skin biopsies of patients, photophysical to characterize reactive intermediates and ultraperformance liquid chromatography coupled to tandem mass spectrometry.