Unión fotoquímica irreversible de ligandos a albúminas séricas

  1. Molins Molina, Óscar
Dirigida por:
  1. Raúl Pérez Ruiz Director/a
  2. María Consuelo Jiménez Molero Director/a

Universidad de defensa: Universitat Politècnica de València

Fecha de defensa: 24 de febrero de 2020

Tribunal:
  1. Uwe Pischel Presidente/a
  2. María Consuelo Cuquerella Alabort Secretario/a
  3. Rafael Alonso Ruiz Vocal

Tipo: Tesis

Resumen

In this thesis a multidisciplinary strategy has been developed that includes irradiation of ligand/protein complexes along with fluorescence and/or transient absorption spectroscopy, size-exclusion chromatography followed by absorption and/or fluorescence spectroscopy, proteomic analysis and modelling (docking and molecular dynamics simulations) in order to deepen and obtain relevant information in processes related to the formation of irreversible ligand-protein complexes. This has made it possible to achieve the description of the molecular recognition centre of serum albumin of different species by the carprofen drug, to deepen in photoallergy processes produced by the metabolite of the triflusal drug and to carry out the la-belling of lysine residues of human serum albumin by photogeneration of latent electrophiles "quinone methide". Each of these aspects is briefly described below. First, the possible existence of a common recognition centre in serum albumin (SA) of different species has been studied using the non-steroidal antiinflammatory drug (S)-carprofen (CPF) as a photoactive probe. Thus, irradiation of the CPF/SA complexes at ¿max = 320 nm has been followed by fluorescence, showing an increase in emission due to dehalogenation. After gel filtration chromatography, the protein fraction presented emission from the ligand, verifying the covalent bonding of the CBZ¿ intermediate photogenerated radical to the SA. Proteomic analysis revealed the incorporation of CBZ¿ in various positions in the different albumins. Modifications in the IB/IIIA interface were observed in all cases (Tyr452 in human, rabbit and rat serum albumin and Tyr451 in bovine, porcine and sheep serum albumin). Docking and molecular dynamics simulation studies in the case of human serum albumin corroborated the experimentally observed covalent modifications. Subsequently, the photochemical binding of HTB, the metabolite of the triflusal platelet antiaggregant to human serum albumin (HSA), has been investigated. Proteomic analysis of the HTB/HSA solutions after being irradiated showed the addition of HTB in the ¿-amino groups of residues Lys137, Lys199, Lys205, Lys352, Lys432, Lys541, Lys545 and Lys525 of the HSA. The reaction mechanism seems to involve replacing the CF3 group of HTB with a new amide residue. Only the Lys199 residue is located in an internal cavity of the protein whilst the rest of the modified residues were found to be located on the outside. Computational studies revealed that supramolecular binding of HTB to HSA occurs in the "V-cleft" region. This photochemical binding may be at the base of the appearance of unwanted photoallergic side effects. Finally, the utility of 4-trifluoromethylphenols as precursors of latent "quinone methide" (QM) type electrophiles for specific binding to lysine residues found at the protein binding sites has been demonstrated. Thus, it has been observed that these photogenerated Michael acceptors, have been able to perform a specific covalent modification of lysine residues in human serum albumin (HSA). Specifically, the QM type reactive intermediates generated after irradiation of the 4-trifluoromethyl-1-naphthol or 4- (4-trifluoromethylphenyl) phenol complexes with HSA exhibited chemical selectivity towards lysine residues giving rise to amide adducts. A detailed study conducted by proteomic analysis confirmed this fact. Thus, for the naphthol derivative the covalent modification of residues Lys106 and Lys414 (located in subdomains IA and IIIA, respectively) was observed, while for the biphenyl derivative the modification occurred in Lys195 (in subdomain IIA). Theoretical studies provided a deeper insight at the molecular level of the experimentally observed selectivity.