Photosensitivity to Triflusal: Formation of a Photoadduct with Ubiquitin Demonstrated by Photophysical and Proteomic Techniques

[EN] Triflusal is a platelet aggregation inhibitor chemically related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacological point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clinical profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint analysis. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradiation, combining proteomic and photophysical (fluorescence and laser flash photolysis) techniques. Photophysical and proteomic analysis demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addition can take place at any of the E-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroaromatic compounds and may be responsible for the appearance of undesired photoallergic side effects.Financial support from the Generalitat Valenciana (Prometeo Program), the Spanish Government (MINECO CTQ2015-70164-P to VL-V and SAF2012-36519 to DP-S) and the Carlos III Institute of Health (Grant RIRAAF, RETICS program, RD12/0013/0009 to MM and RD12/0013/0008 to DP-S, and Miguel Servet Contract CP11/00154 for IA) is gratefully acknowledged.Nuin Pla, NE.; Pérez-Sala, D.; Lhiaubet-Vallet, VL.; Andreu Ros, MI.; Miranda Alonso, MÁ. (2016). Photosensitivity to Triflusal: Formation of a Photoadduct with Ubiquitin Demonstrated by Photophysical and Proteomic Techniques. Frontiers in Pharmacology. 7(277). https://doi.org/10.3389/fphar.2016.00277S727

"Snorkelling" vs. "diving" in mixed micelles probed by means of a molecular bathymeter

[EN] A photoactive bathymeter based on a carboxylic acid moiety covalently linked to a signalling methoxynaphthalene (MNP) fluorophore has been designed to prove the concept of "snorkelling" vs. "diving" in mixed micelles (MM). The carboxylic acid "floats" on the MM surface, while the MNP unit sinks deep in MM. The rate constants of MNP fluorescence quenching by iodide, which remains basically in water, consistently decrease with increasing spacer length, revealing different regions. This is associated with the distance MNP should "dive" in MM to achieve protection from aqueous reactants. Unequivocal proof of the exergonic photoinduced electron transfer was obtained from the UV-visible spectral signature of I-3(-) upon steady-state photolysis. The applicability of the bathymeter was examined upon testing a family of MNP derivatives. The obtained results were validated by comparison with different lipophilicity tests: (i) a modified version of the K-ow partition coefficient and (ii) the retention factor on thin layer chromatography. This concept could potentially be extended to test drugs or pharmacophores exhibiting any photoactive moiety.Financial support from the Spanish Government (SEV-2016-0683), Red RETICS de Investigacion de Reacciones Adversas a Alergenos y Farmacos (RIRAAF), Instituto de Salud Carlos III (RD012/0013, RD16/0006/0030, FIS PI16/01877), VLC-Campus and the Generalitat Valenciana (Prometeo Program) is gratefully acknowledged.Rodríguez Muñiz, GM.; Gomez Mendoza, M.; Nuin Pla, NE.; Andreu Ros, MI.; Marín García, ML.; Miranda Alonso, MÁ. (2017). "Snorkelling" vs. "diving" in mixed micelles probed by means of a molecular bathymeter. Organic & Biomolecular Chemistry. 15(48):10281-10288. https://doi.org/10.1039/c7ob02595eS10281102881548Porter, C. J. H., Trevaskis, N. L., & Charman, W. N. (2007). Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nature Reviews Drug Discovery, 6(3), 231-248. doi:10.1038/nrd2197Hammad, M. ., & Müller, B. . (1998). Increasing drug solubility by means of bile salt–phosphatidylcholine-based mixed micelles. European Journal of Pharmaceutics and Biopharmaceutics, 46(3), 361-367. doi:10.1016/s0939-6411(98)00037-xNagadome, S., Numata, O., Sugihara, G., Sasaki, Y., & Igimi, H. (1995). Solubilization and precipitation of cholesterol in aqueous solution of bile salts and their mixtures. Colloid & Polymer Science, 273(7), 675-680. doi:10.1007/bf00652260Hofmann, A. F. (1999). The Continuing Importance of Bile Acids in Liver and Intestinal Disease. Archives of Internal Medicine, 159(22), 2647. doi:10.1001/archinte.159.22.2647Ding, J., Sun, Y., Li, J., Wang, H., & Mao, S. (2017). Enhanced blood–brain barrier transport of vinpocetine by oral delivery of mixed micelles in combination with a message guider. Journal of Drug Targeting, 25(6), 532-540. doi:10.1080/1061186x.2017.1289541Lasic, D. D. (1992). Mixed micelles in drug delivery. Nature, 355(6357), 279-280. doi:10.1038/355279a0Cheng, L., Kamkaew, A., Sun, H., Jiang, D., Valdovinos, H. F., Gong, H., … Cai, W. (2016). Dual-Modality Positron Emission Tomography/Optical Image-Guided Photodynamic Cancer Therapy with Chlorin e6-Containing Nanomicelles. ACS Nano, 10(8), 7721-7730. doi:10.1021/acsnano.6b03074Lipinski, C. A., Lombardo, F., Dominy, B. W., & Feeney, P. J. (1997). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews, 23(1-3), 3-25. doi:10.1016/s0169-409x(96)00423-1Beaumont, K., Schmid, E., & Smith, D. A. (2005). Oral delivery of G protein-coupled receptor modulators: An explanation for the observed class difference. Bioorganic & Medicinal Chemistry Letters, 15(16), 3658-3664. doi:10.1016/j.bmcl.2005.05.042Al-Abdul-Wahid, M. S., Neale, C., Pomès, R., & Prosser, R. S. (2009). A Solution NMR Approach to the Measurement of Amphiphile Immersion Depth and Orientation in Membrane Model Systems. Journal of the American Chemical Society, 131(18), 6452-6459. doi:10.1021/ja808964eAfri, M., Frimer, A. A., & Cohen, Y. (2004). Active oxygen chemistry within the liposomal bilayer. Chemistry and Physics of Lipids, 131(1), 123-133. doi:10.1016/j.chemphyslip.2004.04.006Cohen, Y., Bodner, E., Richman, M., Afri, M., & Frimer, A. A. (2008). NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer. Chemistry and Physics of Lipids, 155(2), 98-113. doi:10.1016/j.chemphyslip.2008.07.004Cohen, Y., Afri, M., & Frimer, A. A. (2008). NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer. Chemistry and Physics of Lipids, 155(2), 114-119. doi:10.1016/j.chemphyslip.2008.07.007Afri, M., Alexenberg, C., Aped, P., Bodner, E., Cohen, S., Ejgenburg, M., … Frimer, A. A. (2014). NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer. Chemistry and Physics of Lipids, 184, 105-118. doi:10.1016/j.chemphyslip.2014.07.007Afri, M., Naqqash, M. E., & Frimer, A. A. (2011). Using fluorescence to locate intercalants within the lipid bilayer of liposomes, bioliposomes and erythrocyte ghosts. Chemistry and Physics of Lipids, 164(8), 759-765. doi:10.1016/j.chemphyslip.2011.09.002Bodner, E., Afri, M., & Frimer, A. A. (2010). Determining radical penetration into membranes using ESR splitting constants. Free Radical Biology and Medicine, 49(3), 427-436. doi:10.1016/j.freeradbiomed.2010.04.029Laguerre, M., López Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Baréa, B., Weiss, J., … Villeneuve, P. (2009). Chain Length Affects Antioxidant Properties of Chlorogenate Esters in Emulsion: The Cutoff Theory Behind the Polar Paradox. Journal of Agricultural and Food Chemistry, 57(23), 11335-11342. doi:10.1021/jf9026266Laguerre, M., López Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Baréa, B., Weiss, J., … Villeneuve, P. (2010). Relationship between Hydrophobicity and Antioxidant Ability of «Phenolipids» in Emulsion: A Parabolic Effect of the Chain Length of Rosmarinate Esters. Journal of Agricultural and Food Chemistry, 58(5), 2869-2876. doi:10.1021/jf904119vAliaga, C., Bravo-Moraga, F., Gonzalez-Nilo, D., Márquez, S., Lühr, S., Mena, G., & Rezende, M. C. (2016). Location of TEMPO derivatives in micelles: subtle effect of the probe orientation. Food Chemistry, 192, 395-401. doi:10.1016/j.foodchem.2015.07.036Aliaga, C., López de Arbina, A., & Rezende, M. C. (2016). «Cut-off» effect of antioxidants and/or probes of variable lipophilicity in microheterogeneous media. Food Chemistry, 206, 119-123. doi:10.1016/j.foodchem.2016.03.024Lopez de Arbina, A., Rezende, M. C., & Aliaga, C. (2017). Cut-off effect of radical TEMPO derivatives in olive oil-in-water emulsions. Food Chemistry, 224, 342-346. doi:10.1016/j.foodchem.2016.12.058Agostinis, P., Berg, K., Cengel, K. A., Foster, T. H., Girotti, A. W., Gollnick, S. O., … Golab, J. (2011). Photodynamic therapy of cancer: An update. CA: A Cancer Journal for Clinicians, 61(4), 250-281. doi:10.3322/caac.20114Kamkaew, A., Lim, S. H., Lee, H. B., Kiew, L. V., Chung, L. Y., & Burgess, K. (2013). BODIPY dyes in photodynamic therapy. Chem. Soc. Rev., 42(1), 77-88. doi:10.1039/c2cs35216hYano, S., Hirohara, S., Obata, M., Hagiya, Y., Ogura, S., Ikeda, A., … Joh, T. (2011). Current states and future views in photodynamic therapy. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 12(1), 46-67. doi:10.1016/j.jphotochemrev.2011.06.001Dolmans, D. E. J. G. J., Fukumura, D., & Jain, R. K. (2003). Photodynamic therapy for cancer. Nature Reviews Cancer, 3(5), 380-387. doi:10.1038/nrc1071Bronshtein, I., Afri, M., Weitman, H., Frimer, A. A., Smith, K. M., & Ehrenberg, B. (2004). Porphyrin Depth in Lipid Bilayers as Determined by Iodide and Parallax Fluorescence Quenching Methods and Its Effect on Photosensitizing Efficiency. Biophysical Journal, 87(2), 1155-1164. doi:10.1529/biophysj.104.041434Lavi, A., Weitman, H., Holmes, R. T., Smith, K. M., & Ehrenberg, B. (2002). The Depth of Porphyrin in a Membrane and the Membrane’s Physical Properties Affect the Photosensitizing Efficiency. Biophysical Journal, 82(4), 2101-2110. doi:10.1016/s0006-3495(02)75557-4Chattopadhyay, A., & London, E. (1987). Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry, 26(1), 39-45. doi:10.1021/bi00375a006Nuin, E., Gómez-Mendoza, M., Andreu, I., Marin, M. L., & Miranda, M. A. (2012). New Photoactive Compounds To Probe Cholic Acid and Cholesterol inside Mixed Micelles. Organic Letters, 15(2), 298-301. doi:10.1021/ol303201yNuin, E., Gomez-Mendoza, M., Marin, M. L., Andreu, I., & Miranda, M. A. (2013). Influence of Drug Encapsulation within Mixed Micelles on the Excited State Dynamics and Accessibility to Ionic Quenchers. The Journal of Physical Chemistry B, 117(32), 9327-9332. doi:10.1021/jp404353uGomez-Mendoza, M., Nuin, E., Andreu, I., Marin, M. L., & Miranda, M. A. (2012). Photophysical Probes To Assess the Potential of Cholic Acid Aggregates as Drug Carriers. The Journal of Physical Chemistry B, 116(34), 10213-10218. doi:10.1021/jp304708yBoschloo, G., & Hagfeldt, A. (2009). Characteristics of the Iodide/Triiodide Redox Mediator in Dye-Sensitized Solar Cells. Accounts of Chemical Research, 42(11), 1819-1826. doi:10.1021/ar900138mGardner, J. M., Abrahamsson, M., Farnum, B. H., & Meyer, G. J. (2009). Visible Light Generation of Iodine Atoms and I−I Bonds: Sensitized I−Oxidation and I3−Photodissociation. Journal of the American Chemical Society, 131(44), 16206-16214. doi:10.1021/ja905021cSangster, J. (1989). Octanol‐Water Partition Coefficients of Simple Organic Compounds. Journal of Physical and Chemical Reference Data, 18(3), 1111-1229. doi:10.1063/1.55583