Abstract
Thioredoxins are multifunctional oxidoreductase proteins implicated in the antioxidant cellular apparatus and oxidative stress. They are involved in several pathologies and are promising anticancer targets. Identification of noncatalytic binding sites is of great interest for designing new allosteric inhibitors of thioredoxin. In a recent work, we predicted normal mode motions of human thioredoxin 1 and identified two major putative hydrophobic binding sites. In this work we investigated noncovalent interactions of human thioredoxin 1 with three phenotiazinic drugs acting as prooxidant compounds by using molecular docking and circular dichroism spectrometry to probe ligand binding into the previously predicted allosteric hydrophobic pockets. Our in silico and CD spectrometry experiments suggested one preferred allosteric binding site involving helix 3 and adopting the best druggable conformation identified by NMA. The CD spectra showed binding of thioridazine into thioredoxin 1 and suggested partial helix unfolding, which most probably concerns helix 3. Taken together, these data support the strategy to design thioredoxin inhibitors targeting a druggable allosteric binding site.
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Abbreviations
- FP:
-
Fluphenazine
- hTrx1:
-
Human thioredoxin 1
- PTZ:
-
Phenothiazine
- TFP:
-
Trifluoperazine
- TR:
-
Thioridazine
- Trx:
-
Thioredoxin
- hTrx:
-
Human Thioreoxin 1
- H1H3:
-
hTrx1 structure 0.4 Å displaced along mode 16
- H2H4:
-
hTrx1 structure 1 Å displaced along mode 8
References
Adolph O, Köster S, Georgieff M et al (2012) Promethazine inhibits NMDA-induced currents—new pharmacological aspects of an old drug. Neuropharmacology 63:280–291. doi:10.1016/j.neuropharm.2012.03.006
Amaral L, Viveiros M, Molnar J (2004) Antimicrobial activity of phenothiazines. In Vivo 18:725–731
Arnér ES, Holmgren A (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 267:6102–6109
Arnér ESJ, Holmgren A (2006) The thioredoxin system in cancer. Semin Cancer Biol 16:420–426. doi:10.1016/j.semcancer.2006.10.009
Borges MBD, Dos Santos CG, Yokomizo CH et al (2010) Characterization of hydrophobic interaction and antioxidant properties of the phenothiazine nucleus in mitochondrial and model membranes. Free Radic Res 44:1054–1063. doi:10.3109/10715762.2010.498826
Brooks BR, Brooks CL, Mackerell AD et al (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30:1545–1614. doi:10.1002/jcc.21287
Craig JC, Tate ME, Warwick GP, Rogers WP (1960) Chemical constitution and anthelmintic activity. IV. Substituted phenothiazines. J Med Pharm Chem 2:659–668
Cruz TS, Faria PA, Santana DP et al (2010) On the mechanisms of phenothiazine-induced mitochondrial permeability transition: thiol oxidation, strict Ca2+ dependence, and cyt c release. Biochem Pharmacol 80:1284–1295. doi:10.1016/j.bcp.2010.06.052
Cruzeiro-Silva C, Gomes-Neto F, Machado LESF et al (2014) Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange. Biochemistry 53:2890–2902. doi:10.1021/bi401542v
De Faria PA, Bettanin F, Cunha RLOR et al (2015) Cytotoxicity of phenothiazine derivatives associated with mitochondrial dysfunction: a structure-activity investigation. Toxicology 330C:44–54. doi:10.1016/j.tox.2015.02.004
DiRaimondo TR, Plugis NM, Jin X, Khosla C (2013) Selective inhibition of extracellular thioredoxin by asymmetric disulfides. J Med Chem 56:1301–1310. doi:10.1021/jm301775s
Floquet N, Marechal J-D, Badet-Denisot M-A et al (2006) Normal mode analysis as a prerequisite for drug design: application to matrix metalloproteinases inhibitors. FEBS Lett 580:5130–5136. doi:10.1016/j.febslet.2006.08.037
Forman-Kay JD, Clore GM, Wingfield PT, Gronenborn AM (1991) High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution. Biochemistry 30:2685–2698. doi:10.1021/bi00224a017
Holmgren A (1995) Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3:239–243
Holmgren A, Lu J (2010) Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun 396:120–124. doi:10.1016/j.bbrc.2010.03.083
Kirkpatrick DL, Ehrmantraut G, Stettner S et al (1997) Redox active disulfides: the thioredoxin system as a drug target. Oncol Res 9:351–356
Lee S, Kim SM, Lee RT (2013) Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. Antioxid Redox Signal 18:1165–1207. doi:10.1089/ars.2011.4322
Mahmood DFD, Abderrazak A, El Hadri K et al (2013) The thioredoxin system as a therapeutic target in human health and disease. Antioxid Redox Signal 19:1266–1303. doi:10.1089/ars.2012.4757
Maurer H, Pfleger K (1988) Identification of phenothiazine antihistamines and their metabolites in urine. Arch Toxicol 62:185–191
Meyer Y, Buchanan BB, Vignols F, Reichheld J-P (2009) Thioredoxins and glutaredoxins: unifying elements in redox biology. Annu Rev Genet 43:335–367. doi:10.1146/annurev-genet-102108-134201
Milnes JT, Witchel HJ, Leaney JL et al (2006) hERG K+ channel blockade by the antipsychotic drug thioridazine: an obligatory role for the S6 helix residue F656. Biochem Biophys Res Commun 351:273–280. doi:10.1016/j.bbrc.2006.10.039
Miteva MA, Guyon F, Tufféry P (2010) Frog2: efficient 3D conformation ensemble generator for small compounds. Nucleic Acids Res 38:W622–W627. doi:10.1093/nar/gkq325
Murray CW, Baxter CA, Frenkel AD (1999) The sensitivity of the results of molecular docking to induced fit effects: application to thrombin, thermolysin and neuraminidase. J Comput Aided Mol Des 13:547–562. doi:10.1023/A:1008015827877
Ngan C-H, Hall DR, Zerbe B et al (2012) FTSite: high accuracy detection of ligand binding sites on unbound protein structures. Bioinformatics 28:286–287. doi:10.1093/bioinformatics/btr651
Oblong JE, Berggren M, Gasdaska PY et al (1994) Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin. J Biol Chem 269:11714–11720
Ohlow MJ, Moosmann B (2011) Phenothiazine: the seven lives of pharmacology’s first lead structure. Drug Discov Today 16:119–131. doi:10.1016/j.drudis.2011.01.001
Park IY, Kim EJ, Park H et al (2005) Interaction between cardiac calsequestrin and drugs with known cardiotoxicity. Mol Pharmacol 67:97–104. doi:10.1124/mol.104.005744
Pessoto FS, Yokomizo CH, Prieto T et al (2015) Thiosemicarbazone p-substituted acetophenone derivatives promote the loss of mitochondrial Δψ, GSH depletion, and death in K562 cells. Oxid Med Cell Longev 2015:394367. doi:10.1155/2015/394367
Philot EA, Perahia D, Braz ASK et al (2013) Binding sites and hydrophobic pockets in Human Thioredoxin 1 determined by normal mode analysis. J Struct Biol 184:293–300. doi:10.1016/j.jsb.2013.09.002
Qin J, Clore GM, Gronenborn AM (1994) The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin. Structure 2:503–522
Ramanathan RK, Kirkpatrick DL, Belani CP et al (2007) A Phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors. Clin Cancer Res 13:2109–2114. doi:10.1158/1078-0432.CCR-06-2250
Ramanathan RK, Abbruzzese J, Dragovich T et al (2011) A randomized phase II study of PX-12, an inhibitor of thioredoxin in patients with advanced cancer of the pancreas following progression after a gemcitabine-containing combination. Cancer Chemother Pharmacol 67:503–509. doi:10.1007/s00280-010-1343-8
Rodrigues T, Santos AC, Pigoso AA et al (2002) Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis–potentially implicated mechanisms. Br J Pharmacol 136:136–142. doi:10.1038/sj.bjp.0704672
Seeliger D, de Groot BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24:417–422. doi:10.1007/s10822-010-9352-6
Sharma A, Sharma A, Dixit S, Sharma A (2011) Structural insights into thioredoxin-2: a component of malaria parasite protein secretion machinery. Sci Rep 1:179. doi:10.1038/srep00179
Shen WW (1999) A history of antipsychotic drug development. Compr Psychiatry 40:407–414. doi:10.1016/S0010-440X(99)90082-2
Subra AK, Nissen MS, Lewis KM et al (2012) Molecular mechanisms of pharmaceutical drug binding into calsequestrin. Int J Mol Sci 13:14326–14343. doi:10.3390/ijms131114326
Tonissen KF, Di Trapani G (2009) Thioredoxin system inhibitors as mediators of apoptosis for cancer therapy. Mol Nutr Food Res 53:87–103. doi:10.1002/mnfr.200700492
Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461. doi:10.1002/jcc.21334
Weichsel A, Kem M, Montfort WR (2010) Crystal structure of human thioredoxin revealing an unraveled helix and exposed S-nitrosation site. Protein Sci 19:1801–1806. doi:10.1002/pro.455
Acknowledgments
Support from Institut national de la santé et de la recherche médicale-Inserm, Centre National de la Recherche Scientifique-CNRS, Universidade Federal do ABC-UFABC, Comissão de Aperfeiçoamento de Pessoal do Nível Superior-CAPES, Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2011/11857-4, 2012/12247-8, 2012/07456-7) and Conselho Nacional de Pesquisa-CNPq (486067/2013-9) are greatly appreciated.
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Philot, E.A., da Mata Lopes, D., de Souza, A.T. et al. Binding of phenothiazines into allosteric hydrophobic pocket of human thioredoxin 1. Eur Biophys J 45, 279–286 (2016). https://doi.org/10.1007/s00249-016-1113-6
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DOI: https://doi.org/10.1007/s00249-016-1113-6