[EN] Colchicine (COL) is a bioactive molecule with antitumor properties. When COL binds to tubulin (TU), it inhibits microtubule assembly dynamics. We have investigated COL-TU interactions using laser flash photolysis (LFP) technique and performing fully flexible molecular dynamics simulations. Excitation of COL at 355 nm in aqueous medium did not lead to any transient absorption spectrum. By contrast, in the presence of TU a transient peaking at lambda(max) ca. 420 nm was registered and assigned as triplet excited COL complexed with TU ((COL)-C-3*@TU). In aerated medium, the lifetime was tau ca. 160 mu s and the quantum yield was 0.138. Likewise, when the bicyclic COL analog MTC was submitted to LFP in the presence of TU, (MTC)-M-3@TU* was detected with a lifetime of ca. 62 ms and a quantum yield of 0.296, Aqueous solutions of MTC did not produce any signal in the microsecond timescale. The triplet energy of MTC was obtained by means of emission measurements and found to be ca. 200 kJ mol(-1), a value that matches with that previously reported for COL (188 kJ mol(-1)). Molecular dynamic simulations, both with the ground and triplet excited state, reveal a strong interaction between COL and TU to give stabilized complexes with restricted mobility inside the protein binding site. These results demonstrate that LFP is a useful methodology to study the binding of COL derivatives to TU and open a new way to evaluate the interactions of non-fluorescent anticancer drugs with this protein.Financial support from the Spanish Government (grants CTQ2010-19909; BFU2011-23416 and SEV 2012-0267), the Generalitat Valenciana (Prometeo II/2013/005) and Comunidad de Madrid (S2010/BMD-2353) is gratefully acknowledged. G.S. thanks ASIC-UPV for computing time.Bosca Mayans, F.; Sastre Navarro, GI.; Andreu, JM.; Jornet, D.; Tormos Faus, RE.; Miranda Alonso, MÁ. (2015). Drug-tubulin interactions interrogated by transient absorption spectroscopy. RSC Advances. 5(61):49451-49458. https://doi.org/10.1039/C5RA05636ES4945149458561Mitchison, T., & Kirschner, M. (1984). Dynamic instability of microtubule growth. Nature, 312(5991), 237-242. doi:10.1038/312237a0Margolis, R. L., & Wilson, L. (1978). Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell, 13(1), 1-8. doi:10.1016/0092-8674(78)90132-0Desai, A., & Mitchison, T. J. (1997). MICROTUBULE POLYMERIZATION DYNAMICS. Annual Review of Cell and Developmental Biology, 13(1), 83-117. doi:10.1146/annurev.cellbio.13.1.83Howard, J., & Hyman, A. A. (2003). Dynamics and mechanics of the microtubule plus end. Nature, 422(6933), 753-758. doi:10.1038/nature01600Jordan, M. A., & Wilson, L. (2004). Microtubules as a target for anticancer drugs. Nature Reviews Cancer, 4(4), 253-265. doi:10.1038/nrc1317Ravelli, R. B. G., Gigant, B., Curmi, P. A., Jourdain, I., Lachkar, S., Sobel, A., & Knossow, M. (2004). Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature, 428(6979), 198-202. doi:10.1038/nature02393Cormier, A., Marchand, M., Ravelli, R. B. G., Knossow, M., & Gigant, B. (2008). Structural insight into the inhibition of tubulin by vinca domain peptide ligands. EMBO reports, 9(11), 1101-1106. doi:10.1038/embor.2008.171Prota, A. E., Bargsten, K., Diaz, J. F., Marsh, M., Cuevas, C., Liniger, M., … Steinmetz, M. O. (2014). A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs. Proceedings of the National Academy of Sciences, 111(38), 13817-13821. doi:10.1073/pnas.1408124111Prota, A. E., Bargsten, K., Zurwerra, D., Field, J. J., Díaz, J. F., Altmann, K.-H., & Steinmetz, M. O. (2013). Molecular Mechanism of Action of Microtubule-Stabilizing Anticancer Agents. Science, 339(6119), 587-590. doi:10.1126/science.1230582Prota, A. E., Bargsten, K., Northcote, P. T., Marsh, M., Altmann, K.-H., Miller, J. H., … Steinmetz, M. O. (2014). Structural Basis of Microtubule Stabilization by Laulimalide and Peloruside A. Angewandte Chemie International Edition, 53(6), 1621-1625. doi:10.1002/anie.201307749Brossi, A., Yeh, H. J. C., Chrzanowska, M., Wolff, J., Hamel, E., Lin, C. M., … Silverton, J. (1988). Colchicine and its analogues: Recent findings. Medicinal Research Reviews, 8(1), 77-94. doi:10.1002/med.2610080105Imazio, M., Trinchero, R., & Adler, Y. (2008). Colchicine for the treatment of pericarditis. Future Cardiology, 4(6), 599-607. doi:10.2217/14796678.4.6.599Fakih, M., Replogle, T., Lehr, J. E., Pienta, K. J., & Yagoda, A. (1995). Inhibition of prostate cancer growth by estramustine and colchicine. The Prostate, 26(6), 310-315. doi:10.1002/pros.2990260606Lee, R. M., & Gewirtz, D. A. (2008). Colchicine site inhibitors of microtubule integrity as vascular disrupting agents. Drug Development Research, 69(6), 352-358. doi:10.1002/ddr.20267Abad, A., López-Pérez, J. L., del Olmo, E., García-Fernández, L. F., Francesch, A., Trigili, C., … San Feliciano, A. (2012). Synthesis and Antimitotic and Tubulin Interaction Profiles of Novel Pinacol Derivatives of Podophyllotoxins. Journal of Medicinal Chemistry, 55(15), 6724-6737. doi:10.1021/jm2017573Álvarez, R., Puebla, P., Díaz, J. F., Bento, A. C., García-Navas, R., de la Iglesia-Vicente, J., … Peláez, R. (2013). Endowing Indole-Based Tubulin Inhibitors with an Anchor for Derivatization: Highly Potent 3-Substituted Indolephenstatins and Indoleisocombretastatins. Journal of Medicinal Chemistry, 56(7), 2813-2827. doi:10.1021/jm3015603Panda, D., Daijo, J. E., Jordan, M. A., & Wilson, L. (1995). Kinetic Stabilization of Microtubule Dynamics at Steady State in Vitro by Substoichiometric Concentrations of Tubulin-Colchicine Complex. Biochemistry, 34(31), 9921-9929. doi:10.1021/bi00031a014Andreu, J. M., & Timasheff, S. N. (1982). Interaction of tubulin with single ring analogs of colchicine. Biochemistry, 21(3), 534-543. doi:10.1021/bi00532a019Roesner, M., Capraro, H.-G., Jacobson, A. E., Atwell, L., Brossi, A., Iorio, M. A., … Chignell, C. F. (1981). Biological effects of modified colchicines. Improved preparation of 2-demethylcolchicine, 3-demethylcolchicine, and (+)-colchicine and reassignment of the position of the double bond in dehydro-7-deacetamidocolchicines. Journal of Medicinal Chemistry, 24(3), 257-261. doi:10.1021/jm00135a005Pérez-Ramírez, B., Gorbunoff, M. J., & Timasheff, S. N. (1998). Linkages in Tubulin-Colchicine Functions: The Role of the Ring C (C‘) Oxygens and Ring B in the Controls†. Biochemistry, 37(6), 1646-1661. doi:10.1021/bi971344dDUMORTIER, C., YAN, Q., BANE, S., & ENGELBORGHS, Y. (1997). Mechanism of tubulin–colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine. Biochemical Journal, 327(3), 685-688. doi:10.1042/bj3270685Andreu, J. M., Gorbunopff, M. J., Lee, J. C., & Timasheff, S. N. (1984). Interaction of tubulin with bifunctional colchicine analogs: an equilibrium study. Biochemistry, 23(8), 1742-1752. doi:10.1021/bi00303a025Nguyen, T. L., McGrath, C., Hermone, A. R., Burnett, J. C., Zaharevitz, D. W., Day, B. W., … Gussio, R. (2005). A Common Pharmacophore for a Diverse Set of Colchicine Site Inhibitors Using a Structure-Based Approach. Journal of Medicinal Chemistry, 48(19), 6107-6116. doi:10.1021/jm050502tTorin Huzil, J., Winter, P., Johnson, L., Weis, A. L., Bakos, T., Banerjee, A., … Tuszynski, J. A. (2010). Computational Design and Biological Testing of Highly Cytotoxic Colchicine Ring A Modifications. Chemical Biology & Drug Design, 75(6), 541-550. doi:10.1111/j.1747-0285.2010.00970.xCao, R., Liu, M., Yin, M., Liu, Q., Wang, Y., & Huang, N. (2012). Discovery of Novel Tubulin Inhibitors via Structure-Based Hierarchical Virtual Screening. Journal of Chemical Information and Modeling, 52(10), 2730-2740. doi:10.1021/ci300302cLaing, N., Dahllöf, B., Hartley-Asp, B., Ranganathan, S., & Tew, K. D. (1997). Interaction of Estramustine with Tubulin Isotypes†. Biochemistry, 36(4), 871-878. doi:10.1021/bi961445wGireesh, K. K., Rashid, A., Chakraborti, S., Panda, D., & Manna, T. (2012). CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells. Biochemical Pharmacology, 84(5), 633-645. doi:10.1016/j.bcp.2012.06.008Gunasekera, N., Xiong, G., Musier-Forsyth, K., & Arriaga, E. (2004). A capillary electrophoretic method for monitoring the presence of α-tubulin in nuclear preparations. Analytical Biochemistry, 330(1), 1-9. doi:10.1016/j.ab.2004.03.059Medrano, F. J., Andreu, J. M., Gorbunoff, M. J., & Timasheff, S. N. (1991). Roles of ring C oxygens in the binding of colchicine to tubulin. Biochemistry, 30(15), 3770-3777. doi:10.1021/bi00229a026Morrison, K. C., & Hergenrother, P. J. (2012). Whole cell microtubule analysis by flow cytometry. Analytical Biochemistry, 420(1), 26-32. doi:10.1016/j.ab.2011.08.020Hastie, S. B., & Rava, R. P. (1989). Analysis of the near-ultraviolet absorption band of colchicine and the effect of tubulin binding. Journal of the American Chemical Society, 111(18), 6993-7001. doi:10.1021/ja00200a015Bhattacharyya, B., Kapoor, S., & Panda, D. (2010). Fluorescence Spectroscopic Methods to Analyze Drug–Tubulin Interactions. Microtubules, in vitro, 301-329. doi:10.1016/s0091-679x(10)95017-6Sardar, P. S., Maity, S. S., Das, L., & Ghosh, S. (2007). Luminescence Studies of Perturbation of Tryptophan Residues of Tubulin in the Complexes of Tubulin with Colchicine and Colchicine Analogues†. Biochemistry, 46(50), 14544-14556. doi:10.1021/bi701412kBhattacharyya, B., & Wolff, J. (1974). Promotion of Fluorescence upon Binding of Colchicine to Tubulin. Proceedings of the National Academy of Sciences, 71(7), 2627-2631. doi:10.1073/pnas.71.7.2627Lhiaubet-Vallet, V., Sarabia, Z., Boscá, F., & Miranda, M. A. (2004). Human Serum Albumin-Mediated Stereodifferentiation in the Triplet State Behavior of (S)- and (R)-Carprofen. Journal of the American Chemical Society, 126(31), 9538-9539. doi:10.1021/ja048518gVayá, I., Lhiaubet-Vallet, V., Jiménez, M. C., & Miranda, M. A. (2014). Photoactive assemblies of organic compounds and biomolecules: drug–protein supramolecular systems. Chem. Soc. Rev., 43(12), 4102-4122. doi:10.1039/c3cs60413fBosca, F., & Tormos, R. (2013). Behavior of Drug Excited States within Macromolecules: Binding of Colchicine and Derivatives to Albumin. The Journal of Physical Chemistry B, 117(25), 7528-7534. doi:10.1021/jp402489jFltzgerald, T. J. (1976). Molecular features of colchicine associated with antimitotic activity and inhibition of tubulin polymerization. Biochemical Pharmacology, 25(12), 1383-1387. doi:10.1016/0006-2952(76)90108-8Andreu, J. M. (2007). Large Scale Purification of Brain Tubulin With the Modified Weisenberg Procedure. Microtubule Protocols, 17-28. doi:10.1007/978-1-59745-442-1_2S. L. Murov , I.Carmichael and G. L.Hug, Handbook of Photochemistry, Marcel Dekker, Inc., New York, 2nd edn, 1993Silva, J. N., Bosca, F., Tomé, J. P. C., Silva, E. M. P., Neves, M. G. P. M. S., Cavaleiro, J. A. S., … Santus, R. (2009). Tricationic Porphyrin Conjugates: Evidence for Chain-Structure-Dependent Relaxation of Excited Singlet and Triplet States. The Journal of Physical Chemistry B, 113(52), 16695-16704. doi:10.1021/jp907930wLand, E. J. (1980). Pulse radiolysis and flash photolysis: some applications in biology and medicine. Biochimie, 62(4), 207-221. doi:10.1016/s0300-9084(80)80395-6Bensasson, R. V., & Gramain, J.-C. (1980). Benzophenone triplet properties in acetonitrile and water. Reduction by lactams. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases, 76(0), 1801. doi:10.1039/f19807601801Plimpton, S. (1995). Fast Parallel Algorithms for Short-Range Molecular Dynamics. Journal of Computational Physics, 117(1), 1-19. doi:10.1006/jcph.1995.1039In ’t Veld, P. J., Plimpton, S. J., & Grest, G. S. (2008). Accurate and efficient methods for modeling colloidal mixtures in an explicit solvent using molecular dynamics. Computer Physics Communications, 179(5), 320-329. doi:10.1016/j.cpc.2008.03.005Rappe, A. K., Casewit, C. J., Colwell, K. S., Goddard, W. A., & Skiff, W. M. (1992). UFF, a full periodic table force field for molecular mechanics and molecular dynamics simulations. Journal of the American Chemical Society, 114(25), 10024-10035. doi:10.1021/ja00051a040MOPAC2009, James J. P. Stewart, Stewart computational chemistry, version 13.207L; web: http://OpenMOPAC.netMaia, J. D. C., Urquiza Carvalho, G. A., Mangueira, C. P., Santana, S. R., Cabral, L. A. F., & Rocha, G. B. (2012). GPU Linear Algebra Libraries and GPGPU Programming for Accelerating MOPAC Semiempirical Quantum Chemistry Calculations. Journal of Chemical Theory and Computation, 8(9), 3072-3081. doi:10.1021/ct3004645Chai, J.-D., & Head-Gordon, M. (2008). Long-range corrected hybrid density functionals with damped atom–atom dispersion corrections. Physical Chemistry Chemical Physics, 10(44), 6615. doi:10.1039/b810189bSchäfer, A., Huber, C., & Ahlrichs, R. (1994). Fully optimized contracted Gaussian basis sets of triple zeta valence quality for atoms Li to Kr. The Journal of Chemical Physics, 100(8), 5829-5835. doi:10.1063/1.467146Jacquemin, D., Wathelet, V., Perpète, E. A., & Adamo, C. (2009). Extensive TD-DFT Benchmark: Singlet-Excited States of Organic Molecules. Journal of Chemical Theory and Computation, 5(9), 2420-2435. doi:10.1021/ct900298eJacquemin, D., Perpète, E. A., Ciofini, I., & Adamo, C. (2010). Assessment of Functionals for TD-DFT Calculations of Singlet−Triplet Transitions. Journal of Chemical Theory and Computation, 6(5), 1532-1537. doi:10.1021/ct100005dPeach, M. J. G., Benfield, P., Helgaker, T., & Tozer, D. J. (2008). Excitation energies in density functional theory: An evaluation and a diagnostic test. The Journal of Chemical Physics, 128(4), 044118. doi:10.1063/1.2831900Bartovský, P., Tormos, R., & Miranda, M. A. (2009). Colchicine–protein interactions revealed by transient absorption spectroscopy after in situ photoisomerization to lumicolchicines. Chemical Physics Letters, 480(4-6), 305-308. doi:10.1016/j.cplett.2009.09.023Vayá, I., Bueno, C. J., Jiménez, M. C., & Miranda, M. A. (2008). Determination of Enantiomeric Compositions by Transient Absorption Spectroscopy using Proteins as Chiral Selectors. Chemistry - A European Journal, 14(36), 11284-11287. doi:10.1002/chem.200801657Marcus, Y. (1993). The properties of organic liquids that are relevant to their use as solvating solvents. Chemical Society Reviews, 22(6), 409. doi:10.1039/cs9932200409Nery, A. L. P., Quina, F. H., Moreira, Jr, P. F., Medeiros, C. E. R., Baader, W. J., Shimizu, K., … Bechara, E. J. H. (2001). Does the Photochemical Conversion of Colchicine into Lumicolchicines Involve Triplet Transients? A Solvent Dependence Study¶. Photochemistry and Photobiology, 73(3), 213. doi:10.1562/0031-8655(2001)0732.0.co;2Perdew, J. P., Burke, K., & Ernzerhof, M. (1996). Generalized Gradient Approximation Made Simple. Physical Review Letters, 77(18), 3865-3868. doi:10.1103/physrevlett.77.3865Perdew, J. P., Burke, K., & Ernzerhof, M. (1997). Generalized Gradient Approximation Made Simple [Phys. Rev. Lett. 77, 3865 (1996)]. Physical Review Letters, 78(7), 1396-1396. doi:10.1103/physrevlett.78.139
