Abstract
The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser–phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.
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References
Amitai G, Adani R, Yacov G, Yishay S, Teitlboim S, Tveria L, Limanovich O, Kushnir M, Meshulam H (2007) Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity. Toxicology 233(1–3):187–198
Barone V, Cossi M, Tomasi J (1998) Geometry optimization of molecular structures in solution by the polarizable continuum model. J Comp Chem 19:404–417
Carletti E, Colletier JP, Dupeux F, Trovaslet M, Masson P, Nachon F (2010) Structural evidence that human acetylcholinesterase inhibited by tabun ages through O-dealkylation. J Med Chem 53(10):4002–4008
Cochran R, Kalisiak J, Küçükkilinç T, Radic Z, Garcia E, Zhang L, Ho KY, Amitai G, Kovarik Z, Fokin VV, Sharpless KB, Taylor P (2011) Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging. J Biol Chem 286(34):29718–29724
Doorn JA, Schall M, Gage DA, Talley TT, Thompson CM, Richardson RJ (2001) Identification of butyrylcholinesterase adducts after inhibition with isomalathion using mass spectrometry: difference in mechanism between (1R)- and(1S)-stereoisomers. Toxicol Appl Pharmacol 176(2):73–80
Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95
Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JA Jr, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill P. MW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA (2004) Gaussian 03, Revision C.02. Gaussian, Inc., Wallingford, CT
Gonzalez C, Schlegel CB (1989) An improved algorithm for reaction path following. J Chem Phys 90:2154–2159
Grigoryan H, Schopfer LM, Peeples ES, Duysen EG, Grigoryan M, Thompson CM, Lockridge O (2009) Mass spectrometry identifies multiple organophosphorylated sites on tubulin. Toxicol Appl Pharmacol 240(2):149–158
Harris LW, Fleisher JH, Clark J, Cliff WJ (1966) Dealkylation and loss of capacity for reactivation of cholinesterase inhibited by sarin. Science 154(3747):404–407
Hehre WJ, Deppmeier BJ, Klunzinger PE (1999) PC spartan pro. Wavefunction Inc., Irvine
Hobbiger F (1963) Cholinesterases and anticholinesterase agents. In: Koelle GB (ed) Handbuch der Experimentellen Pharmakologie, Erganzungswerk, vol 15. Springer, Berlin, pp 799–832
Jennings LL, Malecki M, Komives EA, Taylor P (2003) Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry. Biochemistry 42(37):11083–11091
Kropp TJ, Richardson RJ (2006) Aging of mipafox-inhibited human acetylcholinesterase proceeds by displacement of both isopropylamine groups to yield a phosphate adduct. Chem Res Toxicol. 19:334–339
Kropp TJ, Richardson RJ (2007) Mechanism of aging of mipafox-inhibited butyrylcholinesterase. Chem Res Toxicol 20(3):504–510
Kropp TJ, Glynn P, Richardson RJ (2004) The mipafox-inhibited catalytic domain of human neuropathy target esterase ages by reversible proton loss. Biochemistry 43(12):3716–3722
Masson P, Froment MT, Bartels CF, Lockridge O (1997) Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase. Biochem J 325(Pt 1):53–61
Masson P, Nachon F, Lockridge O (2010) Structural approach to the aging of phosphylated cholinesterases. Chem Biol Interact 187(1–3):157–162
Milatovic D, Johnson MK (1993) Reactivation of phosphorodiamidated acetylcholinesterase and neuropathy target esterase by treatment of inhibited enzyme with potassium fluoride. Chem Biol Interact 87(1–3):425–430
Millard CB, Kryger G, Ordentlich A, Greenblatt HM, Harel M, Raves ML, Segall Y, Barak D, Shafferman A, Silman I, Sussman JL (1999) Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level. Biochemistry 38(22):7032–7039
Radić Z, Sit RK, Kovarik Z, Berend S, Garcia E, Zhang L, Amitai G, Green C, Radić B, Fokin VV, Sharpless KB, Taylor P (2012) Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases. J Biol Chem 287(15):11798–11809
Radić Z, Sit RK, Garcia E, Zhang L, Berend S, Kovarik Z, Amitai G, Fokin VV, Sharpless BK, Taylor P (2013) Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates. Chem Biol Interact 203(1):67–71
Ramalho TC, Taft CA (2005) Thermal and solvent effects on the NMR and UV parameters of some bioreductive drugs. J Chem Phys 123:0543191–0543197
Ramalho TC, da Cunha EFF, Figueroa-Villar JD, Peixoto FC (2008) Computational NMR investigation of Radiosensitizer in solution. J Theor Comput Chem 7:37–52
Schroeder MJ, Shabanowitz J, Schwartz JC, Hunt DF, Coon JJ (2004) A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal Chem 76(13):3590–3598
Segall Y, Waysbort D, Barak D, Ariel N, Doctor BP, Grunwald J, Ashani Y (1993) Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by 31P NMR spectroscopy. Biochemistry 32(49):13441–13450
Sit RK, Radić Z, Gerardi V, Zhang L, Garcia E, Katalinić M, Amitai G, Kovarik Z, Fokin VV, Sharpless KB, Taylor P (2011) New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterases. J Biol Chem 286(22):19422–19430
Sit RK, Fokin VV, Amitai G, Sharpless KB, Taylor P, Radić Z (2014) Imidazole aldoximes effective in assisting butyrylcholinesterase catalysis of organophosphate detoxification. J Med Chem 57(4):1378–1389
Sultatos LG (1994) Mammalian toxicology of organophosphorus pesticides. J Toxicol Environ Health 43(3):271–289
Taylor P, Radić Z (1994) The cholinesterases: from genes to proteins. Annu Rev Pharmacol Toxicol 34:281–320
Acknowledgments
These studies were supported by NIH GM18360 36 and NINDS, NIH to EK, CAPES/BRASIL and Spanish funds support.
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Mangas, I., Taylor, P., Vilanova, E. et al. Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches. Arch Toxicol 90, 603–616 (2016). https://doi.org/10.1007/s00204-015-1481-1
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DOI: https://doi.org/10.1007/s00204-015-1481-1