Reactivation potency of fluorinated pyridinium oximes for acetylcholinesterases inhibited by paraoxon organophosphorus agent

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 4, 2009, Pages 1214-1217

  Jeong, Hee Chun ^a,b   Kang, Nam Sook ^a   Park, No Joong ^a   Yum, Eul Kyun ^b   Jung, Young Sik ^a  

^a Korea Research Institute of Chemical Technology   (South Korea)

^b Chungnam National University   (South Korea)

Author keywords

Acetylcholinesterase; Fluorinated pyridinium oxime; Organophosphorus agents; Oxime reactivators

Indexed keywords

1 (4 CARBAMOYLPYRIDINIO) 1' (2 HYDROXYIMINOMETHYLPYRIDINIO)DIMETHYL ETHER; ACETYLCHOLINESTERASE; ALDOXIME; DYFLOS; OBIDOXIME; ORGANOPHOSPHORUS COMPOUND; OXIME DERIVATIVE; PARAOXON; PRALIDOXIME; PYRIDINIUM DERIVATIVE;

ARTICLE; BLOOD BRAIN BARRIER; CATTLE; DRUG DESIGN; DRUG PENETRATION; DRUG SOLUBILITY; DRUG SYNTHESIS; ENZYME REACTIVATION; ERYTHROCYTE; FLUORINATION; HOUSE FLY; HYDROPHILICITY; LIPID SOLUBILITY; NONHUMAN; PREDICTION; QUANTUM MECHANICS; REACTION ANALYSIS;

ANIMALS; BLOOD-BRAIN BARRIER; CHOLINESTERASE INHIBITORS; HOUSEFLIES; MOLECULAR STRUCTURE; ORGANOPHOSPHORUS COMPOUNDS; OXIMES; PARAOXON;

EID: 59649085049     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.12.070     Document Type: Article

References (18)

1. Taylor P. Anticholinergic agents. In: Hardman J.G., Limbird L.E., and Gilman A.G. (Eds). Goodman & Gilman's The Pharmacological Basis of Therapeutics. 2nd ed. (1996), McGraw Hill, New York 175-191
2. Wilson I.B. J. Biol. Chem. 190 (1951) 111
3. Kim T.H., Kuca K., Jun D., and Jung Y.S. Bioorg. Med. Chem. Lett. 15 (2005) 2914
4. Oh K.A., Yang G.Y., Jun D., Kuca K., and Jung Y.S. Bioorg. Med. Chem. Lett. 16 (2006) 2914
5. Yang G.Y., Oh K.A., Park N.J., and Jung Y.S. Bioorg. Med. Chem. 15 (2007) 7704
6. Oh K.A., Park N.J., Park N.S., Kuca K., Jun D., and Jung Y.S. Chem-Bio. Int. 175 (2008) 365
7. Wilson I.B., and Ginsburg S. Biochem. Biophys. 18 (1955) 168
8. Poziomek E.J., Hackley B.E., and Steinberg G.M. J. Org. Chem. 23 (1958) 714
9. Luttringhaus A., and Hargendorn I. Arzneimittl. Forsch. 14 (1964) 1
10. Sakurada K., Matsubara K., Shimizu K., Shiono H., Seto Y., Tsuge K., Yoshino M., Sakai I., Mukoyama H., and Takatori T. Neurochem. Res. 28 (2003) 1401
11. In: Bohm H.J., and Schneider G. (Eds). Protein-Ligand Interactions (2003), Wiley-VCH, Weinhim
12. Mueller K., Faeh C., and Diederich F. Science 317 (2007) 1881
13. Kirk K.L. J. Fluorine Chem. 127 (2006) 1013
14. Wildman S.A., and Crippen G.M. J. Chem. Inf. Comput. Sci. 39 (1999) 868
15. Dwyer D.S. Structure, Function, and Bioinformatics 63 (2006) 939
16. Musilek K., Jun D., Cabal J., Kassa J., Gunn-Moore F., and Kuca K. J. Med. Chem. 50 (2007) 5514
17. In vitro determination of AChE activity. The enzyme activity was measured in a 96-well microplate using a microplate reader (Benchmark Microplate Reader, Bio-Rad) at 415 nm and 37 °C with acetylthiocholine (1 mM) as substrate and DTNB (1 mM) as chromogen in 0.05 M Tris-HCl buffer, pH 7.8. Total reaction volume was adjusted to 250 μL with slight modification from Ellman's AChE assay method. The enzyme concentrations of both AChEs in 250 μL of final reacting solutions were 0.05 mg/ml for HF AChE and 0.02 U/mL for RBC AChE, respectively. For RBC AChE, 1% of Triton X-100 (Sigma-Aldrich) was added in the Tris-HCl buffer to preserve enzyme activity. AChE activity was measured by using the change of optical density per minute (OD/min). The percentage of reactivation of AChE activity was calculated by comparison with the AChE activity without inhibitor showing 0.2 OD/min.
18. AChE inhibition and reactivation. DFP and paraoxon were used as organophosphorus inhibitors and the AChE reactivating capability of 2-PAM and HI-6 were examined against OP-inhibited HF and RBC AChE, respectively. AChE was inhibited with the minimum quantity of inhibitor necessary to inactivate 99% of activity for 10 min at room temperature. The concentration of DFP was 12.5 μM for HF AChE and 25 μM for RBC AChE, respectively, and 20 μM of paraoxon for both AChEs. To remove excess inhibitor after reaction with enzyme, the enzyme solution was mixed with 2-fold volume of hexane and vigorously shaken with a vortex mixer for 1 min. The aqueous phase was separated by centrifugation at 3000g for 10 min at 3 °C. The solution containing phosphorylated AChE was incubated with various concentration of 2-PAM or HI-6 for various reactivation periods. To remove small molecules such as reactivator and phosphorylated oxime, the reactivating mixture was filtered through a micro spin-column packed with Sephadex-G50 (Bio-Rad) in a centrifuge at 300g for 1 min at 3 °C. The AChE activity of the filtrate was measured in a 96-well microplate.AChE reactivation with newly synthesized oxime compounds. The reactivating abilities of the newly synthesized oximes were evaluated against DFP or paraoxon-inhibited HF or RBC AChE, respectively. The inhibited AChE was extracted with hexane, and then the aqueous phase was reacted with 5 mM of each oxime compound for 30 min for DFP-inhibited AChE and for 1 h for paraoxon-inhibited AChE, respectively.