B. Tasso et al. / European Journal of Medicinal Chemistry 46 (2011) 2170e2184
Chem. 51 (2008) 347e372;
2183
1B41) and hBChE (PDB code 1P0I) were retrieved from the Protein
Data Bank. The target proteins were prepared by adding hydrogen
atoms, completing and optimizing missing residues, removing
water and the cocrystallized molecules. As well known [55], the
histidine side chains cannot normally be placed into the electron
density map unambiguously. As a result, the protonation state of
such residues was adjusted according to the formation of HB
networks that was further confirmed upon visual inspection. The
basic amino functional groups were protonated, aromatic amino
functional groups were left uncharged and carboxylic groups were
considered to be deprotonated. Molecular docking resulted 10
poses for inhibitor in a sphere of 10Å radius centered on the
centroid atom of E2020 cocrystallized with TcAChE (PDB code
1EVE) previously aligned to hAChE.
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mined in a reaction cuvette containing 200
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acetylthiocholine iodide (100 mL of 5 mM aqueous solution) was
added as the substrate, and AChE-catalyzed hydrolysis was fol-
lowed by measuring the increase of absorbance at 412 nm for
3.0 min at 25 ꢁC. The concentration of compound which deter-
mined 50% inhibition of the AChE activity (IC50) was calculated by
non-linear regression of the responseelog(concentration) curve,
using GraphPad PrismÒ v. 5. BChE inhibitory activity was assessed
similarly using butyrylthiocholine iodide as the substrate.
Acknowledgments
Financial support from Italian MIUR (PRIN 2007E8CRF and
20085HR5JK to Genoa and Bari Universities, respectively) is
gratefully acknowledged. The authors thank O. Gagliardo for per-
forming elemental analyses.
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