Multi-target effects of PMS1339 1411 Inhibition of PAF-induced platelet aggregation substrate(AcThCh) concentrations ranging from 0.033 lood from the carotid artery of New Zealand White to 0.167 mM in the absence and in the presence of rabbits was collected directly into 5-ml plastic syringes The k and v. values for AChe were calculated containing 3.8%(w/v)sodium citrate as anticoagulant by regression analysis of Lineweaver-Burk plots to achieve a final volume proportion of 9: 1(blood citrate). Platelet-rich plasma(PRP)was obtained from (1/velocity us. 1/[substrateD the supernatant fraction of blood after centrifugation at room temperature for 10 min at 800 rpm, and the Molecular modelling t-poor plasma was obtained by centrifuging To explore the binding mode of PMS1339 in AChE the remaining sedimentation for 10 min at 3000 rpm. active site, a molecular modelling study was per- The final cell count in PRP was adjusted to4×10° platelets/ml with platelet-poor plasma formed employing the docking protocol GOLD (ones o Platelet aggregation was determined by the optical et al. 1997) on the basis of the X-ray crystallographic method of Born Cross(1963) with an aggregometer structure of Torpedo californica AChE (TCAChE) Molecular simulations were performed on an incubated with vehicle or PMs1339(0. 1, 0. 25, 0.5, 0.75, R14000 SGI Fuel workstation with software package 1 uM)for 5 min at 37C. Then platelet aggregation wa induced by the addition of PAF at a final concentration were used unless otherwise indicated. The coordinates of 0.1 uM. Inhibition of aggregation at 5 min was of TcAChE structure were obtained from the donepezil (E2020)/TCAChE complex [Protein Data Bank(PDB the presence of PMS1339 with that in the presence of Hetero-atoms and water molecules were removed vehicle alone. The average inhibition rate for each concentration was calculated in order to determine from the PDB file, and all hydrogen atoms were added the ICso value( the concentration required to inhibit subsequently. The structure of PMS1339 was gener- platelet aggregation by 50%) ated in SYBYL, and Gasteiger-Huckel partial charges were assigned. The resultant input ligand was ob- Cholinesterase inhibitory actiuity in vitro tained after 1000 steps of energy minimization using ipos force field AChE and BuChE activities were determined accord Molecular docking was performed using GOLD 3.0 ing to a modified Ellman's method (Ellman et al. 1961) (Cambridge Crystallographic Data Centre, UK)(ones using mouse forebrain(AChE)and sera(BuChE)of et al. 1997). The active site was defined as residues with Kunming mice. Briefly, mouse forebrain homogenates at least one atom within a radius of 10 A from any [1: 9 w/v in 0.05 M phosphate buffer solution(PBS)] or atom of E2020. Thirty genetic algorithm(GA)runs sera(1: 19 w/v in 0.05 M PBS)were pre-incubated with were performed. For each GA run, the default GA PMS1339 or tacrine for 20 min at 37C in 0.05 M PBS settings were used. All conformations that GOLD (PH7. 2), containing 0.25 mM DTNB. The substrates, generated were evaluated with the scoring function 0.5 mM AcThCh or BTCh, were then quickly added. X-SCORE 1.2. 1(Wang et al. 2002) The reaction was terminated by the addition of 100 ul eserine (1 mM), and the colour production was measured spectrophotometrically at 412 nm. Inhibition of A ChE-induced Ap aggregation The percent inhibition of the enzyme reaction in AB40 peptide was dissolved from the lyophilized he presence of the inhibitors was determined by powder in hexafluoro-2-iso-propanol (HFIP)to make a comparison with control, and the average inhibition solution of 2 mg/ml HFIP was removed by vapor rate for each concentration was calculated in order ation under vacuum. Then AB was dissolved in an- to deduce the ICso value (the inhibitor concentration hydrous dimethyl sulfoxide(DMSO)to make a 2.3 mM required for 50% inhibition of AChE activity) for each stock solution est dru For each experiment, 2 ul of the 2.3 mM AB-DMSO stock solution was added to a final volume of 20 ul of Determination of kinetic parameters samples containing different concentrations of tested To estimate the kinetic parameters Km (Michaelis con- inhibitors and incubated for 48 h at room temperature. stant), apparent Km(Km, app), Vmax(maximum velocity Ap was diluted from the stock solution into 0. 215 of reaction) and apparent Vmax(V max app), the enzyme PBS(pH8.0)(resulting in 10% residual DMSO) of electric eel AChE was determined at make a final concentration of 230 uM AAInhibition of PAF-induced platelet aggregation Blood from the carotid artery of New Zealand White rabbits was collected directly into 5-ml plastic syringes containing 3.8% (w/v) sodium citrate as anticoagulant to achieve a final volume proportion of 9: 1 (blood￾citrate). Platelet-rich plasma (PRP) was obtained from the supernatant fraction of blood after centrifugation at room temperature for 10 min at 800 rpm, and the platelet-poor plasma was obtained by centrifuging the remaining sedimentation for 10 min at 3000 rpm. The final cell count in PRP was adjusted to 4r108 platelets/ml with platelet-poor plasma. Platelet aggregation was determined by the optical method of Born & Cross (1963) with an aggregometer at 37 xC under stirring (900 rpm). PRP was pre￾incubated with vehicle or PMS1339 (0.1, 0.25, 0.5, 0.75, 1 mM) for 5 min at 37 xC. Then platelet aggregation was induced by the addition of PAF at a final concentration of 0.1 mM. Inhibition of aggregation at 5 min was calculated by comparing the extent of aggregation in the presence of PMS1339 with that in the presence of vehicle alone. The average inhibition rate for each concentration was calculated in order to determine the IC50 value (the concentration required to inhibit platelet aggregation by 50%). Cholinesterase inhibitory activity in vitro AChE and BuChE activities were determined accord￾ing to a modified Ellman’s method (Ellman et al. 1961) using mouse forebrain (AChE) and sera (BuChE) of Kunming mice. Briefly, mouse forebrain homogenates [1: 9 w/v in 0.05 M phosphate buffer solution (PBS)] or sera (1: 19 w/v in 0.05 M PBS) were pre-incubated with PMS1339 or tacrine for 20 min at 37 xC in 0.05 M PBS (pH 7.2), containing 0.25 mM DTNB. The substrates, 0.5 mM AcThCh or BTCh, were then quickly added. The reaction was terminated by the addition of 100 ml eserine (1 mM), and the colour production was measured spectrophotometrically at 412 nm. The percent inhibition of the enzyme reaction in the presence of the inhibitors was determined by comparison with control, and the average inhibition rate for each concentration was calculated in order to deduce the IC50 value (the inhibitor concentration required for 50% inhibition of AChE activity) for each test drug. Determination of kinetic parameters To estimate the kinetic parameters Km (Michaelis con￾stant), apparent Km (Km,app), Vmax (maximum velocity of reaction) and apparent Vmax (Vmax,app), the enzyme activity of electric eel AChE was determined at substrate (AcThCh) concentrations ranging from 0.033 to 0.167 mM in the absence and in the presence of two concentrations of PMS1339 (0.33 and 1.33 mM). The Km and Vmax values for AChE were calculated by regression analysis of Lineweaver–Burk plots (1/velocity vs. 1/[substrate]). Molecular modelling To explore the binding mode of PMS1339 in AChE active site, a molecular modelling study was per￾formed employing the docking protocol GOLD (Jones et al. 1997) on the basis of the X-ray crystallographic structure of Torpedo californica AChE (TcAChE). Molecular simulations were performed on an R14000 SGI Fuel workstation with software package SYBYL 6.9 (Tripos Inc., USA). Standard parameters were used unless otherwise indicated. The coordinates of TcAChE structure were obtained from the donepezil (E2020)/TcAChE complex [Protein Data Bank (PDB; Berman et al. 2000) code 1EVE] (Kryger et al. 1999). Hetero-atoms and water molecules were removed from the PDB file, and all hydrogen atoms were added subsequently. The structure of PMS1339 was gener￾ated in SYBYL, and Gasteiger–Hu¨ ckel partial charges were assigned. The resultant input ligand was ob￾tained after 1000 steps of energy minimization using Tripos force field. Molecular docking was performed using GOLD 3.0 (Cambridge Crystallographic Data Centre, UK) (Jones et al. 1997). The active site was defined as residues with at least one atom within a radius of 10 A˚ from any atom of E2020. Thirty genetic algorithm (GA) runs were performed. For each GA run, the default GA settings were used. All conformations that GOLD generated were evaluated with the scoring function X-SCORE 1.2.1 (Wang et al. 2002). Inhibition of AChE-induced Ab aggregation Ab40 peptide was dissolved from the lyophilized powder in hexafluoro-2-iso-propanol (HFIP) to make a solution of 2 mg/ml. HFIP was removed by evapor￾ation under vacuum. Then Ab was dissolved in an￾hydrous dimethyl sulfoxide (DMSO) to make a 2.3 mM stock solution. For each experiment, 2 ml of the 2.3 mM Ab-DMSO stock solution was added to a final volume of 20 ml of samples containing different concentrations of tested inhibitors and incubated for 48 h at room temperature. Ab was diluted from the stock solution into 0.215 M PBS (pH 8.0) (resulting in 10% residual DMSO) to make a final concentration of 230 mM Ab. Multi-target effects of PMS1339 1411