W. Zheng et al Toxicology and Applied Pharmacology 264(2012)65-72 Metal-chelating properties. The ability for Cor ZLA, ZLB or bis-MEP to chelate biometals such as Cu(ll) was studied by Uv-vis Baum and Ng, 2004). The absorption spectra of ZLA, ZLB or bis-MEP, alone (in methanol) or in the presence of CuCl Trp 279 with a molar ratio of 1: 1), were recorded at a duration of 30 min at room temperature in a 1 cm quartz cell using Multiscan MK3 spectro- Tyr 70 photometer(Thermo, Waltham, MA, USA). Additionally, the ratio of metal ion in the complex was determined by molar ratio (Bolognesi et al., 2007). Fixed concentrations of the com- 25 HM)was mixed with ascending doses of CuCl2 (12-40 HM)and the UV-vis absorption spectra were recorded. Tyr 334 \Tyr 121 Aggregation assay by sedimentation. AB1-40, lyophilized from 1 mg/ mL HFIP solution, was dissolved in DMSO to get a 500 uM stock solu- tion, and was brought to 10 HM in 20 mM Hepes buffer, 150 mM Nacl alone or with metal ions(20 HM), in the presence or absence of the tested compounds(100 HM). The reaction mixture(100 uL)was in- cubated for 15 min at 37C, and then centrifuged at 13,000xg for 15 min to sediment aggregated proteins. Peptide concentration in the supernatant was determined by the bradford method using the commercial protein assay Coomassie Brilliant Blue solution(Thermo) Trp 84 His 440 and was represented as a percentage of recovery relative to the con- trol without metal ions and tested compounds(Atwood et al, 1998: Raman et al., 2005). As Ad is complicated by cerebral acidosis with a pH of 6.6(Yates et al, 1990). 20 mM Hepes buffer, 150 mM Nacl Fig. 2. Binding modes of bis-MEP ZLA and ZLB (green, was set to pH 6.6 with hydrochloric acid in this assay ZLA and zlB ar 79 and Tyr 70, while the spacer does not seem to be detrimental to the interactions with the enzyme. Turbidometric assay. Turbidity measurements, also as an assay for Worthy to note, some interactions could be identified for ZLa and ZLB with Tyr121. al- gregation, were performed according to the method described in the though they are not strong enough to increase the AChE inhibitory potency revious studies(Atwood et al, 1998). The stock solution of AB1-40 (500 HM) was brought to 10 HM in 20 mM Hepes buffer, 150 mM Nacl(pH 6.6)alone or with metal ions(20 HM), in the presence absence of the tested compounds(100 HM). The reaction mixture Data analysis and statistics. Values are exp (200 uL)was incubated for 30 min at 37., and absorbance Comparisons among means were performed ANOVA (405 nm) was measured using a Varioskan Flash spectrophotometr and post hoc by Dunnett test. Differences values less than microplate reader(Thermo). Automatic 30-s plate agitation mode 0.05 were considered statistically significant. was selected for the plate reader to evenly suspend the aggregates in the wells before all readings Results Molecular docking studies of zla and ZLB Determination of cell viability. The human neuroblastoma cell line SH-SY5Y cells(American Type Culture Collection, Manassas, VA, USA) The results of molecular docking demonstrate that compounds were cultured in MEM/F-12 (1: 1)medium(Invitrogen )supplemented ZLA and ZLB are able to interact with both the catalytic and peripheral 100 ug/mL streptomycin in a humidified atmosphere containing 5% the nonylene spacer with oxalamide or ethylenediamine may reduce CO2 at 37C. Cells were plated at 5x10 cells /well(100 HL)into AChE binding affinity as compared with their prototype bis-MEP And 96-well plates and allowed to adhere and grow. When cells reached although the derivatives could establish favorable interactions with the required confluence, they were placed into serum-free medium some mid-gorge residues, the increased polarity of ZLA and ZLB may and treated with ZLA or ZLB. Twenty-four hours later the survival of result in a major energy penalty during their desolvation cells was determined by Cell Counting Kit-8(CCK-8: Dojindo, Japan) ssay. Briefly, after incubation with 10 uL of CCK-8(5 mg/mL) at 37c Log P of ZLA and ZlB or 2 h, the absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 655 nm. The absorbance of con- The partition coefficients of ZLA and ZLB in the octanol/phosphate trol cells was set to 100%, and the percentage of viable cells collected buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively )were from each treatment was calculated relative to the control group. determined by the classical shake-flask method using RP-HPLC. As Table 1 pH-dependent partition coefficients(log P) of ZLA and ZLB pH 7.4 0.18±0.001 1.11±0001 0.38±0001 0.39±0.0 1.78±0003 07±0005 422±0.003 artition coefficients of ZLA and ZLB in the octanol /buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method. The represent the mean+ SEM of three independent experiments.Metal-chelating properties. The ability for compounds ZLA, ZLB or bis-MEP to chelate biometals such as Cu(II) was studied by UV–vis spectrometry (Baum and Ng, 2004). The absorption spectra of ZLA, ZLB or bis-MEP, alone (in methanol) or in the presence of CuCl2 (with a molar ratio of 1:1), were recorded at a duration of 30 min at room temperature in a 1 cm quartz cell using Multiscan MK3 spectro￾photometer (Thermo, Waltham, MA, USA). Additionally, the ratio of ligand/metal ion in the complex was determined by molar ratio method (Bolognesi et al., 2007). Fixed concentrations of the com￾pounds (25 μM) was mixed with ascending doses of CuCl2 (12–40 μM) and the UV–vis absorption spectra were recorded. Aggregation assay by sedimentation. Aβ1–40, lyophilized from 1 mg/ mL HFIP solution, was dissolved in DMSO to get a 500 μM stock solu￾tion, and was brought to 10 μM in 20 mM Hepes buffer, 150 mM NaCl alone or with metal ions (20 μM), in the presence or absence of the tested compounds (100 μM). The reaction mixture (100 μL) was in￾cubated for 15 min at 37 °C, and then centrifuged at 13,000×g for 15 min to sediment aggregated proteins. Peptide concentration in the supernatant was determined by the Bradford method using the commercial protein assay Coomassie Brilliant Blue solution (Thermo) and was represented as a percentage of recovery relative to the con￾trol without metal ions and tested compounds (Atwood et al., 1998; Raman et al., 2005). As AD is complicated by cerebral acidosis with a pH of 6.6 (Yates et al., 1990), 20 mM Hepes buffer, 150 mM NaCl was set to pH 6.6 with hydrochloric acid in this assay. Turbidometric assay. Turbidity measurements, also as an assay for ag￾gregation, were performed according to the method described in the previous studies (Atwood et al., 1998). The stock solution of Aβ1–40 (500 μM) was brought to 10 μM in 20 mM Hepes buffer, 150 mM NaCl (pH 6.6) alone or with metal ions (20 μM), in the presence or absence of the tested compounds (100 μM). The reaction mixture (200 μL) was incubated for 30 min at 37 °C, and absorbance (405 nm) was measured using a Varioskan Flash spectrophotometric microplate reader (Thermo). Automatic 30-s plate agitation mode was selected for the plate reader to evenly suspend the aggregates in the wells before all readings. Determination of cell viability. The human neuroblastoma cell line SH-SY5Y cells (American Type Culture Collection, Manassas, VA, USA) were cultured in MEM/F-12 (1:1) medium (Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were plated at 5×104 cells/well (100 μL) into 96-well plates and allowed to adhere and grow. When cells reached the required confluence, they were placed into serum-free medium and treated with ZLA or ZLB. Twenty-four hours later the survival of cells was determined by Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. Briefly, after incubation with 10 μL of CCK-8 (5 mg/mL) at 37 °C for 2 h, the absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 655 nm. The absorbance of con￾trol cells was set to 100%, and the percentage of viable cells collected from each treatment was calculated relative to the control group. Data analysis and statistics. Values are expressed as mean± SEM. Comparisons among means were performed using one-way ANOVA and post hoc by Dunnett test. Differences with P values less than 0.05 were considered statistically significant. Results Molecular docking studies of ZLA and ZLB The results of molecular docking demonstrate that compounds ZLA and ZLB are able to interact with both the catalytic and peripheral anionic sites of AChE (Fig. 2). It is worth noting that replacement of the nonylene spacer with oxalamide or ethylenediamine may reduce AChE binding affinity as compared with their prototype bis-MEP. And, although the derivatives could establish favorable interactions with some mid-gorge residues, the increased polarity of ZLA and ZLB may result in a major energy penalty during their desolvation. Log P of ZLA and ZLB The partition coefficients of ZLA and ZLB in the octanol/phosphate buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method using RP-HPLC. As Fig. 2. Binding modes of bis-MEP, ZLA and ZLB (green, purple and yellow, respectively) at the TcAChE gorge. ZLA and ZLB are able to properly contact with both sites of the en￾zyme. The two MEP moieties establish π–π stacking with Trp84, Trp279 and Tyr70, while the spacer does not seem to be detrimental to the interactions with the enzyme. Worthy to note, some interactions could be identified for ZLA and ZLB with Tyr121, al￾though they are not strong enough to increase the AChE inhibitory potency. Table 1 pH-dependent partition coefficients (log P) of ZLA and ZLB. Compounds Log P pH 4 pH 5 pH 6 pH 7 pH 7.4 pH 8 ZLA 0.18±0.001 0.22±0.002 1.11±0.001 2.69±0.003 3.71±0.003 4.06±0.006 ZLB 0.38±0.001 0.39±0.001 1.78±0.003 3.07±0.005 4.00±0.004 4.22±0.003 The partition coefficients of ZLA and ZLB in the octanol/buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method. The data represent the mean± SEM of three independent experiments. 68 W. Zheng et al. / Toxicology and Applied Pharmacology 264 (2012) 65–72