Pharmacodynamic Evaluation of novel Catechol-O-methyltransferase Inhibitors
TThe incubation of liver MB-COMT and S-COMT and brain MB-COMT samples with increasing concentrations of adrenaline resulted in the concentration-dependent formation of metanephrine (Figure 1). The kinetic parameters for liver S-COMT and MB-COMT and brain MB-COMT samples are given in Table 1. The novel COMT tight-binding inhibitors were then tested against liver MB- and S-COMT and brain MB-COMT samples using a fixed amount of protein (2 mg/ml), in the presence of a saturating concentration of adrenaline (5 times the corresponding Km; 1000 µM for liver S-COMT and 10 µM for liver and brain MB-COMT) (Figure 2). The IC50 values for inhibition of MB- and S-COMT activity in liver and brain are given in Tables 2 and 3. Exposure of SK-HEP-1 and SK-N-SH cells to 1, 10 and 50 µM tolcapone or CNCAPE for 24 h reduced cell viability in a concentration-dependent manner (Figures 3 and 4). This pattern was apparent in both cell lines during their linear growth phase (at 48 h post seeding) and when reaching monolayer confluence (72 h post seeding). In general, the cytotoxicity was greater for SK-N-SH cells, in comparison to SK-HEP-1 cells. SK-N-SH cells also showed a greater reduction in cell viability with different concentrations of tolcapone and CNCAPE, in comparison to SK-HEP-1 cells. Cells in the linear growth phase were generally more susceptible to the cytotoxic effects of tolcapone and CNCAPE, in comparison to cells in monolayer. The cytotoxicity of tolcapone and CNCAPE was quite similar for each concentration in liver SK-HEP-1 cells during their linear growth phase (Figure 3). Only the highest concentration (50 µM) produced a statistically significant decrease in cell viability. In SK-HEP-1 cells when reaching monolayer confluence, as shown in Figure 3, only the highest concentration of CNCAPE produced a statistically significant decrease in cell viability, although there was a concentration-dependent decrease in cell viability for both COMT inhibitors. As shown in Figure 4, all concentrations of tolcapone and CNCAPE used on SK-N-SH cells in growth phase produced a statistically significant decrease in cell viability. In SK-N-SH cells when reaching monolayer confluence, only 50 µM of tolcapone and 10 and 50 µM of CNCAPE produced a statistically significant decreases in cell viability (Figure 4).
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COMT activity was evaluated by the ability to methylate adrenaline (AD) to metanephrine (MN) in the presence of a saturating concentration of S-adenosyl-L-methionine (SAM), the methyl donor. Aliquots of 100 µL of brain MB-COMT and liver S and MB-COMT enzyme preparations were preincubated for 20 min with increasing concentrations of COMT inhibitors. The reaction mixtures were then incubated for 5 min with 1000 µM AD (liver S-COMT) and with 10 µM AD (liver MB-COMT) and 15 min with 10 µM AD (brain MB-COMT), in the presence of SAM (100 µM for brain and 500 µM for liver), pargyline (100 µM), MgCl2 (100 µM) and EGTA (1mM). The preincubation and incubation were carried out at 37ºC, with light protection, continuous shaking and without oxygenation. At the end of the incubation period the reaction was stopped using 50µL of 2M PCA and the tubes were transferred to ice. The samples were then centrifuged at 200 X g for 4 min at 4ºC and filtered on 0.22 µm pore size Spin-X filter tubes. 500 µL aliquots of the supernatant were later used in the metanephrine assay, which was done using high pressure liquid chromatography with electrochemical detection. To evaluate the effects of different concentrations of Tolcapone and CNCAPE on SK-N-SH neuroblastoma and SK-HEP-1 liver adenocarcinoma cell proliferation, cells were cultured in growth medium for 48 h (growth phase) and 72 h (cells in monolayer) and then tolcapone (1, 10 and 50 µM) or CNCAPE (1, 10 and 50 µM) were added to the growth medium for another 24h. Cell viability was later accessed using the calcein-AM assay. The non-fluorescent calcein-AM is highly lipophilic and therefore quickly penetrates the cell plasma membrane. Intracellular esterases then convert the calcein-AM to a hydrophilic and intensely green fluorescent free acid form through acetoxymethyl ester hydrolysis. Microplates (96 wells) were washed with Hank’s balanced salt solution pH 7.4 (Gibco™) and the cells were then incubated with 2 µM of calcein-AM for 30 min at 37 ºC. After this, fluorescence was measured at 0, 15, 30, 45 and 60 min using a scanning microplate spectrofluorometer (Spectramax Gemini EM; Molecular Devices, Sunnyvale, CA), alternating between 485 nm excitation and 530 nm emission, with a cutoff filter of 530 nm.