In vitro activity: A 922500 inhibits the phylogenetic family members acyl coenzyme A cholesterol acyltransferase-1 and -2 with IC50 of 296 μM. A 922500 potently inhibits huDGAT-1 and mseDGAT-1.

Kinase Assay: COMT activity was evaluated by the ability of homogenates to methylate adrenaline to metanephrine. Aliquots of 0.5 ml of the homogenate were preincubated for 20 min with 0.5 ml of phosphate buffer (0.5 mM); thereafter, the reaction mixture was incubated for 30 min with increasing concentrations of adrenaline (5 to 500 uM) in the presence of a saturating concentration (100 /M) of the methyl donor (S-adenosyl-L-methionine); the incubation medium contained also pargyline (100 gM), MgCl2 (100 gM) and EGTA (1 mM). The preincubation and incubation were carried out at 37°C, in conditions of light protection, with continuous shaking and without oxygenation. In experiments conducted with the aim of studying the inhibitory effect of tolcapone on COMT activity, tissue homogenates were preincubated for 15 min with increasing concentrations of tolcapone (0.5 to 10,000 nM); the incubation was performed in the presence of a concentration of adrenaline three times the corresponding Km value, as determined in saturation experiments for each age group. At the end of the incubation period the tubes were transferred to ice and the reaction was stopped by the addition of 100 ul of perchloric acid (2 M). The samples were then centrifuged (200 g, 4 min, 4C), and 500 Ml aliquots of the supernatant filtered on Millipore microfilters (MF1) were used for the assay of metanephrine. This procedure allows 99% extraction of catecholamines and their methylated metabolites.

Cell Assay: Tolcapone as an inhibitor of COMT shows a protective effect against HIV associated dendritic and synaptic damage.

Adv Neurol. 1990;53:497-503; Br J Pharmacol. 1996 Feb;117(3):516-520; Neurology. 1992 Mar;42(3 Pt 1):683-5; J Neural Transm Suppl. 1990;32:375-80.