Kinase experiment:

Rat liver microsomes and cytosol are as sources of carboxylesterase to screen activity. Assays are conducted in 96-well microtiter plates at room temperature. Briefly, 0.5 mg liver microsomes or 2 mg cytosol are incubated with 10 mM SA72 for 30 min and then substrate p-nitrophenyl acetate is added to a final concentration of 1 mM. The plate is read 10 min after substrate addition at 405 nm for the appearance of the p-nitrophenol. The carboxylesterase activities in liver extracts without inhibitor added are set at 100% and the remaining carboxylesterase activities after incubation with inhibitors are calculated relative to the control[1].

SA72 is a highly selective fatty acid amide hydrolase (FAAH) inhibitor.

Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. SA-72 is also a carbamate inhibitor. However, SA-72 shows exceptional selectivity for FAAH. Tested carboxylesterases are not off-targets for SA-72. SA-72 does not have major effects on the level of carboxylesterase activities in liver microsomes[1].

[1]. Zhang D, et al. Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off-targets. Neuropharmacology. 2007 Mar;52(4):1095-105.