Novel tools for the investigation of carboxylesterase inhibitor potency and substrate selectivity.

摘要

Carboxylesterases are important enzymes responsible for the hydrolysis and metabolism of numerous pharmaceuticals and xenobiotics. These enzymes are vital in mediating insecticide toxicity and in activating many prodrugs. Knowledge of the mechanism of carboxylesterase-induced hydrolysis will enable us to predict insecticide toxicity and understand the activation and metabolism of pharmaceuticals. To investigate the catalytic mechanisms of carboxylesterase-activity, novel inhibitors as well as novel fluorescence-based reporter substrates were developed and evaluated.;Trifluoromethyl ketone (TFK)-containing compounds are marginally soluble, potent esterase inhibitors. Sulfone and sulfoxide analogs of a series of substituted thioether TFKs were synthesized and their potency and solubility were evaluated. These new compounds have increased water solubility, varying potency and provide further evidence of the differences between esterase orthologs, suggesting that additional development of esterase inhibitors may ultimately provide a battery of isoform selective inhibitors.;The TFK hydration state was related to inhibitor potency, with inhibitors that favored the gem-diol conformation exhibiting greater potency. A series of ab initio calculations supported a correlation between the energy of hydration of the ketone and esterase inhibition. In all cases, the extent of ketone hydration correlated with biological potency. This study provides insight into the mechanism of carboxylesterase inhibition and indicates that inhibitors that too strongly favor the gem-diol could exhibit decreased potency due to a low rate of ketone formation.;To further enhance our knowledge of carboxylesterase-dependent insecticide metabolism, a series of fluorescent substrates were synthesized and evaluated as high-throughput reporters of pyrethroid hydrolysis in human liver microsomes. The routine analysis of enzymatic pyrethroid hydrolysis is time-consuming and laborious. The new substrates were found to be appropriate reporters for the hydrolysis of some Type II pyrethroids.;These tools give us the ability to study carboxylesterase activity in multiple enzyme systems and provide insight into the mechanism of inhibition. Together, these techniques will allow for the rational design of carboxylesterase inhibitors of predetermined potency for potential use in controlling prodrug or softdrug metabolism and insecticide specificity and toxicity.