Analysis of Butyrylcholinesterase Interactions with Novel Inhibitors and Reactivators
The inhibition of the enzyme butyrylcholinesterase (BChE) in human tissues by binding of compounds to its active site serine is important for the detoxification and scavenging of xenobiotics such as organophosphates (OP) as well as for the metabolism of pro-drugs and drugs such as the carbamate bambuterol and the phenothiazine ethopropazine. Despite of the importance of BChE, its kinetic reactions were investigated mostly as comparative studies on the related enzyme acetylcholinesterase (AChE), which has a vital function in cholinergic neurotransmission. Moreover, reactivators of inhibited BChE, as well as drugs for the treatment of neurodegenerative diseases were empirically synthesized before the BChE crystal structure was resolved. Due to specific structural requirements, its binding affinity, inhibition and reactivation rates have not been rigorously investigated. It is known from our recent analyses that reactivation rates are influenced by experimental design and reactivation assays need to account for side reactions – oximolysis, reversible inhibition, and adequate dilution in Ellman reaction in order to effectively quench the reactivation reaction. Therefore, this project utilizes known and new compounds to gain a better understanding of the mechanistic basis of cholinesterase family interactions and their limitations. The biochemical mechanism of enzyme interactions will be comprehensively studied on a molecular level with in silico, in vitro, and ex vivo methods. Kinetic constants of the studied interactions will be determined based on known kinetic models, while in need of unusual regression analysis new kinetic models will be developed. These comprehensive analyses will explain structural requirements for compounds interacting with BChE and gain a platform for synthesis of reactivators of phosphylated BChE and potentially active drugs in disorders that involve BChE inhibition. Many of the findings that should arise from this project will impact the mechanisms of hydrolytic catalysis, extending beyond the field of cholinesterases.