Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study.

In this work an activity-based probe containing a carbamate group was designed to isolate human butyrylcholinesterase (hBChE), a metabolic serine hydrolase (mSH), from complex proteomes. The method took advantage of the native interaction mechanism of mSHs with carbamate pseudo-substrates for temporarily capturing the enzyme on a resin functionalized with the carbamate probe and releasing the enzyme in active form after removal of the contaminating proteins. The isolation relied on the possibility of manipulating the carbamylation and decarbamylation kinetics favoring the former during the capture and wash steps and the latter in the release step. The designed probe captured and released all the active hBChE isoenzymes present in plasma with high selectivity (up to ∼2000-fold purification) and reasonable yields (17% to 36%). The parameters affecting the performance were the incubation time used in the load and elution steps, the plasma to resin volumetric ratio, the elution temperature and the nature and concentration of the eluting agent. The carbamate resin could be prepared either by coupling a fully synthesized probe with an activated resin or by building the probe onto the resin by a step-by-step procedure, without major differences in performance between the two routes. The prepared resins allowed to process up to about 8.5 mL of plasma per g of resin with constant performance. Since the method was based on the general catalytic cycle of mSHs, we expect this approach to be applicable to other enzymes of the family, by selecting a suitable target-selective feature to link to the carbamate group.

A new method to characterize the kinetics of cholinesterases inhibited by carbamates.

The inhibition of cholinesterases (ChEs) by carbamates includes a carbamylation (inhibition) step, in which the drug transfers its carbamate moiety to the active site of the enzyme and a decarbamylation (activity recovery) step, in which the carbamyl group is hydrolyzed from the enzyme. The carbamylation and decarbamylation kinetics decide the extent and the duration of the inhibition, thus the full characterization of candidate carbamate inhibitors requires the measurement of the kinetic constants describing both steps. Carbamylation and decarbamylation rate constants are traditionally measured by two separate set of experiments, thus making the full characterization of candidate inhibitors time-consuming. In this communication we show that by the analysis of the area under the inhibition-time curve of cholinesterases inhibited by carbamates it is possible to calculate the decarbamylation rate constant from the same data traditionally used to characterize only the carbamylation kinetics, therefore it is possible to obtain a full characterization of the inhibition with a single set of experiments. The characterization of the inhibition kinetics of human and dog plasma butyrylcholinesterase and of human acetylcholinesterase by bambuterol and bambuterol monocarbamate enantiomers was used to demonstrate the validity of the approach. The results showed that the proposed method provides reliable estimations of carbamylation and decarbamylation rate constants thus representing a simple and useful approach to reduce the time required for the characterization of carbamate inhibitors.

Stereoselective inhibition of human butyrylcholinesterase by the enantiomers of bambuterol and their intermediates.

This work describes the sequential hydrolysis of bambuterol enantiomers and their monocarbamate metabolites (MONO) catalyzed by human butyrylcholinesterase (BChE) as well as the enzyme inhibition resulting from this process. Particular emphasis is given to the contribution given by MONO to the enzyme inhibition because it was not fully characterized in previous works. Bambuterol and MONO enantiomers displayed the same time- and concentration-dependent mechanism of interaction with the enzyme. The hydrolysis kinetics of both bambuterol and MONO was enantioselective, and the (R)-enantiomer of each compound was hydrolyzed fourfold faster than the respective (S)-enantiomer. Even though the enzyme inhibition rates of (R)- and (S)-MONO were much slower than those of their respective bambuterol enantiomers (∼15-fold), both MONO enantiomers showed a significant BChE inhibition when physiologically relevant concentrations of enzyme and inhibitors were used (∼50% of their respective bambuterol enantiomers). The kinetic constants obtained by testing each single compound were used to model the contribution given by MONO to the enzyme inhibition observed for bambuterol. The hydrolysis of MONO enantiomers enhanced the inhibitory power of bambuterol enantiomers of about 27.5% (R) and 12.5% (S) and extended more than 1 hour the duration of inhibition. The data indicate that MONO contribute significantly to the inhibition of BChE occurring in humans upon administration of normal doses of bambuterol. In addition, the hydrolysis of MONO resulted in the rate-limiting step in the conversion of bambuterol in its pharmacologically active metabolite terbutaline; therefore, MONO concentrations should always be monitored during pharmacokinetic studies of bambuterol.