A New Method To Characterize The Kinetics Of Cholinesterases Inhibition

Background and objective: The inhibition of cholinesterases(Ch Es)by molecules bearing carbamate groups has attracted considerable attention in the past,with applications spanning from pest control to the treatment of Alzheimer’s disease.The determination of the kinetic constants describing the inhibition of Ch Es by carbamate drugs has great importance because it allows to predict the extent and the duration of the inhibitory effect which is helpful for the optimization of their dosage.The interaction of Ch Es and carbamates starts with an inhibition step including the reversible binding of the carbamate to the active site of the enzyme and a carbamylation step,in which the drug transfers its carbamate moiety to the active site according to the carbamylation rate constant k2.The carbamylation is followed by a relatively slow decarbamylation(activity recovery)step,in which the carbamyl group is hydrolyzed from the enzyme according to the decarbamylation rate constant k3.Usually the carbamylation is observed at concentrations far below the affinity(KD)of the carbamate-enzyme reversible complex therefore the inhibition is often well described by a bimolecular carbamylation rate constant(k I)which is related to k2 and KD by the relationship k I = k2/KD.Carbamylation and decarbamylation rate constants are traditionally measured by two separate set of experiments.The traditional method used for the characterization of the decarbamylation step requires relatively high concentrations of enzyme,a condition that sometimes cannot be met due to low enzyme concentrations in the source tissues.Thus,a method that allows the full characterization of the inhibition with a single set of experiments requiring low concentrations of enzyme,could help to save time and resources and could also be used when the traditional decarbamylation experiments cannot be performed.The aim of this thesis was to explore a new method to characterize the kinetics of Ch Es inhibitited by carbamates.Methods:1)We deduced a new linear equation involving the use of the area under the concentration-time curve(AUC)of active and inhibited enzymes from the ordinary differential equations that describe the reaction kinetics.The new equation provides an estimation of both carbamylation and decarbamylation rate constants from data obtained in a single set of experiments performed in pseudo-first order conditions(i.e.with low concentrations of enzyme).The proposed equation represents a simple and useful approach to reduce the time required for the characterization of carbamate inhibitors.2)In order to demonstrate the validity of the approach,bambuterol(BMB)and bambuterol monocarbamate(MONO)enantiomers were chosen as model carbamate drugs.Human and dog plasma butyrylcholinesterase(h BCh E and d BCh E)and human and dog erythrocyte acetylcholinesterase(h ACh E and d ACh E)were used as model Ch Es.The inhibition kinetics of h ACh E by MONO enantiomers and dog Ch Es by BMB and MONO enantiomers were characterized in this work for the first time.3)All the four cholinesterases were inhibited by BMB and MONO enantiomers,with a timeand concentration-dependent mechanism.The molecules tested were potent inhibitors of human and dog BCh Es and much weaker inhibitors of the ACh Es from both species.BMB and MONO enantiomers showed the same enantioselectivity for human and dog BCh Es,the order of k I values being R-BMB > S-BMB > R-MONO > S-MONO,and the(R)-enantiomers inhibited the enzyme about 3-to 6-fold faster than their(S)-enantiomers.The inhibition of the ACh Es from both species by BMB enantiomers showed the same enantioselectivity observed for the BCh Es.Conversely,MONO enantiomers showed an opposite enantioselectivity and in general they were more efficient than BMB enantiomers in inhibiting the ACh Es,the order of k I values being S-MONO > R-BMB ≥ R-MONO > S-BMB for h ACh E and S-MONO > R-MONO > R-BMB > S-BMB for d ACh E.The ACh Es form both species showed to recover from the inhibition faster than BCh Es and according to their k3 values,human Ch Es showed 2-to 4-fold faster activity recovery than the corresponding dog enzymes.4)The inhibition of Ch Es by BMB and MONO enantiomers was characterized by both the traditional approach and our new method and the comparison of the results showed good agreement.Conclusions: The new method proposed in this work allows the simultaneous determination of all the parameters that describe the interaction between Ch Es and carbamates with a single set of experiments and,compared with the traditional method which need two separate experiments,this new method reduces the time required for the characterization of carbamate inhibitors,thus representing an useful tool for the screening of libraries of molecules.In addition,the method presented here is not necessarily limited to the study of Ch Es inhibition,but can be applied to any enzyme-inhibitor system that follow the same reaction schemes.