A SIMPLE APPROACH FOR PILOT ANALYSIS OF TIME-DEPENDENT ENZYME INHIBITION: DISCRIMINATION BETWEEN MECHANISM-BASED INACTIVATION AND TIGHT BINDING INHIBITOR BEHAVIOR

The increase in enzyme inhibition developed during prolonged incubation of an enzyme preparation with a chemical substance may be associated with both the non-covalent and also with covalent enzyme-inhibitor complex formation. The latter case involves catalytic conversion of a mechanism-based irreversible inhibitor (a poor substrate) into a reactive species forming covalent adduct(s) with the enzyme and thus irreversibly inactivating the enzyme molecule. Using a simple approach, based on comparison of enzyme inhibition after preincubation with a potential inhibitor at 4oC or 37oC we have analyzed inhibition of monoamine oxidase A (MAO A) by known MAO inhibitors pargyline and pirlindole (pyrazidol). MAO A inhibitory activity of pirlindole (reversible tight binding inhibitor of MAO A) assayed after mitochondrial wash was basically the same for the incubation at both 4oC and 37oC. In contrast to pirlindole, the effect of pargyline (mechanism based irreversible MAO inhibitor) strongly depended on the temperature of the incubation medium. At 37oC the residual activity MAO A in the mitochondrial fraction after washing was significantly lower than in the mitochondrial samples incubated with pargyline at 4oC. Results of this study suggest that using analysis of both timeand temperature-dependence of inhibition it is possible to discriminate mechanism-based irreversible inhibition and reversible tight binding inhibition of target enzym


INTRODUCTION
The increase in enzyme inhibition developed during prolonged incubation of an enzyme preparation with a chemical substance may be associated with both the non-covalent enzymeinhibitor complex formation via an induced-fit mechanism and also with covalent enzyme-inhibitor complex formation proceeding via the mechanism based inhibition. The latter case involves catalytic conversion of a mechanism-based irreversible inhibitor (a poor substrate) into a reactive species forming covalent adduct(s) with the enzyme and thus irreversibly inactivating the enzyme molecule. Since catalytic activity usually demonstrates higher temperature-dependence than noncovalent enzyme-inhibitor complex formation, it appears that preincubation of enzyme preparations with studied inhibitors at different temperatures will help to discriminate tight binding from mechanism-based inhibition.

MATERIALS AND METHODS
The radiolabeled substance [ 14 C]5-hydroxytryptamine creatinine sulfate (a MAO A substrate) was from Amersham (England). Other reagents were purchased from Sigma-Aldrich (USA). Monoamine oxidase inhibitors were from the Collection of chemical substances (Laboratory of Pharmacoproteomics, Institute of Biomedical Chemistry).
Rat liver mitochondria were isolated by the conventional method of differential centrifugation [7]. The final mitochondrial pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.4 and stored at -20ºC until analysis.
MAO inhibition by pargyline and pirlindole was investigated by incubating the thawed mitochondrial suspension (protein concentration 3 mg/ml) with pirlindole or pargyline (final concentrations of 5 µM and 2 µM, respectively) at 4ºC or 37ºC. The incubations were performed in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (0.1 mM). The mitochondrial samples were incubated with MAO inhibitors for 0-30 min. After centrifugation (13000 rpm, Eppendorf Centrifuge 5415R, 30 min) the mitochondrial pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.4 (protein concentration of 1 mg/ml) and used for the MAO A assay. Control samples were treated under the same conditions, but in the absence of pargyline or pirlindole.
The activity of MAO A was determined radiometrically using 0.1 mM [ 14 C]5-hydroxytryptamine creatinine sulfate (specific  [7]. Mitochondrial protein content was determined by the method of Bradford [8].
Results were statistically treated using two-tailed Student's t test. Differences were considered as statistically significant at p < 0.05.

RESULTS
In accordance with results of previous studies (e.g. [6,9]) pirlindole was a potent MAO A inhibitor (Table 1). Its MAO A inhibitory activity assayed after mitochondrial wash was basically the same for the incubation at both 4ºC and 37ºC (Table 1).
In contrast to pirlindole, the effect of pargyline strongly depended on the temperature of the incubation medium (Table 2). At 37ºC the residual activity MAO A in the mitochondrial fraction after washing was significantly lower than in the mitochondrial samples incubated with pargyline at 4ºC (p < 0.01).

DISCUSSION
A study of a mode of drug-target enzyme interaction is an important step during characterization of mechanisms of the drug action in the context of both its effectiveness and also safety. (In the case of MAO inhibitors this was proved in numerous studies [10,11].) Frequently, initial suggestion on the mode of enzyme inhibition (reversible or irreversible) comes from a time dependent increase in the enzyme inhibition resulted in a IC 50 decrease, which is used for calculation of k inact [12], However, some reversible inhibitors (e.g. befloxatone, a reversible MAO A inhibitor [13]) also demonstrate time-dependent inhibition.
Results of this study indicate that using a simple approach, analysis of both time-and temperature-dependence of inhibition it is possible to discriminate mechanism-based irreversible inhibition and reversible tight binding inhibition. The latter demonstrates less pronounced (if any) temperature dependence of enzyme inhibition.

COMPLIANCE WITH ETHICAL STANDARDS
The experiments were carried out in compliance with generally accepted norms of the humane care of laboratory animals.

FUNDING
This work performed within the framework of the State Academies of Sciences Fundamental Scientific Research Program for 2013-2020.