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 time- and temperature-dependence of inhibition it is possible to discriminate mechanism-based irreversible inhibition and reversible tight binding inhibition of target enzym
- Anderson M., Tipton K., Estimation of monoamine oxidase concentrations in soluble and membrane-bound preparations by inhibitor binding. J. Neural Transm. 1994, 41 (Suppl), 47-53. DOI
- Buneeva O.A., Medvedeva M.V., Medvedev A.E. (2008) Analysis of ubiquitin-dependent regulation of the monoamine oxidase sensitivity to proteolysis and specific inhibition by pargyline. Biomed. Khim., 54(6), 720-726.
- Mashkovskii M.D., Gorkin V.Z., Andreeva N.I., Verevkina I.V., Siniukhin V.N. (1975) Study of the mechanism of action of the new antidepressant pyrazidol. Farmakol. Toksikol. 38(5), 531-536
- Macedo, A., Leiria, E. and Filipe, A. (2011). Pirlindole in the Treatment of Depression. Clin. Drug Investig. 31, 61–71 DOI
- Gorkin V.Z. (1985) Studies on the nature and specific inhibition of monoamine oxidases. In: Neuropharmacology-85, Kelemen K., Magyar K., Vizi E.S, Budapest: Akamediai Kiado, pp. 9-14.
- Medvedev A.E., Shvedov V.I., Chulkova T.M., Fedotova O.A., Saederup E., Squires R.F. The influence of antidepressant pirlindole and its dehydro-derivative on the activity of monoamine oxidase A and GABAA receptor binding. J. Neural Transm. 1998, 52 (Suppl), 337-342. DOI
- Medvedev A.E., Kirkel A.Z., Kamyshanskaya N.S., Axenova L.N., Moskvitina T.A., Gorkin V.Z., Andreeva N.I., Golovina S.M., Mashkovsky M.D. (1994) Inhibition of monoamine oxidase by novel antidepressant tetrindole. Biochem. Pharmacol., 47, (2), 303-308. DOI
- Bradford, M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1–2), 248–254. DOI
- Medvedev A.E., Veselovsky A.V., Shvedov V.I., Tikhonova O.V., Moskvitina T.A., Fedotova O.A., Axenova L.N., Kamyshanskaya N.S., Kirkel A.Z., Ivanov A.S. (1998) Inhibition of monoamine oxidase by pirlindole analogues: 3D-QSAR and CoMFA analysis. J. Chem. Inf. Comput. Sci., 38, 1137-1144. DOI
- Youdim M.B., Edmondson D., Tipton K.F. (2006) The therapeutic potential of monoamine oxidase inhibitors. Nat. Rev. Neurosci., 7(4), 295-309. DOI
- Flockhart DA. (2012) Dietary restrictions and drug interactions with monoamine oxidase inhibitors: an update. J. Clin. Psychiatry;73 (Suppl 1), 17-24. DOI
- Krippendorff B-F., Neuhaus R., Lienau P., Reichel A. and Huisinga W. (2009) Mechanism-based inhibition: deriving Ki and kinact directly from time-dependent IC50 values. J. Biomol. Screen., 14(8), 913-923. DOI
- Curet O., Damoiseau G., Aubin N., Sontag N., Rovei V., Jarreau F.X. (1996) Befloxatone, a new reversible and selective monoamine oxidase-A inhibitor. I. Biochemical profile. J. Pharmacol. Exp. Ther. 277(1), 253-264.