An improved approach to steady-state analysis of monoamine oxidases

The search for new monoamine oxidaseinhibitors aims to identify potential lead compounds thatare more potent and selective than current drugs for use intreating a variety of neuropsychiatric and neurodegenerativeconditions. An integral part of this process is a kineticexamination of monoamine oxidases in the presence of theinhibitor, to determine potency and selectivity and toobtain information on mechanism. To date, kinetic dataobtained with a probe substrate have been analysed byfitting to the Michaelis–Menten equation which describes aunireactant process in which velocity is related to substrateconcentration in a rectangular hyperbolic manner. In thisstudy, we present evidence that monoamine oxidaseactivity is often not adequately described by this approach.We outline a novel equation strategy that takes account ofsubstrate and inhibitor binding to oxidised and reducedenzyme forms, and quantifies differences between substratesand inhibitors in this regard. When combined withplate reader-based experimental techniques that allow largenumbers of substrate and inhibitor concentrations to beused, and the global nonlinear regression facilities ofGraphPad Prism software, this straightforward approachallows more appropriate analyses of monoamine oxidaseby non-experts than has previously been possible.Keywords Monoamine oxidase Steady-state kinetics Equations Inhibition GraphPad PrismIntroductionMonoamine oxidases (MAOs) are targets for therapeuticsdesigned to treat numerous neuropsychiatric and neurodegenerativeconditions (Youdim et al. 2006). To date,drugs targeting MAOs have been developed as inhibitorsof enzymatic activity, designed to increase the availabilityof biogenic amine transmitters and neuromodulators,to decrease formation of reactive oxygen speciesand also for in vitro use to probe mechanisms of MAOcatalysis. Preclinical development and experimental useof MAO inhibitors require determination of inhibitorpotency and mechanism in steady-state assays of enzymeactivity. Such assays typically involve inclusion of theinhibitor, at several concentrations, in assays of MAOactivity versus a range of substrate concentrationsextending either side of the KM value. Historically, initialrate data have then been plotted and analysed by fittingto a hyperbolic or linearised version of the Michaelis–Menten equation. Analyses are rapid and easy to do withregression software, and interpretation of results appearsquit