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On the practical aspects of characterising monoamine oxidase inhibition in vitro

  • Psychiatry and Preclinical Psychiatric Studies - Review Article
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Abstract

The development of novel inhibitors of human monoamine oxidase enzymes with improved pharmacodynamic and pharmacokinetic profiles has, in the past, been hampered by limited access to enzyme, by assay protocols offering limited throughput, and by inappropriate analyses of kinetic data. More recently, high-level expression of human enzymes in yeast has facilitated thorough examinations of steady-state enzyme behaviour that have led to improvements in our understanding of the mathematical underpinnings of kinetic analyses of monoamine oxidases. However, with these improvements have come a realisation that to be useful, more data points across wider concentration ranges are required. In turn, many discontinuous assay approaches, such as those involving radiolabelled substrates or chromatographic separation of product from substrate, have been rendered somewhat obsolete. Justification for the use of a platereader-based approach to assess the effects of novel inhibitors on monamine oxidases is provided, along with details of experimental design optimised to address the unexpectedly complex kinetics followed by these enzymes. Potential sources of error are discussed, and comments provided on techniques that may enhance the quality of experimental data.

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Fig. 1

Adapted from Ramsay et al. (2011)

Fig. 2
Fig. 3

Adapted from McDonald et al. (2010)

Fig. 4
Fig. 5

Adapted from McDonald et al. (2010). Inset: expanded view of curves at low substrate concentrations

Fig. 6
Fig. 7
Fig. 8
Fig. 9

Adapted from Silverman (1995)

Fig. 10

Similar content being viewed by others

Abbreviations

2-BFI:

2-(2-Benzofuranyl)-2-imidazoline

ADME:

Absorption, distribution, metabolism and elimination

DMSO:

Dimethyl sulfoxide

[E]T :

Total enzyme concentration

FAD:

Flavin adenine dinucleotide

FADH2 :

Reduced flavin adenine dinucleotide

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IC50 :

Preincubation concentration of an irreversible inhibitor that reduces enzyme activity by 50% at a point when further preincubation does not increase the degree of inactivation

k cat :

Catalytic rate constant

K D :

Dissociation equilibrium constant for ligand (substrate) interaction with enzyme, representing the ligand concentration at which half of the available enzyme active sites are occupied in the absence of any other ligand

K i :

Dissociation equilibrium constant for inhibitor interaction with enzyme

K I :

Dissociation constant for the initial reversible interaction of a suicide inhibitor with an enzyme active site

k inact :

Rate constant for irreversible inactivation of an enzyme by a suicide inhibitor

K M :

Substrate concentration at which reaction velocity is half of that at VMAX

K Mox :

Substrate concentration at which reaction velocity is half of that at VMAXox, with catalysis following substrate binding only to oxidised enzyme

K Mred :

Substrate concentration at which reaction velocity is half of that at VMAXred, with catalysis following substrate binding only to reduced enzyme

MAO:

Monoamine oxidase

NADH:

Reduced nicotinamide adenine dinucleotide

TSI:

Transition state intermediate

v :

Rate of product formation at a sub-saturating substrate concentration

V MAX :

Rate of product formation at saturating substrate concentration

V MAXox :

Theoretical rate of product formation when substrate binds solely to oxidised MAO, at a substrate concentration that is saturating with respect to affinity for oxidised enzyme

V MAXred :

Theoretical rate of product formation when substrate binds to reduced MAO, at a substrate concentration that is saturating with respect to affinity for both oxidised and reduced enzyme.

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Acknowledgements

The author was supported by an operating Grant (MOP77529) from the Canadian Institutes of Health Research. I thank Professor David Colquhoun and Dr. Rona Ramsay for helpful discussions.

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  1. Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, 1E7.44 Walter Mackenzie Centre, Edmonton, AB, T6G 2B7, Canada

    Andrew Holt

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Holt, A. On the practical aspects of characterising monoamine oxidase inhibition in vitro. J Neural Transm 125, 1685–1705 (2018). https://doi.org/10.1007/s00702-018-1943-8

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