On the practical aspects of characterising monoamine oxidase inhibition in vitro

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.