Label-free LC-HRMS-based enzymatic activity assay for the detection of DDC, MAO and COMT inhibitors

Pharmacological treatment of Parkinson's disease consists of a combined chemotherapy that mostly relies on levodopa (L-DOPA) administration together with inhibitors of dopa-decarboxylase (DDC), monoamine oxidase (MAO) and catechol-methyltransferase (COMT). Identification of inhibitors specifically targeting these enzymes is still a significative part of the development of new alternative antiparkinsonian drugs. Most of the available methods use measurement of enzymatic reactions through radioactive labeling, antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach as it allows direct and specific detection and quantification of enzymatic reactions. We describe the development of a simple, reliable, label-free assay based on high-resolution mass spectrometry (HRMS) for the detection and relative quantification of three different enzymatic reactions using non-isolated enzymes. The assay was applied both to reference drugs and plant crude extracts. This method can be used to detect hits in extracts libraries as well as determine relative IC₅₀ of inhibitors.