Monoamine oxidase (MAO) is a mitochondrial membrane enzyme that catalyses the oxidative deamination reaction and is found in various tissues of the human body. MAO decreases the concentration of neurotransmitters and is therefore associated with the development and progression of diseases, e.g., Parkinson's and Alzheimer's diseases and numerous other neurodegenerative disorders. Two isoforms of the enzyme are known, MAO-A and MAO-B, which differ in the shape of the active site and in the amino acid loop at the entrance to the active site cavity. Considering the structural differences of the active sites in human (h)MAO-B, which is flat and elongated, and hMAO-A, which is spherical, we can design selective inhibitors for a particular isoenzyme.
In our Master’s thesis, we designed and synthesised targeted covalent hMAO-A inhibitors that could potentially covalently bind to amino acid residues Cys321 or Cys323. Targeted covalent inhibitors are usually designed based on an already known inhibitor for a selected biological target and have an electrophilic warhead attached to an appropriate site in the structure. As a structural basis for attaching electrophilic warheads, we chose the reversible inhibitor of hMAO-A, toloxatone, and bound various α-halomethyl ketones and oxoaldehyde to the meta and para positions of benzene, relative to oxazolidin-2-one. In the biochemical assays, we determined the inhibitory potencies of the compounds for both MAO isoforms and indirectly verified whether the compounds exhibited features of covalent inhibition by the time-dependent inhibition and reversibility assay.
We synthesized 12 compounds, confirmed their identity and purity using various spectroscopic and chromatographic techniques, and evaluated them in a coupled biochemical assay. This showed that 11 compounds inhibited at least one isoform of MAO, with 9 compounds having more potent inhibitory activity compared with parent toloxatone. In terms of structure-activity relationship, we can claim that para derivates are more potent inhibitors than their meta analogues. The results of the reversibility assay and the absence of time-dependent inhibition of hMAO indicate that the compounds are most likely reversible and noncovalent inhibitors of both, hMAO-A/B enzymes. By selecting alternative covalent warheads typically used for targeting cysteine residues, we can by using computational approaches, design additional targeted covalent inhibitors on the structural basis of toloxatone.
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