HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases using novel phenyltetrazolylalkanamine substrates

Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO) using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this compound or homologues of it can also function as substrate for related amine oxidases, namely diamine oxidase (DAO), monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B). Among these substances, 4 was converted by DAO with the highest rate. The best substrate for MAO A and B was 4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine (2). To validate the new assays, the inhibition values of known enzyme inhibitors were determined and the data were compared with those obtained with the substrate benzylamine, which is often used in amine oxidase assays. For the DAO inhibitor 2-(4-phenylphenyl)acetohydrazide an about 10fold lower IC50-value against DAO was obtained when benzylamine was applied instead of 4, indicating that 4 binds to the enzyme with higher affinity than benzylamine. The IC50-values of clorgiline and selegiline against MAO A and B, respectively, also decreased (two- and 30fold) replacing 2 by benzylamine. The discrepancies largely disappeared, when the enzymes were pre-incubated with the inhibitors for 15 min. This can be explained with the covalent inhibition mechanism of the inhibitors.