Refinement of In Vitro Methods for Identification of Aldehyde Oxidase Substrates Reveals Metabolites of Kinase Inhibitors.

To identify aldehyde oxidase (AO) substrates, an assay procedure was developed that leverages the capabilities of high-resolution mass spectrometry to simultaneously monitor parent loss and formation of hydroxylated metabolite over time in incubations with liver cytosol. By incorporating metabolite monitoring, false positives resulting from metabolism by other cytosolic enzymes or processes were decreased. A diverse set of 34 kinase inhibitors containing AO-substrate motifs was screened, and 35% of the compounds were identified as human AO substrates. Confirmation was obtained through determination of the site of metabolism. Human AO substrates identified contained unsubstituted diazanaphthalene moieties (A77-01, INCB 28060, ML-347, LDN-193189, and SB-525334), 4-aminoquinazoline cores (lapatinib, lapatinib M1, and CL-387785), and terminal pyridine and pyrimidine groups (imatinib, bafetinib, and AMG 900). Rat and cynomolgus monkey AO displayed substrate specificities that overlapped moderately with human; rates of metabolism were often higher and lower for cynomolgus monkey and rat, respectively, compared with human. A subset of novel AO substrates identified in this study was also subjected to two other methods for AO substrate determination: comparison of human liver microsome and hepatocyte stability, and the effect of hydralazine, an AO-specific inhibitor, on hepatocyte stability. These methods appeared to correlate and be capable of identifying AO substrates when more than one-third of metabolism in hepatocytes was AO-mediated; however, significant limitations exist. Considering the sensitivity, efficiency, and definitiveness of the cytosol assay with metabolite monitoring, its use is recommended as a primary screen for AO substrates.