Mouse Red Blood Cell-Mediated Rare Xenobiotic Phosphorylation Of A Drug Molecule Not Intended To Be A Kinase Substrate

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., (S)-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin3- yl) phenyl)-8-(hydroxymethyl) quinazolin-4(3H)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of O-18 incorporation into the phosphorylated metabolite when inorganic O-18(4)-phosphate and gamma-O-18(4)-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.