Fludarabine metabolism by cytosolic 5'-nucleotidase III and its tissue-specific localization mapping using mass spectrometry imaging.

Nucleotidases are enzymes that play vital roles in nucleotide pool balance and purine and pyrimidine metabolism across various tissues. Two major forms of nucleotidases, 5'-nucleotidases (5'-NTs) and nucleoside triphosphate diphosphohydrolases, dephosphorylate nucleoside monophosphates and triphosphates, respectively. Recently, our laboratory reported the dephosphorylation action of these nucleotidases toward the metabolites of clinically used nucleoside analog drugs, including gemcitabine, emtricitabine, tenofovir, and acyclovir. Here, we extended investigating the role of 5'-NTs in disposition of fludarabine, a drug used to treat B-cell chronic lymphocytic leukemia. In vitro incubations carried out using 5 human recombinant 5'-NTs, including cytosolic 5'-nucleotidase 1A (NT5C1A), NT5C2, NT5C3, NT5C, and mitochondrial 5' (3')-deoxyribonucleotidase revealed that NT5C3 catalyzed the dephosphorylation of fludarabine. Although nucleotidases have critical roles in metabolism of endogenous nucleotides and xenobiotics, their spatial localization in tissues is not fully elucidated yet. In the present work, we employed matrix-assisted laser desorption/ionization mass spectrometry imaging to ascertain localizations of tryptic peptides corresponding to major nucleotidases in mouse kidney, colon, and spleen tissues. First, in silico trypsin digestions were performed to determine the trypsin digestion patterns of the above proteins. Then, recombinant nucleotidases were used to characterize tryptic peptides of major nucleotidases. Following this, matrix-assisted laser desorption/ionization mass spectrometry imaging analyses were carried out to localize tryptic peptides corresponding to major nucleotidases in mouse colon, kidney, and spleen tissues. From tissue imaging experiments, we observed localizations of NT5C3 peptides in distinct regions such as the kidney cortex and colonic mucosa. SIGNIFICANCE STATEMENT: Nucleotidases, including cytosolic 5'-nucleotidase (NT5C) 3, have important roles in the endogenous nucleotide metabolism. Additionally, they may catalyze the dephosphorylation reactions of nucleoside analog drugs and their metabolites due to the structural similarities. Using in vitro incubations and enzyme kinetics, we demonstrate the involvement of NT5C3 in the dephosphorylation of an important antineoplastic agent, fludarabine. Furthermore, we employed mass spectrometry imaging to visualize peptides corresponding to NT5C3 and other major nucleotidases in the kidney cortex and colonic mucosa.