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Two Nonsynonymous Single Nucleotide Polymorphisms of Human Carbonyl Reductase 1 Demonstrate Reduced in Vitro Metabolism of Daunorubicin and Doxorubicin

Division of Pharmaceutics and Biopharmaceutics (O.S.B., M.J.K., K.W.R.); and Division of Biomolecular and Pharmaceutical Chemistry (R.E.R.), Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada; and Life Sciences Institute, Department of Zoology, Faculty of Science, University of British Columbia, Vancouver, British Columbia, Canada (T.A.G.)

Carbonyl reductases (CBRs) are a group of metabolic enzymes belonging to the short-chain dehydrogenase family with NADPH-dependent oxidoreductase activity. These enzymes are known to metabolize the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Both DOX and DAUN are highly effective in cancer therapy; however, there is considerable interpatient variability in adverse effects seen in patients undergoing treatment with these drugs. This may be attributed to altered metabolism associated with nonsynonymous single nucleotide polymorphisms (ns-SNPs) in the genes encoding for CBRs. In this study, we examine the effect of the V88I and P131S mutations in the human CBR1 gene on the metabolism of anthracyclines to their respective major metabolites, doxorubicinol and daunorubicinol. Kinetic studies using purified, histidine-tagged, recombinant enzymes in a high-performance liquid chromatography-fluorescence assay demonstrated that the V88I mutation leads to a significantly reduced maximal rate of activity (V_max) (2090 ± 112 and 257 ± 11 nmol/min · mg of purified protein for DAUN and DOX, respectively) compared with that for the wild-type (3430 ± 241 and 364 ± 37 nmol/min · mg of purified protein for DAUN and DOX, respectively). In the case of the P131S mutation, a significant increase in substrate affinity (K_m) was observed for DAUN only (89 ± 13 µM) compared with that for the wild-type (51 ± 13 µM). In the presence of either anthracycline, both variants exhibited a 20 to 40% decrease in catalytic efficiency (k_cat/K_m) compared with that for the wild-type enzyme. Therefore, the ns-SNPs generating both these mutations may alter bioavailability of these anthracyclines in cancer patients and should be examined in clinical studies as potential biomarkers for DAUN- and DOX-induced adverse effects.