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First published on February 14, 2008; DOI: 10.1124/dmd.107.018895

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Onkar S Bains
Ryan H Takahashi
Ronald E Reid
K. Wayne Riggs
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Received for publication September 19, 2007.
Revised February 10, 2008.
Accepted for publication February 12, 2008.

Two allelic variants of aldo-keto reductase 1A1 exhibit reduced in vitro metabolism of daunorubicin

Onkar S Bains 1, Ryan H Takahashi 1, Tom A Pfeifer 1, Thomas A Grigliatti 1, Ronald E Reid 1, K. Wayne Riggs 1*

1 The University of British Columbia

* Address correspondence to: E-mail: riggskw{at}interchange.ubc.ca

Abstract

Aldo-keto reductases (AKRs) are a class of NADPH-dependent oxidoreductases that have been linked to metabolism of the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Although widely used, cardiotoxicity continues to be a serious side effect that may be linked to metabolites or reactive intermediates generated in their metabolism. This study examines the little known effects of non-synonymous single nucleotide polymorphisms (ns-SNPs) of human AKR1A1, on the metabolism of these drugs to their alcohol metabolites. Expressed and purified from bacteria using affinity chromatography, the AKR1A1 protein with a single histidine (6x-His) tag exhibited the greatest activity using two test substrates: p-nitrobenzaldehyde (5.09±0.16 µmol/min/mg purified protein) and DL-glyceraldehyde (1.24±0.17 µmol/min/mg). These activities are in agreement with published literature values of non-tagged human AKR1A1. The 6x-His-tagged AKR1A1 wild-type and allelic variants, E55D and N52S, were subsequently examined for metabolic activity using DAUN and DOX. The tagged variants showed significantly reduced activities (1.10±0.42 and 0.72±0.47 nmol daunorubicinol (DAUNol) formed/min/mg purified protein for E55D and N52S, respectively) compared to the wild-type (2.34±0.71 nmol/min/mg). The wild-type and E55D variant metabolized DOX to doxorubicinol (DOXol), however, the levels fell below the limit of quantitation (25 nM). The N52S variant yielded no detectable DOXol. A kinetic analysis of the DAUN reductase activities revealed that both amino acid substitutions lead to reduced substrate affinity, measured as significant increases in the measured Km for the reduction reaction by AKR1A1. Hence, it is possible that these allelic variants can act as genetic biomarkers for the clinical development of DAUN-induced cardiotoxicity.


Key words: anticancer agents, cardiac toxicity, genetic polymorphism, HPLC, kinetics, pharmacogenetics, polymorphisms, recombinant proteins, reductases


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