Ronald E. Reid

Naturally Occurring Variants of Human Aldo-Keto Reductases with Reduced In Vitro Metabolism of Daunorubicin and Doxorubicin

Doxorubicin (DOX) and daunorubicin (DAUN) are effective anticancer drugs; however, considerable interpatient variability exists in their pharmacokinetics. This may be caused by altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding aldo-keto reductases (AKRs) and carbonyl reductases. This study examined the effect of 27 ns-SNPs, in eight human genes, on the in vitro metabolism of both drugs to their major metabolites, doxorubicinol and daunorubicinol. Kinetic assays measured metabolite levels by high-performance liquid chromatography separation with fluorescence detection using purified, histidine-tagged, human wild-type, and variant enzymes. Maximal rate of activity (Vmax), substrate affinity (Km), turnover rate (kcat), and catalytic efficiency (kcat/Km) were determined. With DAUN as substrate, variants for three genes exhibited significant differences in these parameters compared with their wild-type counterparts: the A106T, R170C, and P180S variants significantly reduced metabolism compared with the AKR1C3 wild-type (Vmax, 23–47% decrease; kcat, 22–47%; kcat/Km, 38–44%); the L311V variant of AKR1C4 significantly decreased Vmax (47% lower) and kcat and kcat/Km (both 43% lower); and the A142T variant of AKR7A2 significantly affected all kinetic parameters (Vmax and kcat, 61% decrease; Km, 156% increase; kcat/Km, 85% decrease). With DOX, the R170C and P180S variants of AKR1C3 showed significantly reduced Vmax (41–44% decrease), kcat (39–45%), and kcat/Km (52–69%), whereas the A142T variant significantly altered all kinetic parameters for AKR7A2 (Vmax, 41% decrease; kcat, 44% decrease; Km, 47% increase; kcat/Km, 60% decrease). These findings suggest that ns-SNPs in human AKR1C3, AKR1C4, and AKR7A2 significantly decrease the in vitro metabolism of DOX and DAUN.

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

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 (Vmax) (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 (Km) 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 (kcat/Km) 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.

Naturally Occurring Variants of Human CBR3 Alter Anthracycline In Vitro Metabolism

Doxorubicin (DOX) and daunorubicin (DAUN) are anthracycline anticancer agents; however, considerable interpatient variability exists in their pharmacokinetics. This interpatient variability is attributed in part to altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding the carbonyl reductases. This study examines the effect of seven naturally occurring ns-SNPs in the CBR3 gene on in vitro metabolism of anthracyclines to doxorubicinol and daunorubicinol. Kinetic assays measure metabolite levels by high-performance liquid chromatography separation with fluorescence detection by use of purified, histidine-tagged, human CBR3 wild type and variant enzymes. The V224M, C4Y, and V93I variants resulted in significantly reduced maximal reaction velocity (Vmax) for both anthracyclines compared with the wild-type enzyme, whereas the M235L variant had significantly reduced Vmax for DOX only. Significant increases in substrate affinity were found for the V244M variant with DAUN, as well as the C4Y and V93I variants with DOX. The catalytic efficiency values for the V244M, C4Y, and V93I variants were significantly lower than the wild type for DAUN and DOX. Furthermore, DOX was observed to be a better substrate than DAUN for the wild-type enzyme and its variants. HapMap analysis indicated that a haplotype carrying the C4Y and V244M mutations may occur in some individuals in the 11 ethnic populations studied in the HapMap project. Our preparation of the double mutant indicated a significant reduction in activity compared with the wild-type enzyme and single-mutant preparations. These findings suggest that commonly occurring ns-SNPs in human CBR3 significantly alter the in vitro metabolism of DOX and DAUN.

A correlation between cytotoxicity and reductase-mediated metabolism in cell lines treated with doxorubicin and daunorubicin

The role of metabolism in daunorubicin (DAUN)- and doxorubicin (DOX)-associated toxicity in cancer patients is dependent on whether the parent drugs or major metabolites, doxorubicinol (DOXol) and daunorubicinol (DAUNol), are the more toxic species. Therefore, we examined whether an association exists between cytotoxicity and the metabolism of these drugs in cell lines from nine different tissues. Cytotoxicity studies using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cell viability assays revealed that four cell lines [HepG2 (liver), HCT-15 (colon), NCI-H460 (lung), and A-498 (kidney)] were more tolerant to DAUN and DOX than the five remaining cell lines [H9c2 (heart), PC-3 (prostate), OVCAR-4 (ovary), PANC-1 (pancreas), and MCF-7 (breast)], based on significantly higher LC50 values at incubation times of 6, 24, and 48 hours. Each cell line was also assessed for its efficiency at metabolizing DAUN and DOX. The four drug-tolerant cell lines converted DAUN/DOX to DAUNol/DOXol more rapidly than the five drug-sensitive cell lines. We also determined whether exposure to DAUN or DOX induced an increase in metabolic activity among any of these nine different cell types. All nine cell types showed a significant increase in their ability to metabolize DAUN or DOX in response to pre-exposure to the drug. Western blot analyses demonstrated that the increased metabolic activity toward DAUN and DOX correlated with a greater abundance of eight aldo-keto and two carbonyl reductases following exposure to either drug. Overall, our findings indicate an inverse relationship between cytotoxicity and DAUN or DOX metabolism in these nine cell lines.

A discovery study of daunorubicin induced cardiotoxicity in a sample of acute myeloid leukemia patients prioritizes P450 oxidoreductase polymorphisms as a potential risk factor

Anthracyclines are very effective chemotherapeutic agents; however, their use is hampered by the treatment-induced cardiotoxicity. Genetic variants that help define patients sensitivity to anthracyclines will greatly improve the design of optimal chemotherapeutic regimens. However, identification of such variants is hampered by the lack of analytical approaches that address the complex, multi-genic character of anthracycline induced cardiotoxicity (AIC). Here, using a multi-SNP based approach, we examined 60 genes coding for proteins involved in drug metabolism and efflux and identified the P450 oxidoreductase (POR) gene to be most strongly associated with daunorubicin induced cardiotoxicity in a population of acute myeloid leukemia (AML) patients (FDR adjusted p-value of 0.15). In this sample of cancer patients, variation in the POR gene is estimated to account for some 11.6% of the variability in the drop of left ventricular ejection fraction (LVEF) after daunorubicin treatment, compared to the estimated 13.2% accounted for by the cumulative dose and ethnicity. In post-hoc analysis, this association was driven by 3 SNPs—the rs2868177, rs13240755, and rs4732513—through their linear interaction with cumulative daunorubicin dose. The unadjusted odds ratios (ORs) and confidence intervals (CIs) for rs2868177 and rs13240755 were estimated to be 1.89 (95% CI: 0.7435–4.819; p = 0.1756) and 3.18 (95% CI: 1.223–8.27; p = 0.01376), respectively. Although the contribution of POR variants is expected to be overestimated due to the multiple testing performed in this small pilot study, given that cumulative anthracycline dose is virtually the only factor used clinically to predict the risk of cardiotoxicity, the contribution that genetic analyses of POR can make to the assessment of this risk is worthy of follow up in future investigations.

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

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. In this study we examine the little known effects of nonsynonymous single nucleotide polymorphisms 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 of purified protein) and dl-glyceraldehyde (1.24 ± 0.17 μmol/min/mg). These activities are in agreement with published literature values of nontagged 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 of daunorubicinol (DAUNol) formed/min/mg of purified protein for E55D and N52S, respectively) compared with 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.

Calcium‐induced folding of a fragment of calmodulin composed of EF‐hands 2 and 3

Calmodulin (CaM) is an EF‐hand protein composed of two calcium (Ca2+)‐binding EF‐hand motifs in its N‐domain (EF‐1 and EF‐2) and two in its C‐domain (EF‐3 and EF‐4). In this study, we examined the structure, dynamics, and Ca2+‐binding properties of a fragment of CaM containing only EF‐2 and EF‐3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca2+‐free CaM2/3 is predominantly unfolded, but upon binding Ca2+, adopts a monomeric structure composed of two EF‐hand motifs bridged by a short antiparallel β‐sheet. Despite having an “even–odd” pairing of EF‐hands, the tertiary structure of CaM2/3 is similar to both the “odd–even” paired N‐ and C‐domains of Ca2+‐ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter‐EF‐hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca2+ binding with a Kd1 = 30 ± 5 μM to EF‐3, and a Kd2 > 1000 μM to EF‐2. Binding of the first equivalent of Ca2+ induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF‐hand domain together, while the loop between EF‐hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its “even–odd” paired EF‐hands. This nonnative hydrophobic core packing may contribute to the weak Ca2+ affinity exhibited by EF‐2 in the context of CaM2/3.

Single nucleotide polymorphisms in aldo-keto and carbonyl reductase genes are not associated with acute cardiotoxicity after daunorubicin chemotherapy.

Background: Evidence suggests that interpatient variability in anthracycline metabolic rate may contribute to the cardiotoxicity associated with anthracycline-based chemotherapy. Therefore, polymorphisms in the anthracycline metabolizing enzymes have been proposed as potential biomarkers of anthracycline-induced cardiotoxicity (AIC). Methods: We have previously shown that 13 of the naturally occurring nonsynonymous single-nucleotide polymorphisms (nsSNP) in the aldo–keto reductases (AKR) and carbonyl reductases (CBR) reduce anthracycline metabolic rate in vitro. Here, we test these SNPs individually and jointly for association with daunorubicin-induced cardiotoxicity in patients with acute myeloid leukemia (AML). Results: Five of the 13 nsSNPs exhibiting an in vitro effect on anthracycline metabolism were detected among the 185 patients with AML. No association was found between the SNPs and daunorubicin-induced cardiotoxicity in either individual or joint effect analyses. Conclusions: Despite the shown in vitro effect of nsSNPs in reductase genes on anthracycline metabolic rate, on their own these SNPs do not explain enough variability in cardiotoxicity to be useful markers of this adverse event. Impact: The results of this study provide important information for biomarker studies on side effects of anthracycline chemotherapy. Cancer Epidemiol Biomarkers Prev; 21(11); 2118–20. ©2012 AACR.

The Effect of Allelic Variation in Aldo-Keto Reductase 1C2 on the in Vitro Metabolism of Dihydrotestosterone

Aldo-keto reductase (AKR) 1C2 is a human, cytosolic enzyme that has an important role in the deactivation of the potent androgen dihydrotestosterone (DHT). AKR1C2 can regulate the extent and duration of activation of the androgen receptor by catalyzing the reduction of DHT to the less potent receptor ligand 3α-diol. In this study, we functionally characterize in vitro the effect of 11 naturally occurring nonsynonymous single nucleotide polymorphisms on the ability of AKR1C2 to reduce DHT to 3α-diol. The wild-type and variant enzymes were expressed using a transfected insect cell system, and their kinetic activities were measured using both a specific fluorogenic probe and DHT as substrates. This functional characterization demonstrates that several variant AKR1C2 proteins have significantly reduced or altered reductase activities as shown by their measured kinetic parameters. Data from our two separate in vitro studies revealed significant reductions in Vmax for two variants (F46Y and L172Q) and significantly lower apparent Km values for three variants (L172Q, K185E, and R258C) compared with the wild type. These results provide evidence that several naturally occurring nonsynonymous single nucleotide polymorphisms in AKR1C2 result in reduced enzyme activities. These variant AKR1C2 alleles may represent one factor involved in the variable degradation of DHT in vivo.

Aldo-Keto Reductase 1C2 Fails to Metabolize Doxorubicin and Daunorubicin in Vitro

The anthracycline drugs are important for the treatment of a number of malignancies; however, their clinical use is associated with dose-dependent severe chronic cardiotoxicity. Although the mechanism for this side effect has not yet been identified, the alcohol metabolites formed during daunorubicin (DAUN) and doxorubicin (DOX) therapies have been implicated. The alcohol metabolites of DAUN and DOX, daunorubicinol (DAUNol) and doxorubicinol (DOXol), respectively, are generated through reduction of the C-13 carbonyl function, which is reportedly mediated by members of the aldo-keto reductase and carbonyl reductase families of proteins. In our search for potential biomarkers for the occurrence of this side effect, we examined the activity of recombinant aldo-keto reductase enzymes, aldo-keto reductase (AKR) 1A1 and AKR1C2, with DAUN and DOX as substrates. Using purified histidine-tagged recombinant proteins and the direct measurement of metabolite formation with a high-performance liquid chromatography-fluorescence assay, we did not observe DAUNol or DOXol generation in vitro by AKR1C2, whereas AKR1A1 did catalyze the reduction reactions. DAUNol was generated by AKR1A1 at a rate of 1.71 ± 0.09 nmol/min/mg protein, and a low level of DOXol was produced by AKR1A1; however, it was below the limits of quantification for the method. These data suggest that the generation of DAUNol or DOXol by AKR1C2 metabolism in vivo is unlikely to occur during anthracycline treatment.