0090-9556/05/3302-254–257$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 2006/1192668 DMD 33:254–257, 2005 Printed in U.S.A.

FUNCTIONAL SIGNIFICANCE OF A NATURAL ALLELIC VARIANT OF HUMAN CARBONYL REDUCTASE 3 (CBR3)

Sukhwinder S. Lakhman, Debashis Ghosh, and Javier G. Blanco

Department of Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, New York (S.S.L., J.G.B.); Department of Structural Biology, Hauptman-Woodward Medical Research Institute, Buffalo, New York, (D.G.); Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York (D.G.); and Therapeutics Program, Roswell Park Cancer Institute, Buffalo, New York (D.G., J.G.B.)

Received September 16, 2004; accepted November 9, 2004

ABSTRACT: ؍ ␮ ⅐ ؎ ؍ Human carbonyl reductase (CBR) activity accounts for a significant versus Vmax CBR3 V244 19.6 2.0 mol/min mg, p 0.002). In fraction of the metabolism of endogenous and xenobiotic carbonyl contrast, both isoforms presented similar Km values (Km CBR3 Downloaded from ؍ ␮ ؎ ؍ ␮ ؎ ؍ compounds. It is possible that genetic polymorphisms in CBR1 and M244 22.9 2.9 M versus Km CBR3 V244 24.6 3.2 M, p

CBR3 are key for the wide interindividual variability in the disposi- 0.43). Assays with NADP(H) demonstrated a higher VmaxNADP(H) tion of CBR drug substrates. We pinpointed a single nucleotide (1.6-fold) and increased catalytic efficiency (VmaxNADP(H)/KmNADP(H)) -Compara .(0.013 ؍ polymorphism in CBR3 (CBR3 V244M) that encodes for a V244 to for CBR3 M244 compared with CBR3 V244 (p M244 change. Blacks showed a higher frequency of the M244 allele tive three-dimensional analyses based on the structure of the ho- ؍ ؍ ؍ ؍ ؍

(q 0.51, n 49) than did whites (q 0.31, n 70; p 0.003). In mologous porcine carbonyl reductase suggested that the V244M dmd.aspetjournals.org addition, DNA variation panels from 10 ethnic groups presented a substitution is positioned in a region critical for interactions with wide range of CBR3 V244M genotype distributions. Kinetic exper- the NADP(H) cofactor. These studies demonstrate that the com- iments with the recombinant CBR3 protein variants and menadi- mon CBR3 V244M polymorphism encodes for CBR3 isoforms with one revealed that CBR3 M244 has significantly higher Vmax than distinctive enzymatic properties. ␮ ⅐ ؎ ؍ does CBR3 V244 (Vmax CBR3 M244 40.6 1.3 mol/min mg at ASPET Journals on April 8, 2015

Carbonyl reductases (CBRs) are members of the family of short- as well as in tumors into their respective alcohol metabolites doxo- chain dehydrogenases/reductases. In humans, there are two mono- rubicinol and daunorubicinol (Forrest et al., 1990). Variable CBR meric carbonyl reductases, carbonyl reductase 1 (CBR1) and carbonyl activity may contribute to the unpredictable pharmacokinetics and reductase 3 (CBR3), which are encoded by different located 62 pharmacodynamics of anthracyclines in adult and pediatric cancer kilobases apart on 21 (CBR1 and CBR3) (Avramopoulos patients (Ratain et al., 1991; Krischer et al., 1997; Singal and Ilisk- et al., 1992; Watanabe et al., 1998; Forrest and Gonzalez, 2000). ovic, 1998; Frost et al., 2002). We hypothesize that genetic polymor- CBR1 and CBR3 have 72% identity and 79% similarity at the amino phism may play a relevant, although thus far uncharacterized role in acid level as indicated by global alignment analysis. CBRs metabolize modulating CBR activity. Thus, we pinpointed a novel nonsynony- a broad spectrum of endogenous and xenobiotic carbonyl compounds mous single nucleotide polymorphism in CBR3 that encodes for a such as prostaglandins, steroids, and various pharmacological agents V244 to M244 change in the protein. We determined the frequencies (Forrest and Gonzalez, 2000). Despite the prominent role of CBR- of the variant CBR3 alleles in genomic DNA samples from white and mediated drug metabolism, there have been no reports on the catalytic black liver donors, and in DNA variation panels representing different properties of CBR3 since the identification of the in 1998 ethnic groups. Furthermore, the recombinant CBR3 V244 and CBR3 (Watanabe et al., 1998). Several studies have described a wide range M244 isoforms were kinetically characterized with menadione and of interindividual variability in the metabolism of drugs that are CBR NADP(H). The distinctive catalytic properties of the CBR3 V244 and substrates (Hermans and Thijssen, 1992; Wong et al., 1992, 1993; CBR3 M244 isoforms support the notion that the common CBR3 Iwata et al., 1993; Ohara et al., 1995; Rady-Pentek et al., 1997; Maser V244M genetic polymorphism may impact CBR-mediated biotrans- et al., 2000). For example, the anticancer anthracyclines doxorubicin formation. and daunorubicin are extensively reduced by CBRs in normal tissues Materials and Methods This work was supported by grants from The Kapoor Foundation and from the Samples. DNA samples were procured from 117 liver donors (whites, n ϭ Lance Armstrong Foundation (principal investigator, Smita Bhatia), and by Na- 70, and blacks, n ϭ 47) from the Liver Tissue Resource (Pharmacogenetics of tional Institutes of Health Grant CA16056. Anticancer Research Group, http://www.pharmacogenetics.org/). Human Article, publication date, and citation information can be found at DNA variation panels were purchased from the Coriell Institute for Medical http://dmd.aspetjournals.org. Research (Camden, NJ). Each panel contained DNA samples from individuals doi:10.1124/dmd.104.002006. representing the following ethnic groups: Chinese, Indo-Pakistani, Japanese,

ABBREVIATIONS: CBR, carbonyl reductase; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; PTCR, porcine testicular carbonyl reductase.

254 CHARACTERIZATION OF POLYMORPHIC HUMAN CARBONYL REDUCTASE 3 255

reverse transcription-PCR from liver RNA samples of individuals with CBR3 V244M homozygous G/G (valine244), and homozygous A/A (methionine244) genotypes, respectively. Total RNA (1 ␮g) was reverse-transcribed with oli- go(dTs) (Invitrogen), and the cDNA was amplified with primers located at the 5Ј- and 3Ј-untranslated regions using the Expand Long Template PCR System (Roche Diagnostics, Indianapolis, IN). Primers were as follows: forward, 5Ј-GCTCAGC- CATGTCATCCT-3Ј; and reverse, 5Ј-CTCCGAAGCAGACGTTTACC-3Ј. Am- plification conditions were 92°C for 2 min, 45 cycles of 92°C for 35 s, 54°C for 40 s, and 68°C for 1:10 min, followed by a final extension at 68°C for 10 min. Purified PCR products were cloned into pET28a expression vectors (Novagen, Madison, WI). The inserts were fully sequenced with vector- and insert- specific primers. The CBR3 V244 and CBR3 M244 constructs were trans- fected into Escherichia coli BL21 (DE3)-competent cells using the heat shock method. Cells were plated on LB agar supplemented with kanamycin (30 ␮g/ml) for selection. Five clones were randomly picked and grown overnight ␮ ϭ ϭ in 100 ml of LB broth supplemented with kanamycin (15 g/ml) to optimize FIG.1.CBR3 V244M genotype distributions in whites (n 70) and blacks (n Ϸ 49). Allelic frequencies for whites were p ϭ 0.69 (G), q ϭ 0.31 (A), and those for expression. Selected cultures were expanded up to 2 liters (A600 nm 0.2) and Ϸ ␤ blacks were p ϭ 0.49 (G), q ϭ 0.51 (A). grown at 37°C (A600 nm 0.6–0.7). Isopropyl- -D-thiogalactoside was added to a final concentration of 1 mM, and the cultures were grown for another 3 to

Mexican, Middle Eastern, South American (Andes region), South East Asian 4 h. Cells were pelleted by centrifugation, washed, and resuspended in lysis Downloaded from (excluding Japanese and Chinese), Pacific, African (North of the Sahara), and buffer (50 mM Na2HPO4, 300 mM NaCl, and 10 mM imidazole) supplemented African (South of the Sahara). with lysozyme (1 mg/ml), DNase I (5 ␮g/ml), and ribonuclease (10 ␮g/ml). Genotyping. We investigated the CBR3 V244M polymorphism with two Cell lysates were obtained by sonication (three pulses of 30 s at 4°C) and genotyping techniques. The first approach involved PCR amplification of CBR3 centrifuged for 20 min (24,000g at 4°C). The resulting cell extracts (superna- exon 3 followed by SNP detection by direct sequencing analysis (Nickerson et al., tants) were filtered (0.45-␮m membrane) and transferred to a vial with equil- 1997). PCR primers were as follows: forward, 5Ј-TGGAGGACACAAAAAAT- ibrated nickel-nitrilotriacetic acid resin. Binding occurred in a rotator at 4°C

GAGG-3Ј; and reverse, 5Ј-GAGAGAGAGAGAGAGAAAAGAAAG-3Ј. Each overnight. Proteins were eluted with buffer containing 250 mM imidazole. The dmd.aspetjournals.org 50-␮l PCR contained 10 ng of genomic DNA, nuclease-free water, 50 pmol of proteins were concentrated using Centricon membranes (YM10; 10,000 mo- each primer, 2.5 units of AmpliTaq Gold DNA polymerase (PerkinElmer Life and lecular weight cut-off) and extensively dialyzed for 24 h in phosphate-buffered Analytical Sciences, Boston, MA), 250 ␮M each deoxynucleotide-5Ј-triphosphate saline at 4°C with four to five buffer changes (ϫ 2.5 liters). After dialysis, (Invitrogen, Carlsbad, CA), and Gene Amp PCR buffer (PerkinElmer Life and protein fractions were desalted by chromatography on a Sephadex G-25 fine Analytical Sciences). Amplification conditions were 92°C for 5 min, 40 cycles of column (Amersham Biosciences Inc., Piscataway, NJ). Different fractions 92°C for 30 s, 56°C for 40 s, and 70°C for 50 s, followed by a final extension at (e.g., before and after sonication, wash, and elutions) were collected for 70°C for 10 min. PCR products (761 base pairs) were incubated with ExoSAP-IT analysis by SDS-polyacrylamide gel electrophoresis followed by Coomassie

(USB, Cleveland, OH) to remove unincorporated nucleotides and primers. PCR staining and Western blotting with anti-His-tag antibodies. CBR3 M244 and at ASPET Journals on April 8, 2015 products were sequenced on an ABI Prism 3700 sequencer (Applied Biosystems, CBR3 V244 were expressed as discrete 39-kDa single bands. Foster City, CA) with the forward PCR primer and with the internal sequencing Kinetic Analysis. CBR3 V244 and CBR3 M244 enzymatic activities were primer 5Ј-AGCAAGCTCCGAAGCAGA-3Ј. The resultant trace files were assem- measured by recording the rate of oxidation of the NADP(H) cofactor at 340 Ϫ Ϫ bled and analyzed using the public National Cancer Institute SNP server (http:// nm (NADPH molar absorption coefficient, 6220 M 1 cm 1). CBR3 concen- lpgws.nci.nih.gov/perl/snp/snp_cgi.pl). The second CBR3 V244M genotyping ap- trations were determined from the absorbance at 280 nm (molar extinction proach utilized the TaqMan technology with specific fluorescent probes labeled coefficient of 15,220 M Ϫ1cmϪ1; http://us.expasy.org). Protein concentrations with FAM and VIC for discrimination of the G and A alleles, respectively and incubation times were standardized to ensure strict obedience to conditions

(Applied Biosystems, Assays-by-Design). Genotyping reactions were performed of initial velocity (Vo). Experiments were performed in a Cary-Varian Bio 300 according to the manufacturer’s specifications in an MX 4000 thermal cycler UV-visible spectrophotometer equipped with thermal control and proprietary (Stratagene, La Jolla, CA). Genotyping results were analyzed with the MX 4000 software for quality control and kinetics data analysis. Reactions were proprietary software. We performed extensive testing to evaluate the performance incubated at 37°C and monitored for 5 min at an acquisition rate of 10/s (3000 of the CBR3 V244M TaqMan assays. There was 100% concordance in the CBR3 readings). Assay mixtures (1.0 ml) contained 0.1 mM NADP(H) (Sigma- V244M genotype cells determined by both direct sequencing and TaqMan, re- Aldrich, St. Louis, MO), potassium phosphate buffer (pH 7.4, 0.1 M), and spectively. enzyme (CBR3 V244 or CBR3 M244). Menadione (Sigma-Aldrich) concen- Cloning and Expression of Human CBR3 V244 and CBR3 M244. CBR3 trations ranged from 3 to 200 ␮M. For the determination of the kinetic V244 and CBR3 M244 full-length cDNAs (1003 base pairs) were obtained by constants of the cofactor, NADP(H) concentrations ranged from 10 to 200 ␮M,

TABLE 1 Distribution of CBR3 V244M genotypes in DNA human diversity panels The percentages corresponding to each genotype category are indicated in parentheses.

Ethnic Group na G/G G/A A/A pb q Africans north of the Sahara 7 1 (14.3) 4 (57.1) 2 (28.6) 0.43 0.57 Africans south of the Sahara 10 5 (50.0) 5 (50.0) 0 (0.0) 0.75 0.25 Chinese 10 5 (50.0) 5 (50.0) 0 (0.0) 0.75 0.25 Indo-Pakistani 9 2 (22.2) 2 (22.2) 5 (55.6) 0.33 0.67 Japanese 10 4 (40.0) 5 (50.0) 1 (10.0) 0.65 0.35 Mexican 10 7 (70.0) 3 (30.0) 0 (0.0) 0.85 0.15 Middle Eastern 10 2 (20.0) 7 (70.0) 1 (10.0) 0.55 0.45 Pacific 7 6 (85.7) 1 (14.3) 0 (0.0) 0.93 0.07 South American Andes 10 6 (60.0) 3 (30.0) 1 (10.0) 0.75 0.25 South East Asia 10 5 (50.0) 4 (40.0) 1 (10.0) 0.70 0.30

a n, number of DNA samples from nonrelated individuals. b p denotes the G allele, and q denotes the A allele. 256 LAKHMAN ET AL.

TABLE 2 Kinetic constants of CBR3 V244 and CBR3 M244

CBR3 V244 CBR3 M244

Km Vmax kcat Vmax/Km Km Vmax kcat Vmax/Km ␮M ␮mol/min ⅐ mg sϪ1 minϪ1 ϫ 103 ␮M ␮mol/min ⅐ mg sϪ1 minϪ1 ϫ 103 Menadionea 24.6 Ϯ 3.2 19.6 Ϯ 2.0 9.9 797 22.9 Ϯ 2.9 40.6 Ϯ 1.3 20.5 1773 NADP(H)b 90.5 Ϯ 2.5 32.4 Ϯ 1.0 16.3 358 85.0 Ϯ 2.0 53.0 Ϯ 1.0 26.7 624

a Each value corresponds to the mean Ϯ S.D. obtained from eight experiments performed with two independent protein preparations for each isoform. b Menadione was held constant at 150 ␮M. Each value represents the mean Ϯ S.D. obtained from four experiments with two independent protein preparations. with menadione at a fixed concentration of 150 ␮M. Enzymatic velocities were direct interactions between the NADP(H) cofactor and the neighbor- ⌬ ⌬ automatically calculated by linear regression of the Abs/ time points. Re- ing amino acids on the CBR3 peptide backbone. Instead, the higher gression coefficients r Ն 0.98 were obtained for all substrate concentrations. apparent VmaxNADP(H) of the CBR3 M244 isoform may be reflecting K and V values were obtained by nonlinear regression analysis with Sigma m max a faster association-dissociation rate for NADP(H)/NADP per cata- Plot version 8.0 (SPSS Inc., Chicago, IL). Excellent agreement between the lytic cycle. To explore whether the three-dimensional structure of the experimental and predicted Km and Vmax values was obtained in all cases with regression coefficients r Ն 0.9 and CV Յ 10%, respectively. enzyme can provide any clues to this possibility, we constructed a model of human CBR3 and examined the effect of the V244M

Results and Discussion polymorphism. The human CBR3 structure was derived from the Downloaded from The CBR3 V244M polymorphism was pinpointed from the dbSNP crystal structure of porcine testicular carbonyl reductase (PTCR) database (refSNP ID: rs1056892). First, we sought to confirm the (Ghosh et al., 2001), with which it shares about 70% sequence identity polymorphism in DNA samples from liver donors. Therefore, we (global alignment analysis; GCG-SeqWeb version 2.0, Accelrys, San analyzed the CBR3 V244M genotype distributions in 70 DNA sam- Diego, CA), by substituting amino acids in the regions surrounding ples from white donors, and in 47 DNA samples from black donors. the coenzyme and the substrate binding sites, and then optimizing In whites, the genotype distribution was as follows: 47.1% were conformations of the backbone and side chains in those regions. The dmd.aspetjournals.org homozygous for the G allele (p ϭ 0.69), 44.3% were heterozygous molecular modeling software CHAIN (Sack, 1988) was used for this G/A, and 8.6% were homozygous for the A allele (q ϭ 0.31). In purpose. Figure 2 shows a modeling of V244 and M244 side chains on contrast, 17.0% of blacks presented the homozygous G/G genotype the three-dimensional structure of the NADP(H) binding region of (p ϭ 0.49), 69.8% were heterozygous G/A, and 19.1% were homozy- human CBR3. The residue at position 244 is a proline in PTCR (P243 gous A/A (q ϭ 0.51). Allele distributions were in Hardy-Weinberg according to the PTCR numbering), which is the amino-terminal ␣ equilibrium in both groups (chi square test, p ϭ 0.98 for whites and residue of the helix G, adjacent to the NADP(H) binding site (Fig. p ϭ 0.14 for blacks). Comparisons of the allelic frequencies indicated 2). The model was obtained first by mutating the proline side chain to at ASPET Journals on April 8, 2015 that the CBR3 V244M genotype distributions differed significantly a valine and then optimizing the conformation of the loop between between blacks and whites (chi square ϭ 11. 7, p ϭ 0.003) (Fig. 1). residues 230 and 235 such that the backbone carbonyl group of K232 We extended our observations by analyzing the CBR3 V244M geno- can now approach the freed backbone amide group of V244 for a type distributions in 10 human variation panels from the Coriell possible hydrogen bond. Small differences between human CBR3 Institute. CBR3 V244M allele frequencies varied widely among the (Fig. 2, shown in yellow) and PTCR (Fig. 2, shown in green) in the groups (Table 1). For example, 85.7% of individuals from the Pacific conformation of this loop are likely, which could also result from group were homozygous for the G allele (V244), whereas 55.6% of several substitutions in the region 236–241 between PTCR and hu- the samples from the Indo-Pakistani panel presented the homozygous man CBR3. Interestingly, this segment of the polypeptide chain has A/A genotype (M244). two polar contacts to the NADP(H) molecule (T233 side chain to the To evaluate the functional impact of the CBR3 V244M polymor- pyrophosphate moiety and V231 carbonyl to the amide group of the phism, we first characterized the catalytic properties of the recombi- nicotinamide ring; Fig. 2) that could be important for catalysis. nant CBR3 V244 and CBR3 M244 isoforms with the prototypical Furthermore, a small movement of the loop could be accompanied by quinone substrate menadione. Menadione or vitamin K3 is considered shifting of the K232 (R231 in PTCR) side chain to a closer proximity to E245, such that a new salt bridge is formed between K232 and a good CBR substrate in humans and other species with Km values in the5to50␮M range (Wermuth et al., 1988; Park et al., 1991; Inazu E245, which already has a salt bridge with R23. Thus, more tightening et al., 1992; Bohren et al., 1994; Schaller and Wermuth, 1999; of this loop is anticipated in human CBR3, in comparison with PTCR.

Sugiyama et al., 2004). The Km values for CBR3 V244 and CBR3 A valine to methionine substitution at this position (the M244 side M244 did not differ (Student’s t test, p ϭ 0.43). However, both chain is shown with gray carbon atoms; Fig. 2) due to the CBR3 SNP ␣ isoforms showed significant differences in Vmax (Table 2). CBR3 is nicely accommodated in a hydrophobic pocket between -helices G M244 presented a 2-fold higher Vmax than did CBR3 V244 (Student’s and B. Thus, a further compacting of the tertiary structure of this ϭ t test, p 0.002). The higher catalytic efficiency (Vmax/Km)ofthe region due to increased hydrophobic interaction could result in CBR3 CBR3 M244 isoform results in an enzyme that is 100% more efficient M244. This structural variation may influence the docking and/or in catalyzing the reduction of substrate per unit of time. release of the cofactor during enzymatic catalysis. Although a detailed To further characterize the impact of the V244M substitution, we analysis of structural differences due to the SNP is impossible in the performed kinetic studies with varying concentrations of NADP(H) absence of experimental CBR3 V244 and CBR3 M244 structures, the (Sciotti and Wermuth, 2001; Sugiyama et al., 2004). Interestingly, modeling exercise suggests alterations in the dinucleotide-binding

KmNADP(H) values were similar for both CBR3 isoforms, but the interactions, which may explain the observed higher catalytic effi- VmaxNADP(H) of CBR3 M244 was 1.6-fold higher than the Vmax- ciency of CBR3 M244. NADP(H) of CBR3 V244 (Student’s t test, p ϭ 0.013; Table 2). Thus, In conclusion, our results indicate that the CBR3 V244M polymor- the kinetic data suggest that the V244M substitution may not affect phism is common among different ethnic groups and encodes for CHARACTERIZATION OF POLYMORPHIC HUMAN CARBONYL REDUCTASE 3 257

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