DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 DMD FastThis Forward.article has not Published been copyedited on and February formatted. The7, 2014final version as doi:10.1124/dmd.113.056044 may differ from this version. DMD #56044 Cloning and characterization of four rabbit aldo-keto reductases featuring broad substrate specificity for xenobiotic and endogenous carbonyl compounds: Relationship with multiple forms of drug ketone reductases Satoshi Endo, Toshiyuki Matsunaga, Yuki Arai, Akira Ikari, Kazuo Tajima, Ossama El-Kabbani, Shigeru Yamano, Akira Hara, Yukio Kitade Gifu Pharmaceutical University, Gifu, Japan (S.E., T.M., Y.A., A.I.); Faculty of Downloaded from Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan (K.T.); Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia (O.E.); Faculty of dmd.aspetjournals.org Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan (S.Y.); and Faculty of Engineering (A.H., Y.K.), Gifu University, Gifu, Japan at ASPET Journals on October 2, 2021 1 Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 This article has not been copyedited and formatted. The final version may differ from this version. DMD #56044 Running title: RABBIT DRUG KETONE REDUCTASES Address correspondence to: Dr. Satoshi Endo, Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan, E-mail: [email protected]. Downloaded from The number of text pages: 39 The number of tables: 7 dmd.aspetjournals.org The number of figures: 4 The number of references: 54 The number of footnote: 1 at ASPET Journals on October 2, 2021 The number of words in the Abstract: 248 The number of words in Introduction: 696 The number of words in Discussion: 1497 ABBREVIATIONS: AKR, aldo-keto reductase; CBR, carbonyl reductase; HSD, hydroxy- steroid dehydrogenase; LR, loxoprofen reductase; MI, 2-(1-hydroxyethyl)-7-(2-hydroxy-3- isopropylaminopropoxy)benzofuran; NR, naloxone reductase; PG, prostaglandin; RT, reverse transcription; SDR, short-chain dehydrogenase/reductase; TBE, 6-tert-butyl-2,3-epoxy-5- cyclohexene-1,4-dione. 2 DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 This article has not been copyedited and formatted. The final version may differ from this version. DMD #56044 ABSTRACT: Multiple forms of reductases for several drug ketones were isolated from rabbit liver, but their interrelationship and physiological roles remain unknown. We isolated cDNAs for four aldo-keto reductases (AKRs: 1C30, 1C31, 1C32 and 1C33), which share high amino acid sequence identity with the partial sequences of two rabbit naloxone reductases. The four recombinant enzymes reduced a variety of carbonyl compounds, including endogenous α-dicarbonyls (such as isatin and diacetyl), aldehydes (such as farnesal and 4-oxo-2-nonenal) and ketosteroids. They differed in specificity for drug ketones and ketosteroids. Although Downloaded from daunorubicin and befunolol were common substrates of all the enzymes, other drugs were reduced specifically by the enzymes shown in parentheses: naloxone (AKR1C30, AKR1C32 dmd.aspetjournals.org and AKR1C33); metyrapone (AKR1C32 and AKR1C33); loxoprofen (AKR1C31 and AKR1C32); ketotifen (AKR1C30), and naltrexone and fenofibric acid (AKR1C33). AKR1C30 reduced only 17-keto-5β-androstanes, whereas the other enzymes were active at ASPET Journals on October 2, 2021 towards 3/17/20-ketosteroids, and AKR1C33 further reduced 3-keto groups of bile acids and 7α-hydroxy-5β-cholestanes. In addition, AKR1C30, AKR1C31, AKR1C32 and AKR1C33 were selectively inhibited by carbenoxolone, baccharin, phenolphthalein and zearalenone, respectively. The mRNAs for the four enzymes were ubiquitously expressed in male rabbit tissues, in which highly expressed tissues were the brain, heart, liver, kidney, intestine, colon and testis (for AKR1C30 and AKR1C31), brain, heart, liver, kidney, testis, lung and adrenal gland (for AKR1C32), and liver and intestine (for AKR1C33). The data suggest that the four enzymes correspond to the multiple drug-ketone reductases in rabbits, and function in the metabolisms of steroids, isatin and reactive carbonyl compounds, and bile acid synthesis. 3 DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 This article has not been copyedited and formatted. The final version may differ from this version. DMD #56044 Introduction Carbonyl reduction into the alcohol metabolites is an important Phase-I metabolism of carbonyl-containing drugs, which include more than 30 pharmacologically relevant drugs (Hoffmann and Maser, 2007; Barski et al., 2008). Various carbonyl compounds in the environment also enter the body as xenobiotics and environmental pollutants. In addition, many endogenous carbonyl compounds are formed through metabolisms of biogenic amines, steroids, prostaglandins (PGs) and sugars, lipid peroxidation and glycation. To excrete and detoxify the chemically divergent carbonyl compounds, several NADPH-dependent Downloaded from reductases with broad substrate specificity for carbonyl compounds exist in the body. The enzymes with known primary structures are grouped into two distinct protein families: the dmd.aspetjournals.org short-chain dehydrogenase/reductase (SDR) (Hoffmann and Maser, 2007) and aldo-keto reductase (AKR) (Barski et al., 2008; Jin and Penning, 2007) superfamilies. Although fifteen of such carbonyl-reducing enzymes are known in humans (Matsunaga et al., 2006; at ASPET Journals on October 2, 2021 Oppermann, 2007), major enzymes responsible for the reduction of drug ketones are carbonyl reductase (CBR) and 11β-hydroxysteroid dehydrogenase (HSD) type 1 in the SDR superfamily (Hoffmann and Maser, 2007), and those belonging to the AKR superfamily are cytosolic HSD isoforms (AKR1C1, AKR1C2, AKR1C3 and AKR1C4) (Barski et al., 2008; Matsunaga et al., 2006), as well as aldehyde reductase, aldose reductase, aldose reductase-like protein (AKR1B10) and/or aflatoxin reductase that reduce daunorubicin and doxorubicin (Bains et al., 2010). AKR1B10, AKR1C1 and AKR1C3 are suggested to be involved in proliferation of several types of cancer, and their selective inhibitors are expected to be useful for cancer treatments (El-Kabbani et al., 2011; Byrns et al., 2011; Matsunaga et al., 2013). In laboratory animal rats or mice, five AKRs similar to the human HSD isoforms have been suggested to reduce xenobiotic carbonyl compounds (Matsunaga et al., 2006; Ishikura et al., 2006a; 2006b), but their reactivity towards drug ketones has been scarcely studied. In 4 DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 This article has not been copyedited and formatted. The final version may differ from this version. DMD #56044 contrast, rabbit reductases for several drug ketones were studied, since they had been reported to be as a class of drug metabolizing enzymes (Bachur, 1976) and extremely active carbonyl-drug reduction potential was found in rabbit liver cytosol (Ahmed et al., 1978；1979). The studies on reductases for daunorubicin, oxisuran, metyrapone and naloxone indicate that each reductase exists in multiple forms in the liver cytosol (Ahmed et al., 1978；1979; Sawada and Hara, 1979; Felsted et al., 1980). Subsequently, two of four multiple forms of rabbit naloxone reductase (NR1 and NR2) were shown to belong to the AKR superfamily by their Downloaded from partial protein sequencing, and differ from each other in their stereospecific reduction of the drug and substrate specificity for naltrexone and 3/17-hydroxysteroids (Yamano et al., 1999). A major form of four loxoprofen reductases (LRs) in rabbit liver cytosol also shows 3- and dmd.aspetjournals.org 17-ketosteroid reductase activity (Tanaka et al., 1989). The presence of multiple forms of befunolol reductase in rabbit liver cytosol was also reported, although one form was identified as CBR (Imamura et al., 1989). However, in addition to the properties of the other multiple at ASPET Journals on October 2, 2021 forms, the entire primary sequences of the enzymes other than CBR (Gonzalez et al., 1995) have not been studied, and the interrelationship among the enzymes remains unknown. Recently, recombinant rabbit 20α-HSD (AKR1C5) and 3-hydroxyhexobarbital dehydrogenase (AKR1C29) have been reported to exhibit NADPH-linked reductase activity towards several drug ketones. AKR1C5 reduces xenobiotic carbonyl compounds including befunolol and ketotifen, although it is expressed specifically in rabbit ovary and uterus (Endo et al., 2013a). AKR1C29 ubiquitously expressed in rabbit tissues reduces befunolol, daunorubicin, naloxone and loxoprofen, as well as endogenous substrates such as 3-, 17- and 20-ketosteroids and PGD2 (Endo et al., 2013c). However, the properties and/or amino acid sequences of AKR1C5 and AKR1C29 are different from those of NR1 and NR2, and the major form of LR. Current rabbit genomic analysis has predicted four genes encoding the AKR1C subfamily proteins that share high sequence identity with the partial sequences of 5 DMD Fast Forward. Published on February 7, 2014 as DOI: 10.1124/dmd.113.056044 This article has not been copyedited and formatted. The final version may differ from this version. DMD #56044 NR1 and NR2. In this study, we cloned the cDNAs for the four proteins that have been assigned as AKR1C30, AKR1C31, AKR1C32 and AKR1C33 in the AKR superfamily (http://www.med.upenn.edu/akr/).