Drug Metab. Pharmacokinet. 29 (6): 463–469 (2014).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Regular Article Human Nitrilase-like Protein Does Not Catalyze the Hydrolysis of Vildagliptin Mitsutoshi A SAKURA 1 , Masataka N AKANO 2, Kohei H AYASHIDA 1 , Hideaki F UJII 3 , Miki N AKA JIMA 2, Koichiro ATSUDA 3 , Tomoo I TOH 3 and Ryoichi F UJIWARA 3, * 1
Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan 3 School of Pharmacy, Kitasato University, Tokyo, Japan
2
Full text and Supplementary materials of this paper are available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. Keywords: vildagliptin; M20.7; LAY151; nitrilase; NIT1; NIT2; hydrolysis
Introduction Nitrilase (EC3.5.5.1), which is found in plants, fungi, and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds (Fig. 1A).1) Nitrilases form a large and diverse superfamily. On the basis of sequence similarity and the presence of additional domains, the superfamily was classified into 13 branches.1) Branch 10 contains two proteins that are found in mammalian tissues, nitrilase-like protein (Nit) 1 and Nit2. NIT is also present in humans.2) While human NIT has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. Vildagliptin (LAF237) is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4, DPP-IV, EC 3.4.14.5) for the treatment of type 2 diabetes mellitus.3,4) The major metabolic pathway of vildagliptin in humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite (M20.7 also called LAY151) (Fig. 1B).5) In rats and dogs, the main metabolite of vildagliptin is M20.7.6) The parent compound and the major metabolite M20.7, which is pharmacologically inactive, account for the majority of vildagliptin-related materials in human plasma (approximately 25.7 and 55%, respectively). Although cytochrome P450 (CYP) is
Fig. 1. Enzymatic reaction of nitrile metabolism (A) Nitrilase catalyzes the hydration of nitriles to the corresponding acid. (B) The major metabolic pathway of vildagliptin in humans, dogs, and rats is hydrolysis at the cyano group to produce a carboxylic acid metabolite (M20.7, also called LAY151).
a superfamily of microsomal hemoproteins, which catalyze the reduced nicotinamide adenine dinucleotide phosphate (NADPH)dependent oxidative metabolism of many drugs,7) M20.7 was not formed by incubating vildagliptin with human liver microsomes or
Received March 5, 2014; Accepted June 26, 2014 J-STAGE Advance Published Date: July 8, 2014, doi:10.2133/dmpk.DMPK-14-RG-027 *To whom correspondence should be addressed: Ryoichi FUJIWARA, Ph.D., School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel. ©81-3-5791-6249, Fax. ©81-3-5791-6249, E-mail: [email protected] 463
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recombinant human CYP in the presence of NADPH.5) On the other hand, M20.7 was formed by incubating vildagliptin with a human liver slice.5) These data suggest that CYP is not involved in the hydrolysis of the cyano group of vildagliptin and that the hydrolysis occurs in the liver, possibly in the non-microsomal fraction (e.g., cytosolic fraction). However, the enzyme involved in the major metabolic pathways of vildagliptin has not been identified. The major metabolic pathway of vildagliptin in humans is similar to nitrilase reaction, which is hydrolysis at the cyano group to produce a corresponding carboxylic acid. Furthermore, it has been demonstrated that mammalian Nit is expressed in the cytosol,2) which is the fraction in which vildagliptin is expected to be metabolized. Therefore, we hypothesized that human NIT is involved in the metabolism of vildagliptin. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of the cyano group of vildagliptin. First, homology analysis for the relevance of the amino acid sequence between human NIT and the nitrilase of other organisms was performed. Second, to assess the distribution of mouse Nit1 and Nit2 expressions, Western blot analysis was performed using mouse tissues. Additionally, the protein expression levels of human NIT1 and NIT2 in human liver fractions were assessed by Western blot. Finally, to investigate whether human NIT1 and NIT2 are involved in the formation of M20.7, we performed hydrolysis assays of the cyano group of vildagliptin using HEK293 cells expressing human NIT1 or NIT2. Materials and Methods Chemicals and reagents: G418 and trichloroacetic acid were purchased from Nacalai Tesque (Kyoto, Japan). Rabbit polyclonal anti-human NIT1 antibody (LS-C164881) was purchased from LifeSpan BioSciences (Seattle, WA). Rabbit polyclonal anti-human NIT2 antibody (PAB23095) was purchased from Abnova (Taipei, Taiwan). Mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (6C5) was purchased from American Research Products, Inc. (Waltham, MA). Pooled human liver S9 fraction, pooled human liver cytosol, and pooled human liver microsomes were obtained from XenoTech, LLC (Lenexa, KS). Dithiothreitol (DTT) was obtained from Wako (Osaka, Japan). Succinamic acid, hydroxylamine-HCl, FeCl3, and rabbit polyclonal anti-actin antibody (A2103) were purchased from Sigma-Aldrich (St. Louis, MO). Vildagliptin was synthesized in our laboratory using the standard technique. Vildagliptin carboxylic acid metabolite (M20.7) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of the highest grade available. Homology analysis for the similarity of the amino acid sequence between human NIT and the nitrilase of other organisms: Homology analysis of human NIT and nitrilase of other organisms was performed using BLAST (http://www.ncbi.nlm. nih.gov/BLAST/). The BLAST searches used the BLASTP algorithm with the BLOSUM62 scoring matrix. BLASTP is an alignment-based search tool for protein sequences.8) The BLASTP bit scores and sequence identities were calculated by the search algorithm. The BLASTP bit scores are dependent upon the scoring system (substitution matrix and gap costs) employed and take into account both the degree of similarity and the length of the alignment between the query and the match sequences.8) The amino acid sequences were retrieved from NCBI database (http:// www.ncbi.nlm.nih.gov). The amino sequences of Bradyrhizobium
japonicum USDA 110 (NCBI accession number BAC48662.1), Pseudomonas putida (WP_003257871.1), Rhodococcus rhodochros (BAA01994.1), and Arabidopsis thaliana (NP_851011) were used as typical nitrilases for this homology analysis. The amino sequences of mouse Nit1 (NP_036179), mouse Nit2 (AAH20153), human NIT1 (NP_005591.1), and human NIT2 (AAH20620.1) were used as mouse Nit or human NIT for this homology analysis. The BLASTP search was performed for typical nitrilases or mouse Nit against all human proteins. Animals and preparation of mouse tissue samples: Male C57BL/6NCrSlc mice aged 4 weeks were purchased from Japan SLC (Shizuoka, Japan). All animals received food and water ad libitum, and mouse handling and experimental procedures were conducted in accordance with our animal care protocol, approved by Kitasato University. For tissue collections, mice were anesthetized by diethyl ether inhalation, and the liver was perfused with ice-cold 1.15% KCl. The mouse tissues (heart, lung, stomach, liver, kidney, spleen, small intestine, and large intestine) were rinsed in cold 1.15% KCl and stored at ¹80°C. Tissue homogenates and S9 fraction were prepared using the following procedure. Each tissue was homogenized in three volumes of a phosphate buffer (1.15% KCl, 0.1 M KH2PO4–K2HPO4 buffer, pH 7.4). The homogenate was centrifuged at 10,000 © g for 10 min at 4°C, and the supernatant was used as the S9 fraction. Liver S9 fraction was further centrifuged at 105,000 © g for 60 min at 4°C to prepare the mouse liver microsomes. The supernatant was used as mouse liver cytosol. Expression of human NIT1 and NIT2 in HEK293 cells: Human NIT1 (NCBI accession number NM_005600.2) and human NIT2 (NM_020202.4) cDNA were prepared by a reverse transcription-polymerase chain reaction (PCR) technique from human liver total RNA with sense and antisense oligonucleotide primers (5A-GGC TAT ATC TTC ATG CTG GGC T-3A and 5A-TGC CTC CAA GTC ACA TGA GC-3A for NIT1; 5A-GAG TCA TGA CCT CTT TCC GCT-3A and 5A-CAC AAA CTG CTT GGA GGC AA-3A for NIT2). The PCR products were subcloned into pTARGET Mammalian Expression Vector (Promega, Madison, WI) and the DNA sequences of the inserts were determined by a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using a 3130 Genetic Analyzer (Applied Biosystems). Human embryonic kidney 293 (HEK293) cells were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The cells were grown in Dulbecco’s modified Eagle’s medium containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum with 5% CO2 at 37°C. The human NIT1-expressing HEK293 cells, human NIT2expressing HEK293 cells, and control cells (Mock) were constructed by transfecting the expression vector and control pTARGET vector, respectively, into cells using a Lipofectamine LTX & Plus Reagent (Invitrogen, Carlsbad, CA). Stable transfectants were selected in medium containing 600 µg/mL G418 and several clones were isolated. Preparation of cell homogenates and S9 fraction: HEK293 cells expressing single NIT isoforms were suspended in cold phosphate buffered saline (PBS) and homogenized with a teflonglass homogenizer for 40 strokes. The total cell homogenate was centrifuged at 600 © g for 10 min at 4°C. The pellet, which contained nuclear materials, was discarded. The supernatant, which contained mostly cell protein, was used as the cell homogenate. Additionally, the cell homogenate was centrifuged at 10,000 © g
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Human NIT Does Not Catalyze the Hydrolysis of Vildagliptin
for 10 min at 4°C. The supernatant was used as the S9 fraction.9) SDS-PAGE and Western blot analysis: The cell homogenates (30 µg of protein), human liver samples (100 µg of protein), and mouse tissue samples (100 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel (Lifetechnologies, Carlsbad, CA) and transferred to a PVDF membrane Immobilon-P (Millipore, Bedford, MA). The membrane was blocked for 1 h with 50 mg/mL skimmed milk in PBS and then incubated with the primary antibody diluted with PBS (1:1,000) overnight. The membrane was washed with PBS three times and incubated with HRP-labeled goat anti-rabbit secondary antibody diluted with PBS (1:10,000) for 1 h. The bands were detected using Chemi-Lumi One Western-blotting detection reagents (Nacalai Tesque). Anti-actin antibody (1:10,000) or Anti-GAPDH antibody (1:5,000) was used as the protein loading control. Deglycosylation of N- and O-linked glycoproteins was achieved with peptide N-glycosidase F (PNGase F) and Oglycosidase/neuraminidase (New England Biolabs, Ipswich, MA), respectively, according to the manufacturers’ protocols. Mouse liver S9 fraction (100 µg) was deglycosylated under native conditions overnight at 37°C. The deglycosylated sample was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody as described above. A 96-well plate assay for Nit2/½-amidase-catalyzed hydroxaminolysis of succinamic acid: This hydroxaminolysis reaction with succinamic acid was carried out as described by Krasnikov et al.9) with slight modification. The reaction mixture (50 µL) contained 20 mM succinamic acid, 5 mM DTT, 100 mM potassium phosphate buffer (pH 7.4), 100 mM hydroxylamine-HCl, and HEK293 cell S9 fraction (5 µg of protein), human liver S9 fraction (20 µg of protein), or mouse liver S9 fraction (20 µg of protein). Stock solutions of succinamic acid and hydroxylamine-HCl were previously neutralized with NaOH and stored at ¹20°C. The reaction was initiated by adding succinamic acid after a 5-min preincubation at 37°C. After incubation at 37°C for 30 min, 150 µL of a reagent containing 0.37 M FeCl3, 0.67 M HCl, and 0.2 M trichloroacetic acid was added. The absorbance of the succinyl hydroxamate ferric complex was determined at 535 nm using a SpectraMax M5 96-well plate spectrophotometer (Molecular Devices, Sunnyvale, CA). We used a value of 920 M¹1 cm¹1 as the extinction coefficient for the ferric complex of succinyl hydroxamate at 535 nm. Hydrolysis assays of the cyano group of vildagliptin: A typical incubaion mixture (200 µL of total volume) contained 50 mM Tris-HCl buffer, pH 7.4, 100 µM vildagliptin, and cell homogenate, human liver samples, or mouse liver samples. The reaction was initiated by adding vildagliptin after a 5-min preincubation at 37°C. After incubation at 37°C for 12 h, the reaction was terminated by an addition of 200 µL of cold acetonitrile. After removal of protein by centrifugation at 15,000 © g for 5 min, a 10-µL portion of the sample was subjected to liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure the concentration of M20.7. The enzymatic activity was expressed as an M20.7 formation rate (pmol/h/mg protein). LC-MS/MS conditions: The LC system was connected to a PE Sciex API 2000 tandem mass spectrometer (Applied Biosystems) operated in the positive electrospray ionization mode. M20.7 was separated on a Polaris 5 µm C18-A 50 © 2.0 mm column (20°C) (Agilent Technologies, Amstelveen, The Netherlands) with a MetaGuard 2.0 mm Polaris 5 µm C18-A guard column (Agilent Technologies). The mobile phase of A/B (1:3, v/v) was
used, where A was methanol/10 mM ammonium acetate, pH 8.0 (5:95, v/v), and B was acetonitrile/methanol (10:90, v/v). The flow rate was adjusted to 0.2 mL/min using an Agilent 1100 series pump (Agilent Technologies), and an eluent between 0 and 5 min was introduced into the mass spectrometer. The turbo gas was maintained at 550°C. Nitrogen was used as the nebulizing, turbo, and curtain gas at 50, 85, and 30 psi, respectively. Parent and/or fragment ions were filtered in the first quadrupole and dissociated in the collision cell using nitrogen as the collision gas. The collision energy was 25 V. The sample was analyzed during the multiple reaction monitoring (MRM) mode of the mass spectrometer using m/z 323.1 > 173.3 as the transition. The analytical data were processed using Analyst software (version 1.5.1; Applied Biosystems) in the API 2000 LC-MS/MS systems. Statistical analysis: Data were presented as means « S.D. and were assessed for statistical significance using the unpaired t-test. A value of p < 0.05 was considered statistically significant. Results Homology analysis for the similarity of the amino acid sequence between human NIT and the nitrilases of other organisms: The plant and bacterial nitrilases are typical nitrilases that exhibit nitrilase activities.10) Homology analysis of typical nitrilases or mouse Nit was performed using the BLASTP program. The BLASTP search was performed for typical nitrilases or mouse Nit against all human proteins. Table 1 shows the result of amino acid sequence homology analysis. Human NIT and typical nitrilases shared 25–31% sequence identity. Although amino acid sequence identity of typical nitrilases against human NIT was low, human NIT was the most similar to typical nitrilases among all human proteins. Mouse Nit1 shared 84% sequence identity to human NIT1. Mouse Nit2 shared 89% sequence identity to human NIT2, indicating that the amino acid sequences of human NIT1 and NIT2 were highly similar to mouse Nit1 and Nit2, respectively. Expression of Nit1 and Nit2 in mouse tissues: To assess the distribution of mouse Nit1 and Nit2 protein expressions, Western blot analysis was performed using mouse tissues. The molecular weights of Nit1 and Nit2 are approximately 36 kDa and 31 kDa, respectively. Mouse Nit1 was detected in all 8 tissues and was most abundant in the liver and kidney (Fig. 2A). Similarly, mouse Nit2 protein expression was most abundant in the liver and kidney. Thus, Nit1 and Nit2 are ubiquitously expressed proteins and are most abundant in the liver and kidney in mice. The two bands reacting with the anti-human NIT1 antibody were observed in mouse liver and kidney S9 fractions (Fig. 2A). To elucidate whether the two bands were different glycosylation forms of Nit, mouse liver S9 fraction was deglycosylated under native conditions and analyzed by Western blotting with the anti-human NIT1 antibody. Glycosidase treatment did not affect the size of the two bands in mouse liver S9 fraction (Fig. 2B). These results suggested that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not different glycosylation forms of Nit. Establishment of single expression systems of human NIT1 and NIT2 in HEK293 cells: To investigate whether human NIT1 and NIT2 are involved in hydrolysis of the cyano group of vildagliptin, stable single expression systems of human NIT1 and NIT2 were constructed in HEK293 cells. The protein expression levels of NIT were assessed by Western blot analysis. We selected the clones with the highest NIT protein levels for the subsequent
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Mitsutoshi ASAKURA, et al. Table 1. Homology analysis of human NIT and nitrilase of other organisms
Protein Nitrilase Nitrilase Nitrilase Nitrilase Nit1 Nit2 NIT1 NIT2
Source Bacteria Bradyrhizobium japonicum USDA 110 Pseudomonas putida Rhodococcus rhodochros Plant Arabidopsis thaliana Mammalia Mouse Mouse Human Human
Human NIT1 Score (bits) Identity
Human NIT2 Score (bits) Identity
Accession No.
Amino acids
BAC48662.1 WP_003257871.1 BAA01994.1
321 176 366
50.8 48.1 —
76/307 (25%) 34/108 (31%) —
51.2 57.4 70.1
72/287 (25%) 47/162 (29%) 78/313 (25%)
NP_851011
346
71.2
81/321 (26%)
83.6
81/298 (27%)
NP_036179 AAH20153 NP_005591.1 AAH20620.1
323 276 327 276
570 189 627 189
276/327 100/274 327/327 100/274
(84%) (36%) (100%) (36%)
191 529 189 573
106/281 (38%) 246/276 (89%) 100/274 (36%) 276/276 (100%)
The scores were obtained using the BLASTP program. The accession number of the protein is derived from the NCBI protein database.
Fig. 2. Characterization of Nit1 and Nit2 expression in mouse tissues and deglycosylation of mouse Nit1 in the liver S9 fraction (A) Mouse tissue S9 fraction (100 µg of protein) was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. (B) Mouse liver S9 fraction (100 µg) was treated with PNGase F, O-glycosidase, and neuraminidase under native conditions overnight at 37°C. The deglycosylated sample was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody. Actin was used as the protein loading control. The positions of the molecular weight markers are indicated.
analyses. Significant NIT1 protein expression was observed in the cell homogenate of the human NIT1-transfectant compared with that of control vector-transfectant (Fig. 3). In addition, the human NIT2-transfectant also showed significant NIT2 protein expression compared with control vector-transfectant. These results demonstrated that stable single expression systems of human NIT1 and NIT2 in HEK293 cells were constructed. Protein expression of human NIT and mouse Nit in liver S9 fraction, cytosol, and microsomes: To determine whether human NIT1 and NIT2 are expressed in human liver, Western blot analysis was performed using human liver S9 fraction, cytosol, and microsomes. Additionally, to compare the protein expression patterns of human NIT and mouse Nit in the liver, Western blot analysis was also performed using mouse liver samples. Human NIT1 and NIT2 were highly detected in liver S9 fraction and cytosol (Fig. 4). Mouse Nit1 and Nit2 were also highly detected in liver S9 fraction and cytosol. Human NIT and mouse Nit were
slightly and moderately detected in liver microsomes, respectively. The protein expression pattern of human NIT1 and NIT2 in human liver fractions was almost consistent with that of mouse Nit1 and Nit2 in mouse liver fractions. These findings indicate that human NIT and mouse Nit are highly expressed in liver cytosol. Nit2/½-amidase-catalyzed hydrolysis of succinamic acid: The enzyme activity of Nit2 was measured in HEK293 cells expressing human NIT2, human liver S9 fraction, or mouse liver S9 fraction. Human NIT2 has been identified as an ½-amidase (½-amidodicarboxylate amidohydrolase, EC 3.5.1.3).11) Thus, the Nit2/½-amidase activity of each sample was assessed by 96-well plate assay for Nit2/½-amidase-catalyzed hydroxaminolysis of succinamic acid. The Nit2/½-amidase activity in Mock-transfected, human NIT1-expressing, and human NIT2-expressing HEK293 cells was 10.2 « 7.2, 11.1 « 2.4, and 250 « 4.4 nmol/min/mg protein, respectively (Fig. 5). The Nit2/½-amidase activity in HEK293 cells expressing human NIT2 was significantly higher than that in
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Human NIT Does Not Catalyze the Hydrolysis of Vildagliptin
Mock-transfected HEK293 cells (p < 0.01). The Nit2/½-amidase activity in human and mouse liver S9 fractions was 104 « 0.6 and 103 « 4.2 nmol/min/mg protein, respectively. These findings indicated that functional Nit2 was expressed in HEK293 cells expressing human NIT2, human liver S9 fraction, and mouse liver S9 fraction. Hydrolysis assays of the cyano group of vildagliptin: To investigate whether human NIT1 and NIT2 are involved in hydrolysis of the cyano group of vildagliptin, M20.7 formation rates in HEK293 cells expressing human NIT1 and NIT2 were measured. Additionally, to assess the formation of M20.7 in human and mouse liver fractions, M20.7 formation rates in human and mouse liver samples were also measured. M20.7 formation rate in human liver S9 fraction, cytosol, and microsomes was 6.4 « 0.3, 10.5 « 0.3, and 11.9 « 0.1 pmol/h/mg protein, respectively (Fig. 6). M20.7 formation rate in human liver microsomes was statistically higher than that in the liver cytosol (p < 0.05). M20.7 formation rate in mouse liver S9 fraction, cytosol, and microsomes was 3.6 « 0.2, 3.3 « 0.2, and 4.6 « 0.1 pmol/h/mg protein, respectively. M20.7 formation rate in mouse liver microsomes was statistically higher than that in the liver cytosol (p < 0.01). M20.7 formation rates in human liver samples were approximately 2- to 3- fold higher than those in mouse liver samples, respectively. Although M20.7 formation rate in liver
Fig. 3. Western blot analysis of human NIT1 and NIT2 expression in stable cell lines The cell homogenates from the Mock-, NIT1-, and NIT2-transfected HEK293 cells (30 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. Actin was used as the protein loading control. The positions of the molecular weight markers are indicated.
microsomes was higher than that in liver S9 fraction and cytosol, the protein expression level of human NIT and mouse Nit in liver microsomes was lower than that in S9 fraction and cytosol (Figs. 4 and 6). Furthermore, the M20.7 formation rate in HEK293 cells expressing human NIT1 and NIT2 (0.4 « 0.2 and 0.3 « 0.1 pmol/h/mg protein, respectively) was comparable with that in the non-transfected HEK293 cells (0.3 « 0.2 pmol/h/mg protein). These results suggested that human NIT1 and NIT2 were not involved in the metabolism of vildagliptin. Discussion Mouse Nit1 and Nit2 were detected in most tissues by Western blot analysis and were particularly abundant in the liver and kidney. These results were consistent with previous reports.2,12) The two bands reacting with the anti-human NIT1 antibody were observed in mouse liver and kidney S9 fractions (Fig. 2A). We investigated whether these upper and lower bands were different
Fig. 5. Nit2/½-amidase activity in human NIT1- and NIT2-expressing HEK293 cells, human liver S9 fraction, and mouse liver S9 fraction The Nit2/½-amidase activity of HEK293 cell, human liver, or mouse liver S9 fractions was measured using a succinamic acid as a substrate. Data represent the means « S.D. of triplicate determinations. **p < 0.01.
Fig. 4. Western blot analysis of human NIT and mouse Nit expression in liver samples Human liver samples (100 µg of protein) and mouse liver samples (100 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. GAPDH was used as the protein loading control. The positions of the molecular weight markers are indicated.
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Fig. 6. Hydrolysis assays of the cyano group of vildagliptin The cyano group hydrolysis activity of HEK293 cells-expressing human NIT, human liver samples, and mouse liver samples was measured. The enzymatic activity was expressed as an M20.7 formation rate (pmol/h/mg protein). Data represent the means « S.D. of triplicate determinations. *p < 0.05; **p < 0.01.
glycosylation forms of Nit. The results of deglycosylation analysis suggested that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not different glycosylation forms of Nit (Fig. 2B). We then suspected that anti-human NIT1 antibody non-selectively reacted with mouse Nit2. We performed homology analysis of the epitope for antihuman NIT antibody, human NIT, and mouse Nit. The amino acid sequence identities of the epitope for anti-NIT1 antibody against mouse Nit1 and Nit2 were high (81%) and low (39%), respectively (Supplemental Table 1). Similarly, the epitope for anti-NIT1 antibody shared 100% (high) and 46% (low) sequence identity to human NIT1 and NIT2, respectively. Indeed, anti-NIT1 antibody did not react with the protein of human NIT2 in human NIT2expressing HEK293 cells (Fig. 3). Because these results demonstrated the selectivity of anti-NIT1 antibody, we believed that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not mouse Nit2. It has been reported that two mouse Nit1 transcript variants, mouse Nit1 variant 1 (NCBI accession number NM_012049.2) and variant 2 (NM_001242580.1) exist. The reported amino acid sequence of mouse Nit1 variant 1 is longer than that of mouse Nit1 variant 2. These findings raise the possibility that the upper and lower bands reacting with the anti-human NIT1 antibody are mouse Nit1 variant 1 and variant 2, respectively. We believed that the densities of the upper and lower bands reacting with the anti-human NIT1 antibody were associated with the expression levels of mouse Nit1 variant 1 and variant 2 in mouse tissues. A previous report has described the liver as being the major site of vildagliptin metabolism.13) Distributions of Nit1 and Nit2 were consistent with a major site of metabolism of vildagliptin. In fact, the protein of human NIT1 and NIT2 was highly expressed in human liver cytosol (Fig. 4). Although amino acid sequence identity of typical nitrilases against human NIT was low, human NIT was the most similar to typical nitrilases among all human proteins (Table 1). Therefore, human NIT1 and NIT2 were expected to
be involved in the hydrolysis of the cyano group of vildagliptin, which is similar to nitrilase reaction. However, the result of measurement of M20.7 formation rate suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin. The exploration of major metabolic enzyme responsible for metabolism of vildagliptin needs to be conducted in the future. The enzyme activity of human NIT2-expressing HEK293 cells could be examined by 96-well plate assay for Nit2/½-amidasecatalyzed hydroxaminolysis of succinamic acid. However, the enzyme activity of human NIT1-expressing HEK293 cells could not be evaluated. This was because substrates of mammalian Nit1 have not been identified.14) We have not determined whether functional Nit1 is expressed in HEK293 cells expressing human NIT1. Therefore, we could not conclude that human NIT1 was not involved in the hydrolysis of the cyano group of vildagliptin. Although the result of measurement of M20.7 formation rate suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin, human NIT may be involved in the metabolism of other drugs with the cyano group. Our current study examined the involvement of human NIT in drug metabolism for the first time. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. In conclusion, human NIT1 and NIT2 were highly expressed in human liver cytosol, which is the fraction in which vildagliptin is expected to be metabolized. Although human NIT1 and NIT2 were expected to be involved in the hydrolysis of the cyano group of vildagliptin, the result of the measurement of the M20.7 formation rate in HEK293 cells expressing human NIT1 or NIT2 suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. References 1) 2) 3)
4) 5)
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7) 8)
9)
10)
Pace, H. C. and Brenner, C.: The nitrilase superfamily: classification, structure and function. Genome Biol., 2: 1–9 (2001). Lin, C. H., Chung, M. Y., Chen, W. B. and Chien, C. H.: Growth inhibitory effect of the human NIT2 gene and its allelic imbalance in cancers. FEBS J., 274: 2946–2956 (2007). Villhauer, E. B., Brinkman, J. A., Naderi, G. B., Burkey, B. F., Dunning, B. E., Prasad, K., Mangold, B. L., Russell, M. E. and Hughes, T. E.: 1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J. Med. Chem., 46: 2774–2789 (2003). Deacon, C. F.: Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: a comparative review. Diabetes Obes. Metab., 13: 7–18 (2011). He, H., Tran, P., Yin, H., Smith, H., Batard, Y., Wang, L., Einolf, H., Gu, H., Mangold, J. B., et al.: Absorption, metabolism, and excretion of [14C] vildagliptin, a novel dipeptidyl peptidase 4 inhibitor, in humans. Drug Metab. Dispos., 37: 536–544 (2009). He, H., Tran, P., Yin, H., Smith, H., Flood, D., Kramp, R., Filipeck, R., Fischer, V. and Howard, D.: Disposition of vildagliptin, a novel dipeptidyl peptidase 4 inhibitor, in rats and dogs. Drug Metab. Dispos., 37: 545–554 (2009). Meunier, B., De Visser, S. P. and Shaik, S.: Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chem. Rev., 104: 3947–3980 (2004). Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25: 3389–3402 (1997). Krasnikov, B. F., Nostramo, R., Pinto, J. T. and Cooper, A. J.: Assay and purification of ½-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the ¡-keto acid analogues of glutamine and asparagine. Anal. Biochem., 391: 144–150 (2009). O’Reilly, C. and Turner, P. D.: The nitrilase family of CN hydrolysing enzymes—a comparative study. J. Appl. Microbiol., 95: 1161–1174
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11)
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(2003). Chien, C. H., Gao, Q. Z., Cooper, A. J., Lyu, J. H. and Sheu, S. Y.: Structural insights into the catalytic active site and activity of human Nit2/½-amidase: KINETIC ASSAY AND MOLECULAR DYNAMICS SIMULATION. J. Biol. Chem., 287: 25715–25726 (2012). Semba, S., Han, S. Y., Qin, H. R., McCorkell, K. A., Iliopoulos, D., Pekarsky, Y., Druck, T., Trapasso, F., Croce, C. M., et al.: Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor. J. Biol. Chem., 281:
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28244–28253 (2006). He, Y. L., Sabo, R., Campestrini, J., Wang, Y., Ligueros-Saylan, M., Lasseter, K. C., Dilzer, S. C., Howard, D. and Dole, W. P.: The influence of hepatic impairment on the pharmacokinetics of the dipeptidyl peptidase IV (DPP-4) inhibitor vildagliptin. Eur. J. Clin. Pharmacol., 63: 677–686 (2007). Sun, J., Okumura, H., Yearsley, M., Frankel, W., Fong, L. Y., Druck, T. and Huebner, K.: Nit1 and Fhit tumor suppressor activities are additive. J. Cell. Biochem., 107: 1097–1106 (2009).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Regular Article Human Nitrilase-like Protein Does Not Catalyze the Hydrolysis of Vildagliptin Mitsutoshi A SAKURA 1 , Masataka N AKANO 2, Kohei H AYASHIDA 1 , Hideaki F UJII 3 , Miki N AKA JIMA 2, Koichiro ATSUDA 3 , Tomoo I TOH 3 and Ryoichi F UJIWARA 3, * 1
Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan 3 School of Pharmacy, Kitasato University, Tokyo, Japan
2
Full text and Supplementary materials of this paper are available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. Keywords: vildagliptin; M20.7; LAY151; nitrilase; NIT1; NIT2; hydrolysis
Introduction Nitrilase (EC3.5.5.1), which is found in plants, fungi, and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds (Fig. 1A).1) Nitrilases form a large and diverse superfamily. On the basis of sequence similarity and the presence of additional domains, the superfamily was classified into 13 branches.1) Branch 10 contains two proteins that are found in mammalian tissues, nitrilase-like protein (Nit) 1 and Nit2. NIT is also present in humans.2) While human NIT has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. Vildagliptin (LAF237) is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4, DPP-IV, EC 3.4.14.5) for the treatment of type 2 diabetes mellitus.3,4) The major metabolic pathway of vildagliptin in humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite (M20.7 also called LAY151) (Fig. 1B).5) In rats and dogs, the main metabolite of vildagliptin is M20.7.6) The parent compound and the major metabolite M20.7, which is pharmacologically inactive, account for the majority of vildagliptin-related materials in human plasma (approximately 25.7 and 55%, respectively). Although cytochrome P450 (CYP) is
Fig. 1. Enzymatic reaction of nitrile metabolism (A) Nitrilase catalyzes the hydration of nitriles to the corresponding acid. (B) The major metabolic pathway of vildagliptin in humans, dogs, and rats is hydrolysis at the cyano group to produce a carboxylic acid metabolite (M20.7, also called LAY151).
a superfamily of microsomal hemoproteins, which catalyze the reduced nicotinamide adenine dinucleotide phosphate (NADPH)dependent oxidative metabolism of many drugs,7) M20.7 was not formed by incubating vildagliptin with human liver microsomes or
Received March 5, 2014; Accepted June 26, 2014 J-STAGE Advance Published Date: July 8, 2014, doi:10.2133/dmpk.DMPK-14-RG-027 *To whom correspondence should be addressed: Ryoichi FUJIWARA, Ph.D., School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel. ©81-3-5791-6249, Fax. ©81-3-5791-6249, E-mail: [email protected] 463
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recombinant human CYP in the presence of NADPH.5) On the other hand, M20.7 was formed by incubating vildagliptin with a human liver slice.5) These data suggest that CYP is not involved in the hydrolysis of the cyano group of vildagliptin and that the hydrolysis occurs in the liver, possibly in the non-microsomal fraction (e.g., cytosolic fraction). However, the enzyme involved in the major metabolic pathways of vildagliptin has not been identified. The major metabolic pathway of vildagliptin in humans is similar to nitrilase reaction, which is hydrolysis at the cyano group to produce a corresponding carboxylic acid. Furthermore, it has been demonstrated that mammalian Nit is expressed in the cytosol,2) which is the fraction in which vildagliptin is expected to be metabolized. Therefore, we hypothesized that human NIT is involved in the metabolism of vildagliptin. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of the cyano group of vildagliptin. First, homology analysis for the relevance of the amino acid sequence between human NIT and the nitrilase of other organisms was performed. Second, to assess the distribution of mouse Nit1 and Nit2 expressions, Western blot analysis was performed using mouse tissues. Additionally, the protein expression levels of human NIT1 and NIT2 in human liver fractions were assessed by Western blot. Finally, to investigate whether human NIT1 and NIT2 are involved in the formation of M20.7, we performed hydrolysis assays of the cyano group of vildagliptin using HEK293 cells expressing human NIT1 or NIT2. Materials and Methods Chemicals and reagents: G418 and trichloroacetic acid were purchased from Nacalai Tesque (Kyoto, Japan). Rabbit polyclonal anti-human NIT1 antibody (LS-C164881) was purchased from LifeSpan BioSciences (Seattle, WA). Rabbit polyclonal anti-human NIT2 antibody (PAB23095) was purchased from Abnova (Taipei, Taiwan). Mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (6C5) was purchased from American Research Products, Inc. (Waltham, MA). Pooled human liver S9 fraction, pooled human liver cytosol, and pooled human liver microsomes were obtained from XenoTech, LLC (Lenexa, KS). Dithiothreitol (DTT) was obtained from Wako (Osaka, Japan). Succinamic acid, hydroxylamine-HCl, FeCl3, and rabbit polyclonal anti-actin antibody (A2103) were purchased from Sigma-Aldrich (St. Louis, MO). Vildagliptin was synthesized in our laboratory using the standard technique. Vildagliptin carboxylic acid metabolite (M20.7) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of the highest grade available. Homology analysis for the similarity of the amino acid sequence between human NIT and the nitrilase of other organisms: Homology analysis of human NIT and nitrilase of other organisms was performed using BLAST (http://www.ncbi.nlm. nih.gov/BLAST/). The BLAST searches used the BLASTP algorithm with the BLOSUM62 scoring matrix. BLASTP is an alignment-based search tool for protein sequences.8) The BLASTP bit scores and sequence identities were calculated by the search algorithm. The BLASTP bit scores are dependent upon the scoring system (substitution matrix and gap costs) employed and take into account both the degree of similarity and the length of the alignment between the query and the match sequences.8) The amino acid sequences were retrieved from NCBI database (http:// www.ncbi.nlm.nih.gov). The amino sequences of Bradyrhizobium
japonicum USDA 110 (NCBI accession number BAC48662.1), Pseudomonas putida (WP_003257871.1), Rhodococcus rhodochros (BAA01994.1), and Arabidopsis thaliana (NP_851011) were used as typical nitrilases for this homology analysis. The amino sequences of mouse Nit1 (NP_036179), mouse Nit2 (AAH20153), human NIT1 (NP_005591.1), and human NIT2 (AAH20620.1) were used as mouse Nit or human NIT for this homology analysis. The BLASTP search was performed for typical nitrilases or mouse Nit against all human proteins. Animals and preparation of mouse tissue samples: Male C57BL/6NCrSlc mice aged 4 weeks were purchased from Japan SLC (Shizuoka, Japan). All animals received food and water ad libitum, and mouse handling and experimental procedures were conducted in accordance with our animal care protocol, approved by Kitasato University. For tissue collections, mice were anesthetized by diethyl ether inhalation, and the liver was perfused with ice-cold 1.15% KCl. The mouse tissues (heart, lung, stomach, liver, kidney, spleen, small intestine, and large intestine) were rinsed in cold 1.15% KCl and stored at ¹80°C. Tissue homogenates and S9 fraction were prepared using the following procedure. Each tissue was homogenized in three volumes of a phosphate buffer (1.15% KCl, 0.1 M KH2PO4–K2HPO4 buffer, pH 7.4). The homogenate was centrifuged at 10,000 © g for 10 min at 4°C, and the supernatant was used as the S9 fraction. Liver S9 fraction was further centrifuged at 105,000 © g for 60 min at 4°C to prepare the mouse liver microsomes. The supernatant was used as mouse liver cytosol. Expression of human NIT1 and NIT2 in HEK293 cells: Human NIT1 (NCBI accession number NM_005600.2) and human NIT2 (NM_020202.4) cDNA were prepared by a reverse transcription-polymerase chain reaction (PCR) technique from human liver total RNA with sense and antisense oligonucleotide primers (5A-GGC TAT ATC TTC ATG CTG GGC T-3A and 5A-TGC CTC CAA GTC ACA TGA GC-3A for NIT1; 5A-GAG TCA TGA CCT CTT TCC GCT-3A and 5A-CAC AAA CTG CTT GGA GGC AA-3A for NIT2). The PCR products were subcloned into pTARGET Mammalian Expression Vector (Promega, Madison, WI) and the DNA sequences of the inserts were determined by a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using a 3130 Genetic Analyzer (Applied Biosystems). Human embryonic kidney 293 (HEK293) cells were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The cells were grown in Dulbecco’s modified Eagle’s medium containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum with 5% CO2 at 37°C. The human NIT1-expressing HEK293 cells, human NIT2expressing HEK293 cells, and control cells (Mock) were constructed by transfecting the expression vector and control pTARGET vector, respectively, into cells using a Lipofectamine LTX & Plus Reagent (Invitrogen, Carlsbad, CA). Stable transfectants were selected in medium containing 600 µg/mL G418 and several clones were isolated. Preparation of cell homogenates and S9 fraction: HEK293 cells expressing single NIT isoforms were suspended in cold phosphate buffered saline (PBS) and homogenized with a teflonglass homogenizer for 40 strokes. The total cell homogenate was centrifuged at 600 © g for 10 min at 4°C. The pellet, which contained nuclear materials, was discarded. The supernatant, which contained mostly cell protein, was used as the cell homogenate. Additionally, the cell homogenate was centrifuged at 10,000 © g
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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for 10 min at 4°C. The supernatant was used as the S9 fraction.9) SDS-PAGE and Western blot analysis: The cell homogenates (30 µg of protein), human liver samples (100 µg of protein), and mouse tissue samples (100 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel (Lifetechnologies, Carlsbad, CA) and transferred to a PVDF membrane Immobilon-P (Millipore, Bedford, MA). The membrane was blocked for 1 h with 50 mg/mL skimmed milk in PBS and then incubated with the primary antibody diluted with PBS (1:1,000) overnight. The membrane was washed with PBS three times and incubated with HRP-labeled goat anti-rabbit secondary antibody diluted with PBS (1:10,000) for 1 h. The bands were detected using Chemi-Lumi One Western-blotting detection reagents (Nacalai Tesque). Anti-actin antibody (1:10,000) or Anti-GAPDH antibody (1:5,000) was used as the protein loading control. Deglycosylation of N- and O-linked glycoproteins was achieved with peptide N-glycosidase F (PNGase F) and Oglycosidase/neuraminidase (New England Biolabs, Ipswich, MA), respectively, according to the manufacturers’ protocols. Mouse liver S9 fraction (100 µg) was deglycosylated under native conditions overnight at 37°C. The deglycosylated sample was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody as described above. A 96-well plate assay for Nit2/½-amidase-catalyzed hydroxaminolysis of succinamic acid: This hydroxaminolysis reaction with succinamic acid was carried out as described by Krasnikov et al.9) with slight modification. The reaction mixture (50 µL) contained 20 mM succinamic acid, 5 mM DTT, 100 mM potassium phosphate buffer (pH 7.4), 100 mM hydroxylamine-HCl, and HEK293 cell S9 fraction (5 µg of protein), human liver S9 fraction (20 µg of protein), or mouse liver S9 fraction (20 µg of protein). Stock solutions of succinamic acid and hydroxylamine-HCl were previously neutralized with NaOH and stored at ¹20°C. The reaction was initiated by adding succinamic acid after a 5-min preincubation at 37°C. After incubation at 37°C for 30 min, 150 µL of a reagent containing 0.37 M FeCl3, 0.67 M HCl, and 0.2 M trichloroacetic acid was added. The absorbance of the succinyl hydroxamate ferric complex was determined at 535 nm using a SpectraMax M5 96-well plate spectrophotometer (Molecular Devices, Sunnyvale, CA). We used a value of 920 M¹1 cm¹1 as the extinction coefficient for the ferric complex of succinyl hydroxamate at 535 nm. Hydrolysis assays of the cyano group of vildagliptin: A typical incubaion mixture (200 µL of total volume) contained 50 mM Tris-HCl buffer, pH 7.4, 100 µM vildagliptin, and cell homogenate, human liver samples, or mouse liver samples. The reaction was initiated by adding vildagliptin after a 5-min preincubation at 37°C. After incubation at 37°C for 12 h, the reaction was terminated by an addition of 200 µL of cold acetonitrile. After removal of protein by centrifugation at 15,000 © g for 5 min, a 10-µL portion of the sample was subjected to liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure the concentration of M20.7. The enzymatic activity was expressed as an M20.7 formation rate (pmol/h/mg protein). LC-MS/MS conditions: The LC system was connected to a PE Sciex API 2000 tandem mass spectrometer (Applied Biosystems) operated in the positive electrospray ionization mode. M20.7 was separated on a Polaris 5 µm C18-A 50 © 2.0 mm column (20°C) (Agilent Technologies, Amstelveen, The Netherlands) with a MetaGuard 2.0 mm Polaris 5 µm C18-A guard column (Agilent Technologies). The mobile phase of A/B (1:3, v/v) was
used, where A was methanol/10 mM ammonium acetate, pH 8.0 (5:95, v/v), and B was acetonitrile/methanol (10:90, v/v). The flow rate was adjusted to 0.2 mL/min using an Agilent 1100 series pump (Agilent Technologies), and an eluent between 0 and 5 min was introduced into the mass spectrometer. The turbo gas was maintained at 550°C. Nitrogen was used as the nebulizing, turbo, and curtain gas at 50, 85, and 30 psi, respectively. Parent and/or fragment ions were filtered in the first quadrupole and dissociated in the collision cell using nitrogen as the collision gas. The collision energy was 25 V. The sample was analyzed during the multiple reaction monitoring (MRM) mode of the mass spectrometer using m/z 323.1 > 173.3 as the transition. The analytical data were processed using Analyst software (version 1.5.1; Applied Biosystems) in the API 2000 LC-MS/MS systems. Statistical analysis: Data were presented as means « S.D. and were assessed for statistical significance using the unpaired t-test. A value of p < 0.05 was considered statistically significant. Results Homology analysis for the similarity of the amino acid sequence between human NIT and the nitrilases of other organisms: The plant and bacterial nitrilases are typical nitrilases that exhibit nitrilase activities.10) Homology analysis of typical nitrilases or mouse Nit was performed using the BLASTP program. The BLASTP search was performed for typical nitrilases or mouse Nit against all human proteins. Table 1 shows the result of amino acid sequence homology analysis. Human NIT and typical nitrilases shared 25–31% sequence identity. Although amino acid sequence identity of typical nitrilases against human NIT was low, human NIT was the most similar to typical nitrilases among all human proteins. Mouse Nit1 shared 84% sequence identity to human NIT1. Mouse Nit2 shared 89% sequence identity to human NIT2, indicating that the amino acid sequences of human NIT1 and NIT2 were highly similar to mouse Nit1 and Nit2, respectively. Expression of Nit1 and Nit2 in mouse tissues: To assess the distribution of mouse Nit1 and Nit2 protein expressions, Western blot analysis was performed using mouse tissues. The molecular weights of Nit1 and Nit2 are approximately 36 kDa and 31 kDa, respectively. Mouse Nit1 was detected in all 8 tissues and was most abundant in the liver and kidney (Fig. 2A). Similarly, mouse Nit2 protein expression was most abundant in the liver and kidney. Thus, Nit1 and Nit2 are ubiquitously expressed proteins and are most abundant in the liver and kidney in mice. The two bands reacting with the anti-human NIT1 antibody were observed in mouse liver and kidney S9 fractions (Fig. 2A). To elucidate whether the two bands were different glycosylation forms of Nit, mouse liver S9 fraction was deglycosylated under native conditions and analyzed by Western blotting with the anti-human NIT1 antibody. Glycosidase treatment did not affect the size of the two bands in mouse liver S9 fraction (Fig. 2B). These results suggested that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not different glycosylation forms of Nit. Establishment of single expression systems of human NIT1 and NIT2 in HEK293 cells: To investigate whether human NIT1 and NIT2 are involved in hydrolysis of the cyano group of vildagliptin, stable single expression systems of human NIT1 and NIT2 were constructed in HEK293 cells. The protein expression levels of NIT were assessed by Western blot analysis. We selected the clones with the highest NIT protein levels for the subsequent
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Protein Nitrilase Nitrilase Nitrilase Nitrilase Nit1 Nit2 NIT1 NIT2
Source Bacteria Bradyrhizobium japonicum USDA 110 Pseudomonas putida Rhodococcus rhodochros Plant Arabidopsis thaliana Mammalia Mouse Mouse Human Human
Human NIT1 Score (bits) Identity
Human NIT2 Score (bits) Identity
Accession No.
Amino acids
BAC48662.1 WP_003257871.1 BAA01994.1
321 176 366
50.8 48.1 —
76/307 (25%) 34/108 (31%) —
51.2 57.4 70.1
72/287 (25%) 47/162 (29%) 78/313 (25%)
NP_851011
346
71.2
81/321 (26%)
83.6
81/298 (27%)
NP_036179 AAH20153 NP_005591.1 AAH20620.1
323 276 327 276
570 189 627 189
276/327 100/274 327/327 100/274
(84%) (36%) (100%) (36%)
191 529 189 573
106/281 (38%) 246/276 (89%) 100/274 (36%) 276/276 (100%)
The scores were obtained using the BLASTP program. The accession number of the protein is derived from the NCBI protein database.
Fig. 2. Characterization of Nit1 and Nit2 expression in mouse tissues and deglycosylation of mouse Nit1 in the liver S9 fraction (A) Mouse tissue S9 fraction (100 µg of protein) was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. (B) Mouse liver S9 fraction (100 µg) was treated with PNGase F, O-glycosidase, and neuraminidase under native conditions overnight at 37°C. The deglycosylated sample was subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody. Actin was used as the protein loading control. The positions of the molecular weight markers are indicated.
analyses. Significant NIT1 protein expression was observed in the cell homogenate of the human NIT1-transfectant compared with that of control vector-transfectant (Fig. 3). In addition, the human NIT2-transfectant also showed significant NIT2 protein expression compared with control vector-transfectant. These results demonstrated that stable single expression systems of human NIT1 and NIT2 in HEK293 cells were constructed. Protein expression of human NIT and mouse Nit in liver S9 fraction, cytosol, and microsomes: To determine whether human NIT1 and NIT2 are expressed in human liver, Western blot analysis was performed using human liver S9 fraction, cytosol, and microsomes. Additionally, to compare the protein expression patterns of human NIT and mouse Nit in the liver, Western blot analysis was also performed using mouse liver samples. Human NIT1 and NIT2 were highly detected in liver S9 fraction and cytosol (Fig. 4). Mouse Nit1 and Nit2 were also highly detected in liver S9 fraction and cytosol. Human NIT and mouse Nit were
slightly and moderately detected in liver microsomes, respectively. The protein expression pattern of human NIT1 and NIT2 in human liver fractions was almost consistent with that of mouse Nit1 and Nit2 in mouse liver fractions. These findings indicate that human NIT and mouse Nit are highly expressed in liver cytosol. Nit2/½-amidase-catalyzed hydrolysis of succinamic acid: The enzyme activity of Nit2 was measured in HEK293 cells expressing human NIT2, human liver S9 fraction, or mouse liver S9 fraction. Human NIT2 has been identified as an ½-amidase (½-amidodicarboxylate amidohydrolase, EC 3.5.1.3).11) Thus, the Nit2/½-amidase activity of each sample was assessed by 96-well plate assay for Nit2/½-amidase-catalyzed hydroxaminolysis of succinamic acid. The Nit2/½-amidase activity in Mock-transfected, human NIT1-expressing, and human NIT2-expressing HEK293 cells was 10.2 « 7.2, 11.1 « 2.4, and 250 « 4.4 nmol/min/mg protein, respectively (Fig. 5). The Nit2/½-amidase activity in HEK293 cells expressing human NIT2 was significantly higher than that in
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Mock-transfected HEK293 cells (p < 0.01). The Nit2/½-amidase activity in human and mouse liver S9 fractions was 104 « 0.6 and 103 « 4.2 nmol/min/mg protein, respectively. These findings indicated that functional Nit2 was expressed in HEK293 cells expressing human NIT2, human liver S9 fraction, and mouse liver S9 fraction. Hydrolysis assays of the cyano group of vildagliptin: To investigate whether human NIT1 and NIT2 are involved in hydrolysis of the cyano group of vildagliptin, M20.7 formation rates in HEK293 cells expressing human NIT1 and NIT2 were measured. Additionally, to assess the formation of M20.7 in human and mouse liver fractions, M20.7 formation rates in human and mouse liver samples were also measured. M20.7 formation rate in human liver S9 fraction, cytosol, and microsomes was 6.4 « 0.3, 10.5 « 0.3, and 11.9 « 0.1 pmol/h/mg protein, respectively (Fig. 6). M20.7 formation rate in human liver microsomes was statistically higher than that in the liver cytosol (p < 0.05). M20.7 formation rate in mouse liver S9 fraction, cytosol, and microsomes was 3.6 « 0.2, 3.3 « 0.2, and 4.6 « 0.1 pmol/h/mg protein, respectively. M20.7 formation rate in mouse liver microsomes was statistically higher than that in the liver cytosol (p < 0.01). M20.7 formation rates in human liver samples were approximately 2- to 3- fold higher than those in mouse liver samples, respectively. Although M20.7 formation rate in liver
Fig. 3. Western blot analysis of human NIT1 and NIT2 expression in stable cell lines The cell homogenates from the Mock-, NIT1-, and NIT2-transfected HEK293 cells (30 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. Actin was used as the protein loading control. The positions of the molecular weight markers are indicated.
microsomes was higher than that in liver S9 fraction and cytosol, the protein expression level of human NIT and mouse Nit in liver microsomes was lower than that in S9 fraction and cytosol (Figs. 4 and 6). Furthermore, the M20.7 formation rate in HEK293 cells expressing human NIT1 and NIT2 (0.4 « 0.2 and 0.3 « 0.1 pmol/h/mg protein, respectively) was comparable with that in the non-transfected HEK293 cells (0.3 « 0.2 pmol/h/mg protein). These results suggested that human NIT1 and NIT2 were not involved in the metabolism of vildagliptin. Discussion Mouse Nit1 and Nit2 were detected in most tissues by Western blot analysis and were particularly abundant in the liver and kidney. These results were consistent with previous reports.2,12) The two bands reacting with the anti-human NIT1 antibody were observed in mouse liver and kidney S9 fractions (Fig. 2A). We investigated whether these upper and lower bands were different
Fig. 5. Nit2/½-amidase activity in human NIT1- and NIT2-expressing HEK293 cells, human liver S9 fraction, and mouse liver S9 fraction The Nit2/½-amidase activity of HEK293 cell, human liver, or mouse liver S9 fractions was measured using a succinamic acid as a substrate. Data represent the means « S.D. of triplicate determinations. **p < 0.01.
Fig. 4. Western blot analysis of human NIT and mouse Nit expression in liver samples Human liver samples (100 µg of protein) and mouse liver samples (100 µg of protein) were subjected to NuPAGE 4–12% Bis-Tris Gel and probed with the anti-NIT1 antibody and anti-NIT2 antibody, respectively. GAPDH was used as the protein loading control. The positions of the molecular weight markers are indicated.
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Mitsutoshi ASAKURA, et al.
Fig. 6. Hydrolysis assays of the cyano group of vildagliptin The cyano group hydrolysis activity of HEK293 cells-expressing human NIT, human liver samples, and mouse liver samples was measured. The enzymatic activity was expressed as an M20.7 formation rate (pmol/h/mg protein). Data represent the means « S.D. of triplicate determinations. *p < 0.05; **p < 0.01.
glycosylation forms of Nit. The results of deglycosylation analysis suggested that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not different glycosylation forms of Nit (Fig. 2B). We then suspected that anti-human NIT1 antibody non-selectively reacted with mouse Nit2. We performed homology analysis of the epitope for antihuman NIT antibody, human NIT, and mouse Nit. The amino acid sequence identities of the epitope for anti-NIT1 antibody against mouse Nit1 and Nit2 were high (81%) and low (39%), respectively (Supplemental Table 1). Similarly, the epitope for anti-NIT1 antibody shared 100% (high) and 46% (low) sequence identity to human NIT1 and NIT2, respectively. Indeed, anti-NIT1 antibody did not react with the protein of human NIT2 in human NIT2expressing HEK293 cells (Fig. 3). Because these results demonstrated the selectivity of anti-NIT1 antibody, we believed that the two bands reacting with the anti-human NIT1 antibody in mouse liver and kidney S9 fractions were not mouse Nit2. It has been reported that two mouse Nit1 transcript variants, mouse Nit1 variant 1 (NCBI accession number NM_012049.2) and variant 2 (NM_001242580.1) exist. The reported amino acid sequence of mouse Nit1 variant 1 is longer than that of mouse Nit1 variant 2. These findings raise the possibility that the upper and lower bands reacting with the anti-human NIT1 antibody are mouse Nit1 variant 1 and variant 2, respectively. We believed that the densities of the upper and lower bands reacting with the anti-human NIT1 antibody were associated with the expression levels of mouse Nit1 variant 1 and variant 2 in mouse tissues. A previous report has described the liver as being the major site of vildagliptin metabolism.13) Distributions of Nit1 and Nit2 were consistent with a major site of metabolism of vildagliptin. In fact, the protein of human NIT1 and NIT2 was highly expressed in human liver cytosol (Fig. 4). Although amino acid sequence identity of typical nitrilases against human NIT was low, human NIT was the most similar to typical nitrilases among all human proteins (Table 1). Therefore, human NIT1 and NIT2 were expected to
be involved in the hydrolysis of the cyano group of vildagliptin, which is similar to nitrilase reaction. However, the result of measurement of M20.7 formation rate suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin. The exploration of major metabolic enzyme responsible for metabolism of vildagliptin needs to be conducted in the future. The enzyme activity of human NIT2-expressing HEK293 cells could be examined by 96-well plate assay for Nit2/½-amidasecatalyzed hydroxaminolysis of succinamic acid. However, the enzyme activity of human NIT1-expressing HEK293 cells could not be evaluated. This was because substrates of mammalian Nit1 have not been identified.14) We have not determined whether functional Nit1 is expressed in HEK293 cells expressing human NIT1. Therefore, we could not conclude that human NIT1 was not involved in the hydrolysis of the cyano group of vildagliptin. Although the result of measurement of M20.7 formation rate suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin, human NIT may be involved in the metabolism of other drugs with the cyano group. Our current study examined the involvement of human NIT in drug metabolism for the first time. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. In conclusion, human NIT1 and NIT2 were highly expressed in human liver cytosol, which is the fraction in which vildagliptin is expected to be metabolized. Although human NIT1 and NIT2 were expected to be involved in the hydrolysis of the cyano group of vildagliptin, the result of the measurement of the M20.7 formation rate in HEK293 cells expressing human NIT1 or NIT2 suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism. References 1) 2) 3)
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Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Human NIT Does Not Catalyze the Hydrolysis of Vildagliptin
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(2003). Chien, C. H., Gao, Q. Z., Cooper, A. J., Lyu, J. H. and Sheu, S. Y.: Structural insights into the catalytic active site and activity of human Nit2/½-amidase: KINETIC ASSAY AND MOLECULAR DYNAMICS SIMULATION. J. Biol. Chem., 287: 25715–25726 (2012). Semba, S., Han, S. Y., Qin, H. R., McCorkell, K. A., Iliopoulos, D., Pekarsky, Y., Druck, T., Trapasso, F., Croce, C. M., et al.: Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor. J. Biol. Chem., 281:
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