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A Potent Inhibitor of Inducible Nitric Oxide Synthase, ONO-1714, a Cyclic Amidine Derivative
Biochemical and Biophysical Research Communications 270, 663– 667 (2000) doi:10.1006/bbrc.2000.2474, available online at http://www.idealibrary.com on
A Potent Inhibitor of Inducible Nitric Oxide Synthase, ONO-1714, a Cyclic Amidine Derivative Masao Naka,* Toshiyuki Nanbu,* Kaoru Kobayashi,* Yoshihisa Kamanaka,* Masaharu Komeno,* Ryo Yanase,* Tatsushi Fukutomi,* Shinsei Fujimura,* Han Geuk Seo,* Noriko Fujiwara,† Shuichi Ohuchida,* Keiichiro Suzuki,† Kigen Kondo,* and Naoyuki Taniguchi† ,1 *Minase Research Institute, Ono Pharmaceutical Company, Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan; and †Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received March 15, 2000
(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K i of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID 50 value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent. © 2000 Academic Press
An inducible type of nitric oxide synthase can be induced in variety of cells (1–3), including macrophage, by various stimuli including lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF␣), and interferon-␥ (IFN-␥). The overproduction of nitric oxide from iNOS could be an important contributor to circulatory failure, such as hypotension and vascular hyporeactivity to vasoconstrictor agents (4 –7). Moreover, iNOS plays a role in the pathogenesis of variety of other diseases, including osteoarthritis (8, 9), diabetes (10, 11) and Parkinson disease (12, 13). Nonselective inhibition of NO formation could lead to side effects, since this could lead to the inhibition of the 1
To whom correspondence should be addressed. Fax: 81-668793429. E-mail: [email protected].
constitutive isoforms of NOS, which is responsible for a number of physiological actions of NO. Based on this scenario, selective inhibitors of iNOS may well have considerable therapeutic potential, particularly if they do not interfere with the protective and physiological roles of ecNOS. Therefore, a considerable effort has been made to develop a novel iNOS inhibitor which would be useful as a biological tool and, more importantly, as a possible therapeutic agent for use in the treatment of various diseases. Some L-arginine analogues show a limited isoform selectivity in terms of the inhibition of NOS (14, 15). Aminoguanidine is a relatively selective inhibitor of iNOS, but its inhibitory activity is very weak (16, 17). Here we report a novel and potent iNOS inhibitor, ONO-1714, which has potent inhibitory activity, both in vitro and in vivo. MATERIALS AND METHODS Materials. ONO-1714, the structure of which is shown in Fig. 1, was synthesized at the Minase Research Institute, Ono Pharmaceutical Co., Ltd. N G-Monomethyl-L-arginine monoacetate (L-NMMA) and aminoguanidine hemisulfate salt (AG) were purchased from Alexis and Sigma, respectively. All other chemicals and regents were from Sigma, Amersham, Dow Chemical, Genzyme, and R&D System unless otherwise indicated. Cell culture reagents were from Sigma, Becton Dickinson and Invitrogen. Cell culture. RAW264.7 and A549 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Culture of Spodoptera frugiperda (Sf21) cells and the manipulation of baculovirus were carried out according to Piwnica-Worms (18). Sf21 cells were maintained in Grace’s medium (Invitrogen B823-01) containing 10% fetal calf serum, 3.3 g/ml yeastolate, 3.3 g/ml lactoalbumin hydrolysate, and 10 g/ml gentamycin at 27°C. Cellular assay. To assess the potency of iNOS inhibition in rodent cell line, cultured RAW264.7 cells were stimulated with 10 ng/mL of LPS in the presence or absence of test compounds. The nitrite plus nitrate (NOx) concentration in the cultured medium at 24 h after the LPS stimulation was determined.
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 270, No. 2, 2000
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FIG. 1. Structure of ONO-1714.
Preparation of partially purified enzyme and determination of K i value. Human ecNOS was overexpressed in Sf-21 cells which had been infected with baculovirus carrying human ecNOS cDNA (19) and human iNOS was overexpressed in A549 by stimulation with LPS (10 g/mL) plus cytokines (10 ng/mL of TNF-␣, 5 ng/mL of IL-1 and 100 ng/mL of interferon-␥). Human ecNOS and iNOS were partially purified by chromatography on 2⬘, 5⬘-ADP-Sepharose gel. NOS activity was determined as the conversion of [ 14C]-L-arginine to L-citrulline (20) with a minor modification (21). The conversion rate for various concentrations of test compounds and L-arginine was measured, and Dixon and Lineweaver–Burk plots were constructed to determine the K i value and the mode of inhibition. Enzyme assay with recombinant mouse iNOS. Recombinant mouse iNOS was purchased from Cayman Chemical (Cat. No. 60862) and inhibitory activities of test compounds were determined by measuring the conversion rate from [ 14C]-L-arginine to [ 14C]-L-citrulline (21), and the IC 50 value was determined. Inhibition of nitrate/nitrite (NOx) accumulation and the maximum tolerated dose (MTD) in mice. Three hours after LPS (10 mg/kg, i.v.) injection into 7 weeks old Balb/c mice (Charles River Japan, Inc.), test compound or saline was administered subcutaneously. Blood was collected by venipuncture from the abdominal aorta under light anesthesia at 6 h after LPS treatment. Plasma was obtained by centrifugation and the accumulated NOx concentration for 3 h was determined by the method described below. To evaluate the acute toxicity, the MTD (i.v. maximum dose where no death was observed within 24 h after the administration) of the test compound was determined. Measurement of nitrite/nitrate. Nitrite and nitrate, the oxidized form of nitric oxide that accumulated in the culture medium and plasma were determined by the use of nitrite/nitrate colorimetric assay kit (Cayman Chemical, Cat. No. 780001). Basically, the nitrate in the sample was reduced to nitrite with a nitrate reductase contained in the assay kit; nitrite levels were then determined spectrophotometrically as the total NOx concentration.
by an analysis of Lineweaver–Burk plot (Fig. 3). The K i values of L-NMMA and AG for human iNOS were 847 nM and 39.9 M, and the K i values of both compounds for human ecNOS were 250 nM and 190 M, respectively (Table 1). These data indicate that ONO-1714 is a 451-fold more potent inhibitor than L-NMMA and ⬎20,000-fold more potent than AG, in terms of inhibiting human iNOS. In a comparison of the inhibitory activities on iNOS and ecNOS, ONO-1714 was 10-fold selective for human iNOS over human ecNOS. The rank order of potency for the inhibition of ecNOS was ONO-1714 ⬎ L-NMMA ⬎ AG. However, the iNOS selectivity of ONO-1714 was about 34-fold higher than L-NMMA and about 2-fold higher than AG, based on the relative iNOS selectivity of ONO-1714 when the ratio of L-NMMA was set as 1 (Table 1). In enzyme assay with recombinant mouse iNOS, ONO-1714 inhibited the conversion of [ 14C]-L-arginine to [ 14C]-L-citrulline with an IC 50 value of 4.0 nM and the IC 50 values of L-NMMA and AG were 3.5 M and 19.6 M, respectively. These data indicate that ONO1714 is 875-fold more potent than L-NMMA and more than 4,900-fold potent than AG (Table 2). In order to assess the potency of ONO-1714 for inhibiting iNOS in vivo, mice were administered with LPS, and ONO-1714 was treated subcutaneously 3 h after LPS injection. Plasma NOx accumulation from 3 h to 6 h after LPS injection was then determined. As shown in Fig. 4, ONO-1714 inhibited NOx accumulation in plasma with a dose-dependent manner, and the ID 50 values were 0.010 mg/kg, s.c. (Table 3). The similar type of inhibition was observed in the case of L-NMMA treatment (Fig. 4), wherein the ID 50 value of L-NMMA was 26 mg/kg, s.c. (Table 3). In a comparison of the potency of inhibiting NOx accumulation in mice, ONO-1714 was 2,600-fold more potent than L-NMMA.
RESULTS ONO-1714 inhibited nitrite accumulation, an indicator of NO production, in RAW264.7 cells stimulated with 10 ng/mL of LPS for 24 h and an IC 50 value was 20 ⫾ 3 nM (Fig. 2). L-NMMA also inhibited nitrite production with an IC 50 value was 44 ⫾ 4 M, indicating that ONO-1714 is 2,200-fold more potent than L-NMMA. ONO-1714 inhibited a conversion of [ 14C]-L-arginine to [ 14C]-L-citrulline by human NOS, and the K i values were 1.88 nM for human iNOS and 18.8 nM for human ecNOS, as determined from Dixon plot analysis (Fig. 3, Table 1). The mode of enzyme inhibition of ONO-1714 was competitive in both iNOS and ecNOS, as evidenced
FIG. 2. Inhibitory activities of ONO-1714 and L-NMMA relative to nitrite production in LPS-stimulated RAW264.7 cells. Cultured RAW264.7 cells were stimulated with 10 ng/ml of LPS in the presence or absence of test compounds. The nitrite concentration in the cultured medium at 24 h after the LPS stimulation was determined by the Greiss method. Each column represents the mean ⫾ SE of three experiments.
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FIG. 3. Representative Dixon and Lineweaver–Burk plots of ONO-1714 on partially purified human iNOS and ecNOS. Dixon plots: (A) human iNOS and (C) human ecNOS. Lineweaver–Burk plots: (B) human iNOS and (D) human ecNOS. S, concentration of substrate (L-arginine); V, velocity (pmol/min).
To assess the acute toxicity, MTD values were determined. The MTD of ONO-1714 and L-NMMA when given a single intravenous dose to normal mice were 30 mg/kg and 3,000 mg/kg, respectively. The MTD of ONO-1714 was much lower than that of L-NMMA, but the ratio of the MTD/ID 50 of NOx accumulation in mice were 3,000 for ONO-1714 and 115 for L-NMMA (Table 3). DISCUSSION The K i value of ONO-1714 for human iNOS was 1.88 nM. This very low K i value indicates that ONO-1714 is
one of the most potent iNOS inhibitors in vitro discovered to date (22–24). This potent iNOS inhibition of ONO-1714 can be supported by three dimensional docking analysis of this compound with human iNOS protein (PDB code. 2NSI) (data not shown). When compared to the inhibitory activity of ONO-1714 on human iNOS vs. human ecNOS, ONO-1714 is 10-fold more selective for iNOS than for ecNOS. A non-selective inhibitor, L-NMMA, showed K i values of 847 nM for human iNOS and 250 nM for human ecNOS, indicating that L-NMMA is more selective (⬇3.4-fold) for ecNOS than for iNOS as reported previously (25). In contrast, the potency of AG for inhibiting NOS isozymes was
TABLE 1
Inhibitory Activities of NOS Inhibitors on Partially Purified Human iNOS and ecNOS K i values
ONO-1714 L-NMMA Aminoguanidine
iNOS
ecNOS
iNOS selectivity
RIS a (L-NMMA ⫽ 1)
1.88 ⫾ 0.05 (nM) 847 ⫾ 92 (nM) 39.9 ⫾ 2.1 (M)
18.8 ⫾ 1.7 (nM) 250 ⫾ 15 (nM) 190 ⫾ 13 (M)
10 0.295 4.76
33.9 1 16.1
Note. Each K i value was determined from three separate experiments and is expressed as the mean ⫾ SE. iNOS selectivity is calculated by the formulation; K i of ecNOS/K i of iNOS. a RIS, relative iNOS selectivity represents the iNOS selectivity of ONO-1714 and AG when the iNOS selectivity of L-NMMA was set as 1. 665
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
TABLE 3
Comparison of Inhibitory Activities of NOS Inhibitors on Mouse iNOS
ID 50 Values of ONO-1714 and L-NMMA on NOx Production and Their Maximum Tolerated Dose (MTD) in Mice
IC 50 values (M)
NOx
ONO-1714
L-NMMA
Aminoguanidine
Compound
ID 50 (mg/kg, s.c.)
MTD (mg/kg, i.v.)
MTD/NOx
0.0040 (875)
3.5 (1)
19.6 (0.18)
ONO-1714 L-NMMA
0.010 26
30 3000
3000 115
Note. Each value represents the mean of two separate experiments. The conversion of [ 14C]-L-arginine (1.68 M) to [ 14C]-Lcitrulline by recombinant iNOS for 10 min was determined. The number in the parentheses indicates the relative activity when the inhibitory activity of L-NMMA is set as 1.
very low (K i; 39.9 M for iNOS, and 190 M for ecNOS), but AG is a fairly selective (4.8-fold) inhibitor for iNOS. These data indicate that of the three compounds tested ONO-1714 is the most potent and selective inhibitor for iNOS in vitro. In an in vivo study, ONO-1714 has also been proved to be a very potent iNOS inhibitor, based on its inhibitory activity on NOx accumulation in mice, which had been treated with LPS, and the ID 50 value was 0.010 mg/kg, s.c. In contrast, the ID 50 value of L-NMMA on the accumulation of NOx in LPS-treated mice was 26 mg/kg, s.c., indicating that ONO-1714 is a 2600-fold more potent than L-NMMA in vivo. This difference in in vivo potencies between ONO-1714 and L-NMMA corresponds to the difference (2,200-fold) in potencies of both compounds for inhibiting nitrite production from LPS-stimulated RAW264.7 cells. However, the enzyme assay with recombinant mouse iNOS showed that ONO-1714 was 875-fold more potent than L-NMMA, indicating that the inhibitory activities of ONO-1714 were more dominant than those of L-NMMA in the cellular assay with RAW264.7 as well
Note. The ID 50 value was determined from a log-logit transformation of the dose–response curves (ONO-1714; 3, 10 and 30 g/kg, s.c. L-NMMA; 10, 30, and 100 mg/kg, s.c.). The ID 50 value was defined as the dose of test compound that produced a 50% inhibition on NOx accumulation by LPS treatment. The MTD was defined as the maximum dose where no death was observed within 24 h after intravenous administration. The doses used were 10, 20, 30, 40, and 50 mg/kg for ONO-1714, and 1000, 2000, 3000, 4000, and 5000 mg/kg for L-NMMA.
as the in vivo experiment in mice. These results suggest that ONO-1714 is capable of being more accessible to the intracellular portions than L-NMMA because of the non L-arginine structure of the molecule. This is in direct contrast to L-NMMA. When given in the form of a single intravenous dose to normal mice, ONO-1714 and L-NMMA were tolerated up to 30 mg/kg and 3,000 mg/kg, respectively. This indicates that the MTD of ONO-1714 is much lower than that of L-NMMA. However, the window of ONO-1714 between biological activity (the inhibition of NOx accumulation in LPS-treated mice) and toxicity (the MTD value for mice) was 3000. This appears to be significant, compared to the corresponding values (⫽115) for L-NMMA, although a true therapeutic index cannot be assigned at present, because of the different mode of administration. ONO-1714 represents a potentially beneficial tool for clarifying the role of iNOS in disease states and may well have considerable therapeutic potential as a novel drug. ACKNOWLEDGMENT The authors thank Dr. Nishizaki for the docking analysis.
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FIG. 4. Inhibitory activities of ONO-1714 and L-NMMA on NOx accumulation in LPS-treated mice. After the administration of 10 mg/kg of LPS, an increase of NOx concentration in plasma was observed as function of time. Test compounds were administered s.c. at 3 h after LPS injection, after which, the accumulation of NOx in plasma for 3 h was determined. Each column represents the mean ⫾ SE of 5 animals. 666
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A Potent Inhibitor of Inducible Nitric Oxide Synthase, ONO-1714, a Cyclic Amidine Derivative Masao Naka,* Toshiyuki Nanbu,* Kaoru Kobayashi,* Yoshihisa Kamanaka,* Masaharu Komeno,* Ryo Yanase,* Tatsushi Fukutomi,* Shinsei Fujimura,* Han Geuk Seo,* Noriko Fujiwara,† Shuichi Ohuchida,* Keiichiro Suzuki,† Kigen Kondo,* and Naoyuki Taniguchi† ,1 *Minase Research Institute, Ono Pharmaceutical Company, Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan; and †Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received March 15, 2000
(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K i of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID 50 value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent. © 2000 Academic Press
An inducible type of nitric oxide synthase can be induced in variety of cells (1–3), including macrophage, by various stimuli including lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF␣), and interferon-␥ (IFN-␥). The overproduction of nitric oxide from iNOS could be an important contributor to circulatory failure, such as hypotension and vascular hyporeactivity to vasoconstrictor agents (4 –7). Moreover, iNOS plays a role in the pathogenesis of variety of other diseases, including osteoarthritis (8, 9), diabetes (10, 11) and Parkinson disease (12, 13). Nonselective inhibition of NO formation could lead to side effects, since this could lead to the inhibition of the 1
To whom correspondence should be addressed. Fax: 81-668793429. E-mail: [email protected].
constitutive isoforms of NOS, which is responsible for a number of physiological actions of NO. Based on this scenario, selective inhibitors of iNOS may well have considerable therapeutic potential, particularly if they do not interfere with the protective and physiological roles of ecNOS. Therefore, a considerable effort has been made to develop a novel iNOS inhibitor which would be useful as a biological tool and, more importantly, as a possible therapeutic agent for use in the treatment of various diseases. Some L-arginine analogues show a limited isoform selectivity in terms of the inhibition of NOS (14, 15). Aminoguanidine is a relatively selective inhibitor of iNOS, but its inhibitory activity is very weak (16, 17). Here we report a novel and potent iNOS inhibitor, ONO-1714, which has potent inhibitory activity, both in vitro and in vivo. MATERIALS AND METHODS Materials. ONO-1714, the structure of which is shown in Fig. 1, was synthesized at the Minase Research Institute, Ono Pharmaceutical Co., Ltd. N G-Monomethyl-L-arginine monoacetate (L-NMMA) and aminoguanidine hemisulfate salt (AG) were purchased from Alexis and Sigma, respectively. All other chemicals and regents were from Sigma, Amersham, Dow Chemical, Genzyme, and R&D System unless otherwise indicated. Cell culture reagents were from Sigma, Becton Dickinson and Invitrogen. Cell culture. RAW264.7 and A549 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Culture of Spodoptera frugiperda (Sf21) cells and the manipulation of baculovirus were carried out according to Piwnica-Worms (18). Sf21 cells were maintained in Grace’s medium (Invitrogen B823-01) containing 10% fetal calf serum, 3.3 g/ml yeastolate, 3.3 g/ml lactoalbumin hydrolysate, and 10 g/ml gentamycin at 27°C. Cellular assay. To assess the potency of iNOS inhibition in rodent cell line, cultured RAW264.7 cells were stimulated with 10 ng/mL of LPS in the presence or absence of test compounds. The nitrite plus nitrate (NOx) concentration in the cultured medium at 24 h after the LPS stimulation was determined.
663
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 270, No. 2, 2000
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FIG. 1. Structure of ONO-1714.
Preparation of partially purified enzyme and determination of K i value. Human ecNOS was overexpressed in Sf-21 cells which had been infected with baculovirus carrying human ecNOS cDNA (19) and human iNOS was overexpressed in A549 by stimulation with LPS (10 g/mL) plus cytokines (10 ng/mL of TNF-␣, 5 ng/mL of IL-1 and 100 ng/mL of interferon-␥). Human ecNOS and iNOS were partially purified by chromatography on 2⬘, 5⬘-ADP-Sepharose gel. NOS activity was determined as the conversion of [ 14C]-L-arginine to L-citrulline (20) with a minor modification (21). The conversion rate for various concentrations of test compounds and L-arginine was measured, and Dixon and Lineweaver–Burk plots were constructed to determine the K i value and the mode of inhibition. Enzyme assay with recombinant mouse iNOS. Recombinant mouse iNOS was purchased from Cayman Chemical (Cat. No. 60862) and inhibitory activities of test compounds were determined by measuring the conversion rate from [ 14C]-L-arginine to [ 14C]-L-citrulline (21), and the IC 50 value was determined. Inhibition of nitrate/nitrite (NOx) accumulation and the maximum tolerated dose (MTD) in mice. Three hours after LPS (10 mg/kg, i.v.) injection into 7 weeks old Balb/c mice (Charles River Japan, Inc.), test compound or saline was administered subcutaneously. Blood was collected by venipuncture from the abdominal aorta under light anesthesia at 6 h after LPS treatment. Plasma was obtained by centrifugation and the accumulated NOx concentration for 3 h was determined by the method described below. To evaluate the acute toxicity, the MTD (i.v. maximum dose where no death was observed within 24 h after the administration) of the test compound was determined. Measurement of nitrite/nitrate. Nitrite and nitrate, the oxidized form of nitric oxide that accumulated in the culture medium and plasma were determined by the use of nitrite/nitrate colorimetric assay kit (Cayman Chemical, Cat. No. 780001). Basically, the nitrate in the sample was reduced to nitrite with a nitrate reductase contained in the assay kit; nitrite levels were then determined spectrophotometrically as the total NOx concentration.
by an analysis of Lineweaver–Burk plot (Fig. 3). The K i values of L-NMMA and AG for human iNOS were 847 nM and 39.9 M, and the K i values of both compounds for human ecNOS were 250 nM and 190 M, respectively (Table 1). These data indicate that ONO-1714 is a 451-fold more potent inhibitor than L-NMMA and ⬎20,000-fold more potent than AG, in terms of inhibiting human iNOS. In a comparison of the inhibitory activities on iNOS and ecNOS, ONO-1714 was 10-fold selective for human iNOS over human ecNOS. The rank order of potency for the inhibition of ecNOS was ONO-1714 ⬎ L-NMMA ⬎ AG. However, the iNOS selectivity of ONO-1714 was about 34-fold higher than L-NMMA and about 2-fold higher than AG, based on the relative iNOS selectivity of ONO-1714 when the ratio of L-NMMA was set as 1 (Table 1). In enzyme assay with recombinant mouse iNOS, ONO-1714 inhibited the conversion of [ 14C]-L-arginine to [ 14C]-L-citrulline with an IC 50 value of 4.0 nM and the IC 50 values of L-NMMA and AG were 3.5 M and 19.6 M, respectively. These data indicate that ONO1714 is 875-fold more potent than L-NMMA and more than 4,900-fold potent than AG (Table 2). In order to assess the potency of ONO-1714 for inhibiting iNOS in vivo, mice were administered with LPS, and ONO-1714 was treated subcutaneously 3 h after LPS injection. Plasma NOx accumulation from 3 h to 6 h after LPS injection was then determined. As shown in Fig. 4, ONO-1714 inhibited NOx accumulation in plasma with a dose-dependent manner, and the ID 50 values were 0.010 mg/kg, s.c. (Table 3). The similar type of inhibition was observed in the case of L-NMMA treatment (Fig. 4), wherein the ID 50 value of L-NMMA was 26 mg/kg, s.c. (Table 3). In a comparison of the potency of inhibiting NOx accumulation in mice, ONO-1714 was 2,600-fold more potent than L-NMMA.
RESULTS ONO-1714 inhibited nitrite accumulation, an indicator of NO production, in RAW264.7 cells stimulated with 10 ng/mL of LPS for 24 h and an IC 50 value was 20 ⫾ 3 nM (Fig. 2). L-NMMA also inhibited nitrite production with an IC 50 value was 44 ⫾ 4 M, indicating that ONO-1714 is 2,200-fold more potent than L-NMMA. ONO-1714 inhibited a conversion of [ 14C]-L-arginine to [ 14C]-L-citrulline by human NOS, and the K i values were 1.88 nM for human iNOS and 18.8 nM for human ecNOS, as determined from Dixon plot analysis (Fig. 3, Table 1). The mode of enzyme inhibition of ONO-1714 was competitive in both iNOS and ecNOS, as evidenced
FIG. 2. Inhibitory activities of ONO-1714 and L-NMMA relative to nitrite production in LPS-stimulated RAW264.7 cells. Cultured RAW264.7 cells were stimulated with 10 ng/ml of LPS in the presence or absence of test compounds. The nitrite concentration in the cultured medium at 24 h after the LPS stimulation was determined by the Greiss method. Each column represents the mean ⫾ SE of three experiments.
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FIG. 3. Representative Dixon and Lineweaver–Burk plots of ONO-1714 on partially purified human iNOS and ecNOS. Dixon plots: (A) human iNOS and (C) human ecNOS. Lineweaver–Burk plots: (B) human iNOS and (D) human ecNOS. S, concentration of substrate (L-arginine); V, velocity (pmol/min).
To assess the acute toxicity, MTD values were determined. The MTD of ONO-1714 and L-NMMA when given a single intravenous dose to normal mice were 30 mg/kg and 3,000 mg/kg, respectively. The MTD of ONO-1714 was much lower than that of L-NMMA, but the ratio of the MTD/ID 50 of NOx accumulation in mice were 3,000 for ONO-1714 and 115 for L-NMMA (Table 3). DISCUSSION The K i value of ONO-1714 for human iNOS was 1.88 nM. This very low K i value indicates that ONO-1714 is
one of the most potent iNOS inhibitors in vitro discovered to date (22–24). This potent iNOS inhibition of ONO-1714 can be supported by three dimensional docking analysis of this compound with human iNOS protein (PDB code. 2NSI) (data not shown). When compared to the inhibitory activity of ONO-1714 on human iNOS vs. human ecNOS, ONO-1714 is 10-fold more selective for iNOS than for ecNOS. A non-selective inhibitor, L-NMMA, showed K i values of 847 nM for human iNOS and 250 nM for human ecNOS, indicating that L-NMMA is more selective (⬇3.4-fold) for ecNOS than for iNOS as reported previously (25). In contrast, the potency of AG for inhibiting NOS isozymes was
TABLE 1
Inhibitory Activities of NOS Inhibitors on Partially Purified Human iNOS and ecNOS K i values
ONO-1714 L-NMMA Aminoguanidine
iNOS
ecNOS
iNOS selectivity
RIS a (L-NMMA ⫽ 1)
1.88 ⫾ 0.05 (nM) 847 ⫾ 92 (nM) 39.9 ⫾ 2.1 (M)
18.8 ⫾ 1.7 (nM) 250 ⫾ 15 (nM) 190 ⫾ 13 (M)
10 0.295 4.76
33.9 1 16.1
Note. Each K i value was determined from three separate experiments and is expressed as the mean ⫾ SE. iNOS selectivity is calculated by the formulation; K i of ecNOS/K i of iNOS. a RIS, relative iNOS selectivity represents the iNOS selectivity of ONO-1714 and AG when the iNOS selectivity of L-NMMA was set as 1. 665
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
TABLE 3
Comparison of Inhibitory Activities of NOS Inhibitors on Mouse iNOS
ID 50 Values of ONO-1714 and L-NMMA on NOx Production and Their Maximum Tolerated Dose (MTD) in Mice
IC 50 values (M)
NOx
ONO-1714
L-NMMA
Aminoguanidine
Compound
ID 50 (mg/kg, s.c.)
MTD (mg/kg, i.v.)
MTD/NOx
0.0040 (875)
3.5 (1)
19.6 (0.18)
ONO-1714 L-NMMA
0.010 26
30 3000
3000 115
Note. Each value represents the mean of two separate experiments. The conversion of [ 14C]-L-arginine (1.68 M) to [ 14C]-Lcitrulline by recombinant iNOS for 10 min was determined. The number in the parentheses indicates the relative activity when the inhibitory activity of L-NMMA is set as 1.
very low (K i; 39.9 M for iNOS, and 190 M for ecNOS), but AG is a fairly selective (4.8-fold) inhibitor for iNOS. These data indicate that of the three compounds tested ONO-1714 is the most potent and selective inhibitor for iNOS in vitro. In an in vivo study, ONO-1714 has also been proved to be a very potent iNOS inhibitor, based on its inhibitory activity on NOx accumulation in mice, which had been treated with LPS, and the ID 50 value was 0.010 mg/kg, s.c. In contrast, the ID 50 value of L-NMMA on the accumulation of NOx in LPS-treated mice was 26 mg/kg, s.c., indicating that ONO-1714 is a 2600-fold more potent than L-NMMA in vivo. This difference in in vivo potencies between ONO-1714 and L-NMMA corresponds to the difference (2,200-fold) in potencies of both compounds for inhibiting nitrite production from LPS-stimulated RAW264.7 cells. However, the enzyme assay with recombinant mouse iNOS showed that ONO-1714 was 875-fold more potent than L-NMMA, indicating that the inhibitory activities of ONO-1714 were more dominant than those of L-NMMA in the cellular assay with RAW264.7 as well
Note. The ID 50 value was determined from a log-logit transformation of the dose–response curves (ONO-1714; 3, 10 and 30 g/kg, s.c. L-NMMA; 10, 30, and 100 mg/kg, s.c.). The ID 50 value was defined as the dose of test compound that produced a 50% inhibition on NOx accumulation by LPS treatment. The MTD was defined as the maximum dose where no death was observed within 24 h after intravenous administration. The doses used were 10, 20, 30, 40, and 50 mg/kg for ONO-1714, and 1000, 2000, 3000, 4000, and 5000 mg/kg for L-NMMA.
as the in vivo experiment in mice. These results suggest that ONO-1714 is capable of being more accessible to the intracellular portions than L-NMMA because of the non L-arginine structure of the molecule. This is in direct contrast to L-NMMA. When given in the form of a single intravenous dose to normal mice, ONO-1714 and L-NMMA were tolerated up to 30 mg/kg and 3,000 mg/kg, respectively. This indicates that the MTD of ONO-1714 is much lower than that of L-NMMA. However, the window of ONO-1714 between biological activity (the inhibition of NOx accumulation in LPS-treated mice) and toxicity (the MTD value for mice) was 3000. This appears to be significant, compared to the corresponding values (⫽115) for L-NMMA, although a true therapeutic index cannot be assigned at present, because of the different mode of administration. ONO-1714 represents a potentially beneficial tool for clarifying the role of iNOS in disease states and may well have considerable therapeutic potential as a novel drug. ACKNOWLEDGMENT The authors thank Dr. Nishizaki for the docking analysis.
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FIG. 4. Inhibitory activities of ONO-1714 and L-NMMA on NOx accumulation in LPS-treated mice. After the administration of 10 mg/kg of LPS, an increase of NOx concentration in plasma was observed as function of time. Test compounds were administered s.c. at 3 h after LPS injection, after which, the accumulation of NOx in plasma for 3 h was determined. Each column represents the mean ⫾ SE of 5 animals. 666
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