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Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy.
Stroke is a major cause of death and disability worldwide (1). Improving the case fatality rates and long term disability after stroke continues to be a challenge (2). During the past decade thrombolytic enzyme tissue plasminogen activator-based treatment has been the standard therapy for acute ischemic stroke (2). However, due to its risk of parenchymal hemorrhage and a limited therapeutic time window (3,–5), only a small fraction of patients (1.8–8.9%) benefits from tissue plasminogen activator-based therapy (6, 7). The development of an alternative therapeutic agent is urgently needed. In this context, suppressing inflammation within the infarction area to rescue penumbral tissue and reduce hemorrhagic transformation is particularly important (8, 9).
SMTPs3 (named after Stachybotrys microspora triprenyl phenols) are a family of novel small molecules produced by the fungus Stachybotrys microspora (10,–12). SMTP-7 (Fig. 1A), one of the SMTP family compounds with profound biological activities, enhances plasminogen activation by modulating plasminogen conformation (10, 13,–15). SMTP-7 thus promotes plasmin formation and clot clearance in vivo (14, 16), and it has been used to treat thrombotic and embolic strokes in experimental models in rodents and nonhuman primates (17,–20). Unexpectedly, SMTP-7 was demonstrated to reduce hemorrhagic transformation and to have a wide therapeutic time window (17, 18, 20). These excellent activities are partly explained by the finding that, unlike tissue plasminogen activator, SMTP-7 suppresses inflammatory/oxidative responses after thrombolytic reperfusion (16, 18, 19, 21). These beneficial properties prompted the development of SMTP-7 as an alternative stroke drug that can treat patients who do not benefit from tissue plasminogen activator-based therapy (20).
The aim of the present study was to explore the anti-inflammatory mechanism of SMTP-7. We first compared the efficacy of two SMTP congeners differing in the potential of thrombolytic activity using three inflammatory disease animal models. The results demonstrate that the anti-inflammatory action of SMTP is independent of its thrombolytic activity. We next searched for an anti-inflammatory target using SMTP affinity beads resulting in the identification of soluble epoxide hydrolase (sEH). sEH is a bifunctional enzyme with an epoxide hydrolase activity at the C-terminal domain (Cterm-EH) and a lipid phosphate phosphatase activity at the N-terminal domain (Nterm-phos) (22). The Cterm-EH catalyzes the hydrolysis of epoxy fatty acids such as epoxyeicosatrienoic acids (EETs), which are potent endogenous signaling molecules implicated in anti-inflammation, vascular dilation, endothelial cell hyperpolarization, angiogenesis, neuroprotection, and analgesia (antihyperalgesia) (23,–27). The Nterm-phos hydrolyzes lipid phosphates such as lysophosphatidic acid and intermediates of the cholesterol biosynthesis (28,–30). SMTP-7 inhibited both of the two activities of sEH: competitively for Cterm-EH and pseudo-noncompetitively for Nterm-phos. The treatment of HepG2 cells with of SMTP-7 or its congeners suppressed the hydrolysis of EET to dihydroxyeicosatrienoic acid (DHET). Here, we describe the anti-inflammatory action of SMTP-7 and its mechanism.
All of the animal protocols were approved by the institutional animal experiment committees at the Tokyo Noko University and Nihon Pharmaceutical. Male C57BL/6J mice (7 weeks old; Japan SLC, Hamamatsu) and male Lewis rats (6 weeks old; Charles River Laboratories Japan, Yokohama) were used after 1 week of preliminary rearing for the inflammatory disease models. Retired male ICR mice (Japan SLC, Hamamatsu) were used to obtain the livers for the sEH purification.
On day 0, male Lewis rats (7 weeks old) were given 0.1 ml of a 1:1 mixture of the synthetic P2 peptide (35) (H-TESPFKNTEISFKLGQEFEETTADNR-OH corresponding to Thr-53–Arg-78 of bovine P2 protein; 2.5 mg ml−1 in saline) and Freund's incomplete adjuvant containing Mycobacterium tuberculosis H37 Ra (Difco) by injection into the foot pads of both of the hind limbs. Sodium salts of SMTP-7 and SMTP-44D dissolved in 5% (wt vol−1) mannitol was intraperitoneally injected at a dose of 10 mg kg−1 through days 7–16. As a standard, sulfonated immunoglobulin formulation (Kaketsuken, Kumamoto, Japan) was given intravenously at a dose of 400 mg kg−1 on days 0, 7, 14, 15, and 16. Control animals received no drug treatment. Normal animals received neither P2 peptide nor any drug. The experimental autoimmune neuritis score (score 0, normal; 0.5, reduced tone of the tail; 1, limp tail; 2, moderate paraparesis; 3; severe paraparesis; 4, tetraparesis or death) was determined on days 7, 9, 11, 13, 15, 17, 20, and 24. There were five animals in each group.
Male C57BL/6J mice (8 weeks old) were given dextran sulfate sodium salt (36–50 kDa) (36) contained in drinking water (2%, wt vol−1) daily through days 0–7. Sodium salt of SMTP-7 or SMTP-44D was intraperitoneally injected at a dose of 10 mg kg−1. As standards, 5-aminosalicylic acid (37) (10 mg ml−1 in 0.5% carboxymethylcellulose) or sodium prednisolone succinate (2 mg ml−1 in phosphate-buffered saline) were given orally at a dose of 100 and 20 mg kg−1, respectively. Drugs were given daily through days 0–7. Control animals received no drug treatment. Normal animals received neither dextran sulfate nor any drug. The disease activity index score (38, 39), determined based on the change in body weight (score 0, <1%; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, >20%), stool inconsistency (score 0, normal; 2, loose; 4, diarrhea), and stool blood (score 0, negative; 2, occult blood; 4, gross bleeding) was measured on days 2, 4, 5, 6, 7, and 8. There were five animals in each group.
Male C57BL/6J mice (8 weeks old) were intrarectally injected with 2,4,6-trinitrobenzene sulfonic acid (TNBS) (40) solution (20 mg ml−1 in 50% ethanol) at a dose of 100 mg kg−1. SMTP-7, SMTP-44D, 5-aminosalicylic acid, or prednisolone was administered as described for the dextran sulfate model. These treatments were made 30 min before the TNBS injection as well as 24, 48, and 72 h after the TNBS injection. Control animals received no drug treatment. Normal animals received neither TNBS nor any drug. The disease activity index score was measured on days 1, 2, 3, and 4 after the TNBS injection. There were five animal in each group.
SMTP-47 was synthesized by the microbial amine feeding method (31). For the preparation of feeding amine, Nα-tert-butoxycarbonyl-Nδ-Fmoc-l-ornithine (60 mg ml−1 in tetrahydrofuran) was treated with equal volume of trifluoroacetic acid, affording Nδ-Fmoc-l-ornithine. The culture of S. microspora IFO 30018 (100 ml) was fed with 100 mg of Nδ-Fmoc-l-ornithine, and the resulting SMTP-47 (89 mg) was purified by reverse-phase HPLC developed with MeOH, 0.1% formic acid (85:15). 1H NMR (acetone-d6, 600 MHz): δ 7.83 (2H, d, J = 7.3 Hz), 7.67 (2H, d, J = 7.3 Hz), 7.38 (2H, m), 7.29 (2H, t, J = 7.3 Hz), 6.80 (1H, d, J = 2.2 Hz), 5.16 (1H, t, J = 6.6 Hz), 5.06 (1H, m), 4.97 (1H, dd, J = 4.4, 11.0 Hz), 4.44 (1H, d, J = 16.1 Hz), ~4.31 (3H, m), 4.20 (1H, t, J = 6.6 Hz), 3.95 (1H, dd, J = 5.9, 7.3 Hz), 3.23 (2H, m), 3.02 (1H, dd, J = 5.9, 17.6 Hz), 2.68 (1H, dd, J = 7.3, 17.6 Hz), 2.20 (2H, m), ~2.06 (4H, m), 1.95 (2H, m), 1.71 (2H, m), 1.62 (3H, s), 1.57 (3H, s), ~1.56 (2H, m), 1.55 (3H, s), 1.28 (3H, s). 13C NMR (acetone-d6, 150 MHz): δ 172.95, 169.71, 157.18, 157.13, 149.88, 145.18, 142.09, 135.70, 132.56, 131.65, 128.43, 127.87, 126.08, 125.19, 125.12, 121.77, 120.73, 112.76, 100.86, 79.93, 67.61, 66.68, 54.30, 48.15, 44.97, 40.90, 40.40, 38.42, ~27.67 (2 signals overlapped), ~27.64, 27.38, 25.80, 22.23, 18.79, 17.70, 15.99. MALDI-TOF-MS (m/z): [M + Na]+ calculated for C43H50N2NaO8, 745.3465; found, 745.3575. UV (MeOH): λmax nm (ϵ) 208 (86, 390), 263 (29, 330), 289 (7, 370), 300 (9, 100). IR (neat): νmax cm−1 3329, 3064, 2968, 2926, 2866, 1701, 1670, 1620, 1533, 1462, 1356, 1252, 1157, 1076, 847, 744, 542.
To prepare SMTP-50, SMTP-47 (90 mg) was treated with piperidine/N,N-dimethylformamide (6:1) for 1 h to afford SMTP-50, which was purified by reverse-phase HPLC developed with a linear gradient of MeOH in 0.1% formic acid (60–100%) for 20 min, yielding 30.3 mg of the purified material. 1H NMR (CD3OD, 600 MHz): δ 6.74 (1H, s), 5.12 (1H, t, J = 6.9 Hz), 5.06 (1H, t, J = 6.9 Hz), 4.75 (1H, dd, J = 4.8, 10.2 Hz), 4.61 (1H, d, J = 16.8 Hz), 4.26 (1H, d, J = 17.4 Hz), 3.88 (1H, t, J = 6.0 Hz), 2.97 (3H, m), 2.64 (1H, dd, J = 7.2, 17.4 Hz), 2.16 (3H, m), 2.05 (2H, m), 1.96 (2H, m), 1.90 (1H, m), 1.68 (1H, m), 1.64 (3H, s), ~1.61 (3H, m), 1.59 (3H, s), 1.57 (3H, s), 1.26 (3H, s). 13C NMR (CD3OD, 150 MHz): δ 177.17, 171.83, 157.82, 150.01, 136.39, 132.66, 132.13, 125.39, 125.36, 122.24, 113.21, 100.91, 80.09, 68.22, 57.09, 45.92, 40.81, 40.01, 38.72, 28.61, 27.73 (2 signals overlapped), 25.89, 25.81, 22.53, 18.95, 17.76, 16.04. Electrospray ionization-TOF-MS (m/z): [M + H]+ calculated for C28H41N2O6, 501.2965; found, 501.3003. UV (MeOH): λmax nm (ϵ): 216 (41, 700), 260 (9, 600), 300 (2, 800). IR (neat): νmax cm−1 3386, 3208, 2967, 2926, 1657, 1611, 1466, 1381, 1245, 1213, 1163, 1080, 1047. [α]D = −12.1° (c. 1.0, MeOH).
One column volume (1 ml for target identification or 5 ml for target purification) of 1.2 mm SMTP-50, dissolved in 10 mm sodium phosphate, pH 8.3, was applied to a HiTrap NHS-activated HP column (GE Healthcare) at room temperature for 30 min. The column was then treated with monoethanolamine to block residual N-hydroxysuccinimidyl group of the matrix.
The livers from male ICR mice were perfused with ice-cold saline and homogenized in 4 volumes of 25 mm Tris-HCl, pH 7.4, containing 150 mm NaCl and 0.2% (wt vol−1) sodium deoxycholate (buffer A). A supernatant fraction was obtained after centrifugation at 1000 × g for 15 min followed by 20,000 × g for 15 min at 4 °C. After filtration, 15 ml of the supernatant was applied to a 1-ml SMTP-affinity column pre-equilibrated with buffer A at 20 °C. The column was washed with 10 ml of buffer A and developed with 10 ml of buffer A containing additional NaCl (500 mm, finally). Aliquots of the eluate were resolved by reduced SDS-polyacrylamide gel electrophoresis. Coomassie Brilliant Blue R250-stained protein bands were excised from the gel and digested with trypsin. The resulting peptides were subjected to chemically assisted fragmentation post-source decay analysis by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry on a Ettan MALDI-TOF Pro (Amersham Biosciences) or LC-MALDI-TOF/TOF analysis on a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Detected masses were subjected to amino acid sequence analysis and to comparison with theoretical peptide masses on MASCOT search engine (Matrixscience, Boston, MA) to identify the protein species.
All of the following operations were carried out at 0–4 °C. The livers from male ICR mice were homogenized in 11.5 volumes of 76 mm sodium phosphate, pH 7.4. A supernatant fraction was obtained after centrifugation at 1000 × g for 10 min followed by 10,000 × g for 25 min and then 100,000 × g for 60 min. The resulting cytosol fraction (250 ml) was applied to a 5-ml SMTP-column pre-equilibrated with 76 mm sodium phosphate, pH 7.4, containing 0.1 mm EDTA (buffer B). After washing with buffer B (200 ml), the column was developed with 60 ml of buffer B containing 10 μm 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). The eluate was dialyzed against buffer B to remove AUDA and ultrafiltered to concentrate and exchange buffer to 100 mm sodium phosphate, pH 7.4, containing 3 mm dithiothreitol. From 3 batches of affinity chromatography, 2.2 mg of homogeneous sEH was purified. The purified sEH had specific activities of 511 nmol min−1 mg−1 for the Cterm-EH and 2850 nmol min−1 mg−1 for the Nterm-phos when we used trans-stilbene oxide and p-nitrophenyl phosphate as respective substrates.
Samples to be analyzed were extracted with ethyl acetate. After centrifugation, supernatant was concentrated to dryness. The resulting materials were dissolved in methanol and subjected to LC-MS analysis for 14,15-EET and 14,15-DHET on a Micromass Quattro Ultima triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface (Waters, Tokyo, Japan). Samples (10 μl) were resolved on a silica-ODS column (100 × 2 mm; Pegasil ODS SP100–3, Senshu Scientific, Tokyo, Japan) developed at 0.2 ml min−1 with a linear gradient of acetonitrile in 0.1% formic acid (60–100%) for 15 min). The electrospray ionization was performed in the negative ion mode with a capillary voltage at 3.0 kV. The cone voltages were set at 35 V for 14,15-DHET/14,15-DHET-d11, and 30 V for 14,15-EET/14,15-EET-d11. Data were acquired in the multichannel analysis mode and analyzed using the MassLynx software (Version 3.5; Waters).
The Cterm-EH activity was assayed with 14,15-EET or (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME) as a substrate. When using PHOME, we preincubated mouse sEH (60 ng) for 10 min in 80 μl of 25 mm Bis-Tris-HCl, pH 7.0, containing 0.1 mm MgCl2 and 0.1 mg ml−1 bovine serum albumin (buffer C) with or without a compound to be tested. The composition of buffer C was based on the method by Tran et al. (28), unless MgCl2 was added to unify the buffer composition with that for Nterm-phos determination, in which MgCl2 is essential. After adding 20 μl of the substrate, fluorescence (excitation, 355 nm; emission, 460 nm) of the reaction product was measured kinetically at 30 °C. The final concentrations of sEH and PHOME in the standard assay conditions were 4.7 nm and 12.5 μm, respectively. When using EET, we incubated mouse sEH (0.18 ng) with 14,15-EET in 60 μl of buffer C at 30 °C for 20 min. After the addition of 14,15-EET-d11 and 14,15-DHET-d11 (25 pmol and 5 pmol, respectively) as internal standards, 14,15-DHET formed was determined by LC-MS as described above. The Nterm-phos activity was assayed with AttoPhos as a substrate. Mouse sEH (30 ng) was preincubated for 10 min in 80 μl of buffer C with or without a compound to be tested. After adding 20 μl of AttoPhos, fluorescence (excitation, 450 nm; emission, 545 nm) of the reaction product was measured kinetically at 30 °C. The final concentrations of sEH and AttoPhos in the standard assay conditions were 2.3 nm and 5 μm, respectively.
HepG2 cells (5 × 104 cells) were seeded on 24-well plates and cultured overnight. Cells were then washed with Hanks' balanced salt solution containing 20 mm Hepes, pH 7.4, 0.1 mg ml−1 bovine serum albumin, and 1 mm MgCl2 (buffer D) and subsequently treated with various concentrations of SMTP-7 (0–30 μm) in 500 μl of buffer D for 10 min. After the addition of 14,15-EET (0.3 μm) to the culture for 40 min, the reaction was stopped by adding 2-propanol (300 μl). After the addition of 14,15-EET-d11 and 14,15-DHET-d11 (200 and 10 pmol, respectively), the amounts of 14,15-EET remaining and 14,15-DHET formed were determined by LC-MS as described above.
Plasma (400 μl) obtained from Guillain-Barré syndrome model rats 2 h after SMTP-7 or saline treatment (n = 8) on day 13 was randomly paired within each group, and the mixture (n = 4 for each group) was centrifuged at 5000 × g for 10 min. The resulting supernatant was mixed with 80 μl of formic acid, and the mixture was applied to Sep-Pak C18 Plus Short Cartridges (Waters, Tokyo, Japan). The column was washed with EtOH-water-formic acid (10:100:1, vol−1 vol−1), and metabolites of interest were eluted with 5 ml of EtOH. The eluate was evaporated and dissolved with 40 μl of 50% aqueous MeOH. Aliquots (10 μl) were subjected to LC-MS/MS analysis on an L-column2 ODS (2 μm, 1 × 150 mm, CERI, Tokyo, Japan) developed at a rate of 0.1 ml−1 with a liner gradient (10–85%) of acetonitrile in 5 mm ammonium formate-formic acid (1000:1, vol−1 vol−1) for 26 min. Eluates were ionized with electrospray ionization, and negative ions of oxylipins were monitored on API 3200 QTRAP (AB SCIEX, Tokyo, Japan). Metabolites to be analyzed were the following 48: (±)-5,6-DHET, (±)-8,9-DHET, (±)-11,12-DHET, (±)-14,15-DHET, (±)-5,6-EET, (±)-8,9-EET, (±)-11,12-EET, (±)-14,15-EET, prostaglandin (PG) A2, PGB2, PGD2, 6-keto-PGE1, PGE2, 6-keto-PGF1α, PGF2α, 15-keto-PGF2α, 2,3-dinor-8-iso-PGF2α, PGJ2, δ-12-PGJ2, 15-deoxy-δ-12,14-PGJ2, thromboxane (TX)B2, 11-dehydro-TXB2, leukotriene (LT) B4, 12-keto-LTB4, 20-COOH-LTB4, LTC4, LTD4, LTE4, LTF4, 20-OH-LTB4, 5(S)-hydroxyeicosatetraenoic acid (HETE), 8(R)-HETE, 9(S)-HETE, 11(R)-HETE, 12(R)-HETE, 15(S)-HETE, 16(R)-HETE, 19(S)-HETE, 20-HETE, 5(S)-hydroperoxyeicosatetraenoic acid (HPETE), 12(S)-HPETE, 15(S)-HPETE, 5-oxo-eicosatetraenoic acid (ETE), 12-oxoETE, 15-oxoETE, 5(S),6(S)-lipoxin (LX) A4, 5(S),14(R)-LXB4, and hepoxilin A3.
Although SMTP-7 reduces inflammatory responses in thromboembolic stroke models, it was unclear whether or not this outcome is a consequence of the recanalization by thrombolytic enhancement. We thus employed inflammatory disease models (Guillain-Barré syndrome, ulcerative colitis, and Crohn disease models) that were irrelevant to thromboembolic complications to directly assess the anti-inflammatory action of SMTP-7. We compared the efficacy of SMTP-7 with that of its congener, SMTP-44D (11) (Fig. 1A), which is essentially inactive in the plasminogen modulation activity (Fig. 1B). In the Guillain-Barré syndrome model in rats, SMTP-7 (10 mg kg−1) and SMTP-44D (10 mg kg−1) both ameliorated neuritis symptoms as did the clinically used immunoglobulin formulation (400 mg kg−1) (Fig. 1C). Both SMTP-7 (10 mg kg−1) and SMTP-44D (10 mg kg−1) alleviated the disease-associated body weight loss, stool inconsistency, and intestinal bleeding in the models of ulcerative colitis and Crohn disease in mice (Fig. 1, D–G and H–K). These effects were comparable with more prominent than the effects of the standard drug 5-aminosalicylic acid (100 mg kg−1) and those of prednisolone (20 mg kg−1). Thus, the anti-inflammatory action of SMTP is independent of the plasminogen modulation activity.
To identify the molecule that is involved in the anti-inflammatory mechanism of SMTP, we designed an affinity matrix that contained an essential part of SMTP, tricyclic γ-lactam with a geranylmethyl side chain (Fig. 2A). To prepare the SMTP congener with a primary amine at the N-linked side chain (SMTP-50), we first synthesized a precursor with the primary amine of SMTP-50 protected by a 9-fluorenylmethyloxycarbonyl group (SMTP-47) (Fig. 2A) by the precursor amine-fed culture method (31). After eliminating the protective group, the resulting SMTP-50 was coupled to cross-linked agarose beads.
Detergent-solubilized homogenates of the mouse liver were subjected to affinity chromatography on the SMTP-coupled beads. The resulting eluate gave several protein bands on SDS-polyacrylamide gel electrophoresis. We subjected four specifically eluted protein species to peptide-mass fingerprinting analysis (Fig. 2B), and every protein species was identified as a full-length form or a fragment of sEH (Fig. 2, C and D, and Table 1). The purification of sEH to homogeneity was achieved when SMTP-affinity chromatography was performed using a cytosol fraction of detergent-free homogenates and an elution buffer containing AUDA, a competitive inhibitor of the Cterm-EH of sEH (41) (Fig. 2E).
SMTP-7 inhibited both the Cterm-EH and the Nterm-phos of sEH (their respective IC50 values were 23 ± 1 and 6 ± 1 μm) when we used the synthetic substrates PHOME and AttoPhos, respectively (Fig. 3, A and B). Similarly, SMTP-44D inhibited Cterm-EH and Nterm-phos (IC50 27 ± 2 and 24 ± 3 μm, respectively) (Fig. 3, A and B). In addition, the structurally simplest congener SMTP-0 (which lacks the N-linked side chain) was inhibitory to both activities (IC50 6 ± 1 and 14 ± 1 μm, respectively) (Fig. 3, A and B). Thus, the structural requirement for sEH inhibition is clearly distinguishable from that for plasminogen modulation activity in which the N-linked side chain plays a crucial role (11, 12, 32).
We performed detailed kinetic analyses of sEH inhibition using SMTP-0 and the natural substrate 14,15-EET. The use of SMTP-0 was to avoid complexity of data analysis: SMTP-0 consists of a single core unit of SMTP (Fig. 3), whereas SMTP-7 has two core units that are asymmetrically configured (Fig. 1A), and each of these may differently interact with the enzyme. The kinetic results revealed a positive cooperativity for the hydrolysis of 14,15-EET (Fig. 4A) (Hill coefficient of 1.9 for the substrate binding; Fig. 4B), suggesting an allosteric interaction between the two monomers of sEH. The data were, therefore, analyzed based on a nonlinear mathematical model that allowed allostericity between the two equivalent catalytic sites of Cterm-EH (Fig. 4C). The pattern of the Cterm-EH inhibition by SMTP-0 fitted well to a competitive model (Fig. 4, A and C). Moreover, Cterm-EH inhibition by SMTP-0 was competed for by AUDA (this class of inhibitor binds to the catalytic site in Cterm-EH (42)) (Fig. 4, D and E). These results are consistent with the observation that sEH is specifically eluted with AUDA in SMTP affinity chromatography. Regarding the Nterm-phos, the kinetic data fitted to a linear mathematical model (Fig. 5, A–C), suggesting no cooperativity between the two Nterm-phos domains (Hill coefficient of 0.91; Fig. 5B). The inhibition of Nterm-phos by SMTP-0 was pseudo-noncompetitive with respect to the substrate AttoPhos (Fig. 5, A and B). The pseudo-noncompetitive mechanism suggests that the Nterm-phos inhibition is mediated by the SMTP-0 binding to an allosteric site other than the substrate binding site in the Nterm-phos. Because there remained a possibility that the binding of SMTP-0 to the substrate binding site in the Cterm-EH might affect the activity of the Nterm-phos, we assessed the inhibitory activity of SMTP-0 toward the Nterm-phos in the presence of AUDA, which competed with SMTP-0 for binding to the catalytic site in the Cterm-EH (Fig. 4D). As a result, the presence of AUDA did not affect the SMTP-0 inhibition of the Nterm-phos (Fig. 5, D and E). Thus, SMTP-0 should bind to two distinct sites in sEH; one is the catalytic site in the Cterm-EH, and the other is an allosteric site that affects the Nterm-phos.
The structure-activity relationships of SMTP congeners differing in the N-linked side chain are summarized in Fig. 6. Although the minimum structural requirement for the sEH inhibition was the tricyclic γ-lactam with a geranylmethyl side chain (represented by SMTP-0), the difference in the N-linked side chain variably affected the potency of the inhibition of the Cterm-EH and Nterm-phos. The congener with a naphthalene (SMTP-16) (32) or a glucose moiety as the N-linked side chain (SMTP-33) (32) was essentially inactive in inhibiting the Cterm-EH and Nterm-phos. SMTP-54 (with a glutamine moiety) (33) and SMTP-55 (with a glutamic acid moiety) (33) were relatively specific for the inhibition of the Cterm-EH. On the other hand, SMTP-5D (with a d-leucine moiety) (34) was relatively specific for the inhibition of the Nterm-phos. The inhibitory activity of the congeners with a phenylamine derivative as the N-linked side chain (such as SMTP-26, SMTP-27, and SMTP-28) (12) were potent with respect to both the Cterm-EH and Nterm-phos inhibitions. The variability of the inhibition selectivity (IC50 for the Cterm-EH versus that for the Nterm-phos) supports the idea that SMTPs bind to two distinct sites in sEH.
HepG2 cells had an ability to hydrolyze 14,15-EET to 14,15-DHET, an inactive diol. SMTP-7 inhibited the formation of 14,15-DHET from 14,15-EET added to the culture medium (IC50 6.5 μm) (Fig. 7A). Along with the inhibition of the 14,15-EET hydrolysis, the level of 14,15-EET was elevated in the presence of SMTP-7 (Fig. 7A). SMTP-0 and SMTP-44D were also active in inhibiting 14,15-DHET formation from 14,15-EET in HepG2 cells (IC50 1.2 and 9.2 μm, respectively) (Fig. 7B).
To confirm the impact of sEH inhibition by SMTP-7 and its selectivity on sEH, we measured the levels of metabolites in the cyclooxygenase, lipoxygenase, and cytochrome P450 pathways using plasma obtained from Guillain-Barré syndrome model rats. A global analysis revealed that SMTP-7 did not significantly affect the levels of 47 out of 48 metabolites examined (see “Experimental Procedures” for metabolites analyzed), resulting in a small change in the distribution of the three classes of arachidonate metabolites (Fig. 8A). The only one metabolite that was significantly affected by the treatment with SMTP-7 was 11,12-DHET. The level (% distribution among the 48 metabolites) of 11,12-DHET in SMTP-7-treated rats was significantly lower than that in saline-treated rats (13.5 ± 0.17% compared with 20.2 ± 0.04% in saline group, p < 0.05; Fig. 8B). The levels of 5,6-, 8,9-, and 14,15-DHETs, however, were not significantly changed by the treatment with SMTP-7 (Fig. 8B). The levels of all regioisomers of EETs were too low to be detected by the method employed. The result that SMTP-7 selectively decreased 11,12-DHET can partly be explained by inhibition of sEH. Details of this interpretation are described under “Discussion.”
SMTP-7 has a plasminogen modulation activity that leads to a thrombolytic enhancement as observed in several animal models (14, 16, 20). SMTP-7 effectively treats thrombotic and embolic stroke models (17, 18, 20), and the involvement of an additional mechanism that leads to anti-inflammation has been suggested (16, 18, 19, 21). In the present study we observed the SMTP anti-inflammatory action that is independent of the plasminogen modulation activity. Although the in vivo models used in this study (Guillain-Barré syndrome, ulcerative colitis, and Crohn disease) are apparently irrelevant to thromboembolic complication, there remained a possibility that a local generation of plasmin might affect disease progress. Therefore, we compared the effect of SMTP-7 with that of the congener SMTP-44D, which is essentially inactive in plasminogen modulation activity (11). The results clearly demonstrate that both compounds are effective in treating these inflammatory disease models (Fig. 1). Thus we conclude that SMTPs have an anti-inflammatory activity independent of their plasminogen modulation activity. A target molecule for the anti-inflammatory action should, therefore, exist. In addition, the structural requirement for the anti-inflammatory action could be different from that for the plasminogen modulation activity.
To identify the anti-inflammatory target, we designed an affinity matrix that can bind a target protein. In our preliminary experiments, we observed anti-inflammatory effects with SMTP congeners with varying N-linked side-chain structures. We, therefore, employed the strategy of coupling the core SMTP structural moiety (tricyclic γ-lactam with a geranylmethyl side-chain) to a bead via an N-linked side chain. The major protein that bound to the resulting affinity matrix was sEH (Fig. 2). We purified a homogeneous sEH preparation by performing a single-step chromatography on this matrix using AUDA, which binds to the catalytic site of the Cterm-EH, as an eluent. This result is consistent with the fact that SMTP-0 competitively inhibits the Cterm-EH (Fig. 4). In addition to its inhibition of Cterm-EH, SMTP-0 inhibits the Nterm-phos pseudo-noncompetitively (Fig. 5). The result that the inhibition of the Cterm-EH, but not the Nterm-phos, by SMTP-0 is competed for by AUDA suggests a mechanism in which SMTP-0 binds to two distinct sites in sEH; one is the substrate binding site in the Cterm-EH and the other is an allosteric site that affects the Nterm-phos. We observed a positive cooperativity in Cterm-EH, whereas no cooperativity (43) or a negative cooperativity in Cterm-EH (44) has been reported in previous studies. These variable findings may be due to the use of enzyme from different sources (native or recombinant) and/or buffer compositions. As expected from the crystal structure, sEH has higher order intradomain and interdomain interactions (45), and environmental conditions would affect the conformational status of sEH to exhibit cooperativity. Nevertheless, kinetic parameters obtained with these three investigations are relatively close (Vmax 11.5 μmol min−1 mg−1 and apparent Km ((KS1 × KS2)1/2] 3.52 μm in this study (Fig. 4); Vmax 9.0 μmol min−1 mg−1 and Km 4 μm for 14(R),15(R)-EET, and Vmax 1.36 μmol min−1 mg−1 and Km 5 μm for 14(S),15(S)-EET by Zeldin et al. (43); Vmax 20 μmol min−1 mg−1 and apparent Km ((KS1 × KS2)1/2) 6.3 μm by Marowsky et al. (44)).
The structure-activity relationship results, which reveal variable inhibition selectivity (the ratio of the IC50 of the Cterm-EH over that of the Nterm-phos) among congeners with different N-linked side chains (Fig. 6), support this idea. Although the physiological function of the Cterm-EH has been extensively characterized by the use of the combination of sEH-deficient animals and specific inhibitors of the Cterm-EH (46,–56), the role of the Nterm-phos remains elusive because of the lack of information about the relevant substrate and a potent specific inhibitor. The existence of an allosteric site that affects the Nterm-phos has not been reported. Our structure-activity relationship data suggest the possibility that a selective Nterm-phos inhibitor can be developed based on the allosteric mechanism.
EETs are signaling molecules implicated in anti-inflammation (57, 58). SMTP-7 and its congeners inhibited the hydrolysis of 14,15-EET to the inactive 14,15-DHET in HepG2 cells (Fig. 7). In addition, the plasma level of 11,12-DHET in Guillain-Barré syndrome model rats was significantly decreased by SMTP-7 treatment (Fig. 8), whereas the levels of 5,6-, 8,9-, and 14,15-DHETs were unaffected. The levels of 5,6- and 8,9-DHETs were low, which is consistent with the finding by Li et al. (59). The lack of effect of SMTP-7 on the levels of these metabolites can be explained by the low catalytic activity of sEH toward these regioisomers (43, 60), letting sEH to contribute lesser to these levels. We speculate the lack of effect on the 14,15-DHET level is due to complex mechanisms of the formation/catabolism (degradation and excretion) of EETs and DHETs under physiological conditions. According to previous literatures (43, 60, 61) the specific activity of human sEH for the hydrolysis of 14,15-EET is approximately >2 times higher than that for 11,12-EET. In our animal model, the circulating level of 14,15-DHET was only 1.1 times higher than that of 11,12-DHET in control animals. Thus, it is likely that the rates of degradation and/or excretion of 14,15-DHET in this model is higher than those of 11,12-DHET. This may make it difficult to reflect the impact of sEH inhibition by SMTP-7. The selective change in the 11,12-DHET level by SMTP-7 suggest a specificity of SMTP-7 in the arachidonate metabolisms. Along with the in vitro data, this in vivo result supports the idea that sEH is an anti-inflammatory target of SMTP-7.
The deficiency of sEH and the inhibition of the Cterm-EH have been reported to be protective against disease progression in animal models of inflammatory bowel diseases (62). In thrombotic and embolic stroke models in rodents and primates, SMTP-7 exhibited excellent activities, with a wide therapeutic time window and a reduced cerebral hemorrhage (17, 18, 20), that were not achieved by the conventional thrombolytic therapy. The finding that SMTP-7 inhibits sEH suggests that this activity, aside from the thrombolytic enhancement by plasminogen modulation, may account for these additional pharmacological potentials of SMTP-7. The observations that the sEH deficiency or the Cterm-EH inhibition is protective against experimental stroke (63,–65) support this hypothesis. The pharmacological significance of the SMTP-7 ability to inhibit the Nterm-phos remains to be investigated. SMTP congeners that selectivity inhibit the Nterm-phos can be useful tools to investigate the physiological role of the Nterm-phos.
In conclusion, our results demonstrated SMTP-7's anti-inflammatory action that is independent of its plasminogen modulation activity. The finding that SMTP-7 inhibits sEH and the resulting hydrolysis of the anti-inflammatory signaling molecule EET suggests that sEH inhibition is involved in the anti-inflammatory action of SMTP-7. The combination of thrombolysis and sEH inhibition explains the excellent activity of SMTP-7 in treating thrombotic and embolic strokes. SMTP-7 is under development as an alternative drug that could be effective in stroke patients who do not benefit from the standard thrombolytic therapy.
We thank Haruki Koide (Tokyo Noko University) for synthesizing SMTP-47 and Naoko Nishimura and Keiko Hasegawa (TMS Co., Ltd.) for SMTP-7 and congeners.
*This work was supported in part by Japan Science and Technology Agency Grant AS2316911G (to K. H.) a grant from the Ministry of Education, Culture, Sports, Science and Technology (Translational Research Network Program C7; to K. H.).
3The abbreviations used are: