Potent Efficacy of 3-Amino-4-hydroxy Benzoic Acid, a Small Molecule Having Anti-fibrotic Activity, in a Mouse Model of Non-alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH), which is on the rise due to the increasing obese population and changing lifestyles, causes fibrosis over time and carries the risk of progression to cirrhosis and hepatocellular carcinoma. However, there are no approved effective treatments for NASH. Recent studies suggest that increased lipid metabolism and reduced nitric oxide content are responsible for NASH; 3-amino-4-hydroxy benzoic acid (AHBA) was identified as an inhibitor for the phosphatase activity of soluble epoxy hydrolase, which in turn inhibits lipid metabolism and endothelial nitric oxide synthase activity. The aim of this study was to assess the efficacy of AHBA in a mouse model of NASH. NASH was induced in mice by streptozotocin administration and a high-fat diet loading. The efficacy of AHBA was determined by measuring liver function using serum and liver samples and conducting a morphological assessment. AHBA considerably attenuated the increase in the liver weight and alkaline phosphatase content, which occurred due to the progression of NASH. Hepatocellular steatosis, inflammatory cell infiltration, and hepatocellular ballooning of hepatocytes remained unaltered. In contrast, AHBA treatment significantly ameliorated the fibrotic alterations within liver tissue that were induced by the onset of NASH. These results demonstrate the potential of AHBA as a therapeutic pharmaceutical compound that can treat NASH.

A first-in-human study of the anti-inflammatory profibrinolytic TMS-007, an SMTP family triprenyl phenol.

AIMS: TMS-007, an SMTP family member, modulates plasminogen conformation and enhances plasminogen-fibrin binding, leading to promotion of endogenous fibrinolysis. Its anti-inflammatory action, mediated by soluble epoxide hydrolase inhibition, may contribute to its efficacy. Evidence suggests that TMS-007 can effectively treat experimental thrombotic and embolic strokes with a wide time window, while reducing haemorrhagic transformation. We aim to evaluate the safety, pharmacokinetics and pharmacodynamics of TMS-007 in healthy volunteers. METHODS: This was a randomized, placebo-controlled, double blind, dose-escalation study, administered as a single intravenous infusion of TMS-007 in cohorts of healthy male Japanese subjects. Six cohorts were planned, but only five were completed. In each cohort (n = 8), individuals were randomized to receive one of five doses of TMS-007 (3, 15, 60, 180 or 360 mg; n = 6) or placebo (n = 2). RESULTS: TMS-007 was generally well tolerated, and no serious adverse events were attributed to the drug. A linear dose-dependency was observed for plasma TMS-007 levels. No symptoms of bleeding were observed on brain MRI analysis, and no bleeding-related responses were found on laboratory testing. The plasma levels of the coagulation factor fibrinogen and the anti-fibrinolysis factor α2 -antiplasmin levels were unchanged after TMS-007 dosing. A slight increase in the plasma level of plasmin-α2 -antiplasmin complex, an index of plasmin formation, was observed in the TMS-007 group in cohort 2. CONCLUSIONS: TMS-007 is generally well tolerated and exhibits favourable pharmacokinetic profiles that warrant further clinical development.

Identification of aminobenzoic acids as selective inhibitors of the N-terminal phosphatase of soluble epoxide hydrolase.

Soluble epoxide hydrolase (EC 3.3.2.10) is a key enzyme in the regulation of inflammation and metabolism, whereas, the role of its N-terminal phosphatase activity (N-phos) has been poorly understood because of a lack of selective inhibitors. Here we report 4-aminobenzoic (Ki 15.3 µm) and 3-amino-4-hydroxy benzoic acid (Ki 11.7 µm) as selective competitive inhibitors of N-phos.

Preclinical safety, toxicokinetics and metabolism of BIIB131, a novel prothrombolytic agent for acute stroke.

BIIB131, a small molecule, is currently in Phase 2 for the treatment of acute ischemic stroke. Safety and metabolism of BIIB131 were evaluated following intravenous administration to rats and monkeys. Exposure increased dose-proportionally in rats up to 60 mg/kg and more than dose-proportionally in monkeys at greater than 10 mg/kg accompanied by prolonged half-life and safety findings. The BIIB131 was poorly metabolized in microsomes with no inhibition of CYPs. BIIB131-glucuronide, formed by UGT1A1, accounted for 21.5% metabolism in human hepatocytes and 28-40% in rat bile. In rats, excretion was primarily via the bile. BIIB131 inhibited the hERG and Nav1.5 cardiac channels by 39% but showed no effect on cardiovascular parameters in monkeys. Toxicology findings were limited to reversable hematuria, changes in urinary parameters and local effects. A MTD of 30 mg/kg was established in monkeys, the most sensitive species, at total plasma Cmax and AUC of 6- and 14-fold, respectively, greater than the NOAEL. The Phase 1 study started with intravenous 0.05 mg/kg and ascended to 6.0 mg/kg which corresponded to safety margins of 147- to 0.9-fold (for Cmax) within the linear drug exposure. Thus, the preclinical profile of BIIB131 has been appropriately characterized and supports its further clinical development.

SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis.

SMTP-44D has been reported to have anti-oxidative and anti-inflammatory reactions, including reduced expression of receptor for advanced glycation end products (RAGE) in experimental diabetic neuropathy. Although activation of RAGE with its ligands, and advanced glycation end products (AGEs), play a crucial role in atherosclerotic cardiovascular disease, a leading cause of death in diabetic patients, it remains unclear whether SMTP-44D could inhibit experimental atherosclerosis by suppressing the AGEs-RAGE axis. In this study, we investigated the effects of SMTP-44D on atherosclerotic plaque formation and expression of AGEs in apolipoprotein-E null (Apoe-/-) mice. We further studied here whether and how SMTP-44D inhibited foam cell formation of macrophages isolated from Apoe-/- mice ex vivo. Although administration of SMTP-44D to Apoe-/- mice did not affect clinical or biochemical parameters, it significantly decreased the surface area of atherosclerotic lesions and reduced the atheromatous plaque size, macrophage infiltration, and AGEs accumulation in the aortic roots. SMTP-44D bound to immobilized RAGE and subsequently attenuated the interaction of AGEs with RAGE in vitro. Furthermore, foam cell formation evaluated by Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and gene expression of RAGE, cyclin-dependent kinase 5 (Cdk5) and CD36 in macrophages isolated from SMTP-44D-treated Apoe-/- mice were significantly decreased compared with those from saline-treated mice. Gene expression levels of RAGE and Cdk5 were highly correlated with each other, the latter of which was also positively associated with that of CD36. The present study suggests that SMTP-44D may inhibit atherosclerotic plaque formation in Apoe-/- mice partly by blocking the AGEs-RAGE-induced ox-LDL uptake into macrophages via the suppression of Cdk5-CD36 pathway.

Direct cell-cell interaction regulates division of stem cells from PC-3 human prostate cancer cell line.

Cancer stem cells (CSCs) are a subpopulation that can drive recurrence and metastasis. Therefore, therapies targeting CSCs are required. Although previous findings have suggested that non-CSCs regulate the proliferation and differentiation of CSCs in the tumor microenvironment, the precise molecular mechanism is largely unknown. In this study, we found that a direct interaction between CSCs and non-CSCs downregulated CSC division in the PC-3 human prostate cancer cell line. We found that the proliferation of PC-3-derived CSCs (PrSCs) was significantly decreased (∼47%) in the presence of non-CSC-rich parental PC-3 cells compared with that in a culture in which they were absent. We observed no differences in PrSC proliferation when we indirectly cocultured them with PC-3 cells across a Transwell insert, and PrSCs that were transiently bound to immobilized PC-3 cells proliferated more slowly than those bound to PrSCs. The frequency of cell division with prior PrSC-PrSC contact was 2.8 times higher in the PrSC monoculture compared with that in the coculture with PC-3 cells. We found that the PrSCs were approximately 1.3 times more closely associated in the monoculture compared with the coculture with PC-3 cells, as determined by a cell proximity assay. The frequency of asymmetric PrSC division was 6.5% in the monoculture compared with 1.0% in the coculture with PC-3 cells (P < 0.045). By analyzing our data, we determined the importance of PrSC-non-CSC contact in regulating the frequency and mode of PrSC division. This regulation might be a valuable target for treating cancer.

Effect of SMTP-7 on Cisplatin-Induced Nephrotoxicity in Mice.

SMTP-7, a fungal metabolite, is reported to have a high degree of availability for the ischemia-reperfusion (IR)-induced acute kidney injury (AKI) model. Cisplatin, a widely used anticancer drug, has serious side effects, such as AKI. Hence, we aimed to examine the effect of SMTP-7 on cisplatin-induced AKI in this study. Significant increases in blood urea nitrogen (BUN) and serum creatinine (Scr) were observed at 72 h after the intravenous infusion of cisplatin (20 mg/kg). Histologically, necrosis and dilatation (hyaline casts) as well as regeneration were observed in proximal tubules. SMTP-7 inhibited the elevation on BUN and Scr caused by cisplatin dose dependently. The efficacy of SMTP-7 was notable when the drug was administered on the day after cisplatin treatment, whereas the repeated administration of the drug did not result in an enhanced efficacy. Moreover, 10 mg/kg of SMTP-7 considerably ameliorated tubular necrosis and dilatation. The cisplatin treatment also caused an up-regulation of tumor necrosis factor-α (TNF-α) mRNA expression prior to the elevation of the levels of BUN and Scr. Administration of SMTP-7 (10 mg/kg) at 24 h after the cisplatin infusion alleviated the up-regulation of TNF-α mRNA expression. These findings suggest that SMTP-7 exhibits a renoprotective effect against cisplatin infusion based on the inhibition of the expression of pro-inflammatory cytokines such as TNF-α and may be expected a new effective drug for the treatment of cisplatin-induced AKI.

SMTP-44D Exerts Antioxidant and Anti-Inflammatory Effects through Its Soluble Epoxide Hydrolase Inhibitory Action in Immortalized Mouse Schwann Cells upon High Glucose Treatment.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. We have previously reported the efficacy of Stachybotrys microspora triprenyl phenol-44D (SMTP-44D) for DN through its potential antioxidant and anti-inflammatory activities. However, the mechanisms underlying the antioxidant and anti-inflammatory activities of SMTP-44D remain unclear. The present study aimed to explore the mechanism of these effects of SMTP-44D in regard to its inhibition of soluble epoxide hydrolase (sEH) in immortalized mouse Schwann cells (IMS32) following high glucose treatment. IMS32 cells were incubated in a high glucose medium for 48 h and then treated with SMTP-44D for 48 h. After incubation, the ratio of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), oxidative stress markers, such as NADPH oxidase-1 and malondialdehyde, inflammatory factors, such as the ratio of nuclear to cytosolic levels of NF-κB and the levels of IL-6, MCP-1, MMP-9, the receptor for the advanced glycation end product (RAGE), and apoptosis, were evaluated. SMTP-44D treatment considerably increased the ratio of EETs to DHETs and mitigated oxidative stress, inflammation, RAGE induction, and apoptosis after high glucose treatment. In conclusion, SMTP-44D can suppress the induction of apoptosis by exerting antioxidant and anti-inflammatory effects, possibly through sEH inhibition. SMTP-44D can be a potential therapeutic agent against DN.

Impact of SMTP Targeting Plasminogen and Soluble Epoxide Hydrolase on Thrombolysis, Inflammation, and Ischemic Stroke.

Stachybotrys microspora triprenyl phenol (SMTP) is a large family of small molecules derived from the fungus S. microspora. SMTP acts as a zymogen modulator (specifically, plasminogen modulator) that alters plasminogen conformation to enhance its binding to fibrin and subsequent fibrinolysis. Certain SMTP congeners exert anti-inflammatory effects by targeting soluble epoxide hydrolase. SMTP congeners with both plasminogen modulation activity and anti-inflammatory activity ameliorate various aspects of ischemic stroke in rodents and primates. A remarkable feature of SMTP efficacy is the suppression of hemorrhagic transformation, which is exacerbated by conventional thrombolytic treatments. No drug with such properties has been developed yet, and SMTP would be the first to promote thrombolysis but suppress disease-associated bleeding. On the basis of these findings, one SMTP congener is under clinical study and development. This review summarizes the discovery, mechanism of action, pharmacological activities, and development of SMTP.

N-Substituted amino acid inhibitors of the phosphatase domain of the soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a bifunctional enzyme implicated in the regulation of inflammation. The N-terminal domain harbors a phosphatase activity (N-phos) with an affinity to lipid phosphomonoesters, and the C-terminal domain has an activity to hydrolyze anti-inflammatory lipid epoxides (C-EH). Although many potent inhibitors of C-EH have been discovered, little is known about inhibitors of N-phos. Here, we identify N-substituted amino acids as selective inhibitors of N-phos. Many of the N-substituted amino acids inhibited differently mouse and human N-phos; phenylalanine derivatives are relatively selective for mouse N-phos, whereas tyrosine derivatives are more selective for human N-phos. The best inhibitors, Fmoc-l-Phe(4-CN) (67) and Boc-l-Tyr(Bzl) (23), inhibited mouse and human N-phos competitively with KI in the low micromolar range. These compounds inhibit the N-phos activity 37- (67) and 137-folds (23) more potently than the C-EH. The differences in inhibitor structure activity suggest different active site structure between species, and thus, probably a divergent substrate preference between mouse and human N-phos.

Neuroprotective effects of SMTP-44D in mice stroke model in relation to neurovascular unit and trophic coupling.

Stachybotrys microspora triprenyl phenol (SMTP)-44D has both anti-oxidative and anti-inflammatory activities, but its efficacy has not been proved in relation to the pathological changes of neurovascular unit (NVU) and neurovascular trophic coupling (NVTC) in ischemic stroke. Here, the present study was designed to assess the efficacies of SMTP-44D, moreover, compared with the standard neuroprotective reagent edaravone in ischemic brains. ICR mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min, SMTP-44D (10 mg/kg) or edaravone (3 mg/kg) was intravenously administrated through subclavian vein just after the reperfusion, and these mice were examined at 1, 3, and 7 d after reperfusion. Compared with the vehicle group, SMTP-44D treatment revealed obvious ameliorations in clinical scores and infarct volume, meanwhile, markedly suppressed the accumulations of 4-HNE, 8-OHdG, nitrotyrosine, RAGE, TNF-α, Iba-1, and cleaved caspase-3 after tMCAO. In addition, SMTP-44D significantly prevented the dissociation of NVU and improved the intensity of NAGO/BDNF and the number of BDNF/TrkB and BDNF/NeuN double positive cells. These effects of SMTP-44D in reducing oxidative and inflammatory stresses were similar to or stronger than those of edaravone. The present study demonstrated that SMTP-44D showed strong anti-oxidative, anti-inflammatory, and anti-apoptotic effects, moreover, the drug also significantly improved the NVU damage and NVTC in the ischemic brain.

Reduction of Ischemia Reperfusion-Related Brain Hemorrhage by Stachybotrys Microspora Triprenyl Phenol-7 in Mice With Antioxidant Effects.

BACKGROUND: Stachybotrys microspora triprenyl phenol-7 (SMTP-7) has both thrombolytic and anti-inflammatory effects, but its neuroprotective effects on cerebral ischemia are still unclear. The present study assessed the antioxidative and neurovascular unit (NVU) protective effects of SMTP-7 using transient middle cerebral artery occlusion (tMCAO) mice. METHODS: After 60 minutes tMCAO, 0.9% NaCl, tissue-type plasminogen activator (tPA), SMTP-7 or tPA + SMTP-7 was intravenously administrated through subclavian vein just before the reperfusion, and these mice were examined at 24 hours after reperfusion. We histologically assessed the hemorrhage and expressive changes of antioxidative markers in brains. RESULTS: SMTP-7 treatment showed a similar antithrombotic effect to tPA, but significantly decreased the hemorrhage volumes and the number of 4-HNE, 3-NT and 8-OHdG positive cells, meanwhile, ameliorated the decrease of collagen IV in the ischemic brains. However, tPA + SMTP-7 treatment did not decrease hemorrhage volumes nor showed NVU protective effect. CONCLUSIONS: The present study suggested that SMTP-7 provided therapeutic benefits for ischemic stroke through antioxidative and NVU protective effects unlike tPA alone or tPA + SMTP-7.

Antineuroinflammatory Effect of SMTP-7 in Ischemic Mice.

BACKGROUND: Stachybotrys microspora triprenyl phenol-7 (SMTP-7) has both potentials of thrombolytic and neuroprotective effects, but its detailed neuroprotective mechanisms in ischemic stroke are still unclear. Here, we assessed the neuroprotective effects of SMTP-7 for anti-inflammatory and antiapoptosis mechanisms after 60 minutes of transient middle cerebral artery occlusion (tMCAO) in mice. METHODS: After 60minutes of tMCAO, 0.9% NaCl, tissue-type plasminogen activator (tPA), SMTP-7 or tPA+SMTP-7 was intravenously administrated through subclavian vein just before the reperfusion, and these mice were examined at 24hours after reperfusion. We histologically assessed the antineuroinflammatory effect of SMTP-7 on the expressive changes of inflammatory markers in ischemic mouse brains. RESULTS: Compared with the vehicle and tPA groups, SMTP-7 treatment significantly improved clinical scores and decreased the infarct volume and the numbers of TNF-α, nuclear factor-κB (NF-κB), nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), and cleaved caspase-3-positive cells in the brain of mice at 24hours after tMCAO but not p62-positive cells. However, tPA+SMTP-7 treatment did not show such effects. CONCLUSIONS: The present study suggested that SMTP-7 provides a therapeutic benefit for ischemic stroke mice through anti-inflammatory and antiapoptotic effects but not antiautophagic effect.

Structure-activity relationship of cyclic pentapeptide malformins as fibrinolysis enhancers.

The formation of blood clots in blood vessels causes severe ischemic diseases such as cerebral infarction and myocardial infarction. While searching for microbial products that increase fibrinolytic activity using an in vitro fibrin degradation assay, we found malformin A1, a disulfide form of cyclo(-d-Cys-d-Cys-l-Val-d-Leu-l-Ile-), as an active compound. In this study, we synthesized malformin derivatives using a solid-phase peptide synthesis method and evaluated their fibrinolytic activity and cytotoxicity. Reduction of the disulfide bond and linearization of the cyclic peptide frame decreased the pro-fibrinolytic activity. Substitution of a branched-chain amino acid with lysine resulted in loss of activity. However, protection of the amino group in the lysine derivatives by the tert-butoxycarbonyl (Boc) group rescued the inactivity. Furthermore, the phenylalanine derivatives also exhibited a similar pro-fibrinolytic effect compared to malformin A1. These results suggest that the disulfide bond, the cyclic peptide frame, and the bulky hydrophobic side chains play a crucial role in the pro-fibrinolytic activity of malformin. The effective dose of the active derivatives for the in vitro fibrin degradation showed similar ranges (1-5µM), while the order of cytotoxic potency for the active derivatives was as follows: Phe-derivatives>BocLys-derivatives>malformin A1>reduced form. These results showed no correlation between pro-fibrinolytic activity and cytotoxicity, suggesting the possibility of the synthesis for non-toxic malformin derivatives possessing the activity.

Soluble epoxide hydrolase as an anti-inflammatory target of the thrombolytic stroke drug SMTP-7.

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.

Altered gene expression in an embolic stroke model after thrombolysis with tissue plasminogen activator and Stachybotrys microspora triprenyl phenol-7.

The present study compares gene expression and infarct area in a mouse model of embolic stroke after thrombolysis with t-PA and SMTP-7. Embolic occlusion was induced by transfer of acetic acid-induced embolus into the brain. t-PA or SMTP-7 was administered 3 h after embolization. Changes in gene expression were evaluated using microarray and RT-PCR analysis. To determine the involvement of reactive oxygen species in the response to t-PA, the free radical scavenger edaravone was infused immediately before t-PA administration. The expressions of 459 genes involved in the inflammatory response, cell-to-cell signaling, cell movement, and inflammatory disease were altered by embolic occlusion. Twenty-two of those genes were upregulated after t-PA but not SMTP-7 administration. Differences between the t-PA- and SMTP-7-treated groups in the expression of genes including the proinflammatory genes Il6, Stat3, S100a8, and Mmp9 were confirmed with RT-PCR. Edaravone ameliorated the overexpression of these genes. Our data demonstrate differences in gene expression following treatment with SMTP-7 or t-PA that likely explain the difference in therapeutic time windows of the two drugs. ROS are involved in the overexpression of proinflammatory genes. The wide therapeutic time window may be achieved through an anti-oxidative effect and inhibition of proinflammatory gene overexpression.

Soluble epoxide hydrolase maintains steady-state lipid turnover linked with autocrine signaling in peritoneal macrophages.

Soluble epoxide hydrolase is a widely distributed bifunctional enzyme that contains N-terminal phosphatase (N-phos) and C-terminal epoxide hydrolase (C-EH) domains. C-EH hydrolyzes anti-inflammatory epoxy-fatty acids to corresponding diols and contributes to various inflammatory conditions. However, N-phos has been poorly examined. In peritoneal macrophages, the N-phos inhibitor amino-hydroxybenzoic acid (AHBA) seemed to primarily interrupt the dephosphorylation of lysophosphatidates and broadly attenuated inflammation-related functions. AHBA activated intrinsic lysophosphatidate and thromboxane A2 receptors by altering lipid-metabolite distribution; downstream the signaling, phospholipase C was facilitated to dampen intracellular Ca2+ stores and AKT kinase (protein kinase B) was activated to presumably inhibit inflammatory gene expression. Our data suggest that N-phos maintains steady-state phospholipid turnover connecting autocrine signaling and is a prospective target for controlling inflammatory responses in macrophages.

SMTP (Stachybotrys microspora triprenyl phenol) enhances clot clearance in a pulmonary embolism model in rats.

BACKGROUND: Stachybotrys microspora triprenyl phenols (SMTPs) are a novel family of small molecules that enhance both activation and fibrin-binding of plasminogen. While their effects on fibrinolysis have been characterized in vitro, little is known about their activity in vivo with respect to plasminogen activation and blood clot clearance. RESULTS: To select a potent SMTP congener for the evaluation of its action in vitro and in vivo, we tested several SMTP congeners with distinct structural properties for their effects on plasminogen activation. As a result, SMTP-7 (orniplabin) was found to have distinguished activity. Several lines of biochemical evidence supported the idea that SMTP-7 acted as a plasminogen modulator. SMTP-7 elevated plasma level of plasmin-α2-antiplasmin complex, an index of plasmin formation in vivo, 1.5-fold in mice after the intravenous injections at doses of 5 and 10 mg kg-1. In a rat pulmonary embolism model, SMTP-7 (5 mg kg-1) enhanced the rate of clot clearance ~3-fold in the absence of exogenous plasminogen activator. Clot clearance was enhanced further by 5 mg kg-1 of SMTP-7 in combination with single-chain urokinase-type plasminogen activator. CONCLUSIONS: Our results show that SMTP-7 is a superior plasminogen modulator among the SMTP family compounds and suggest that the agent enhances plasmin generation in vivo, leading to clearance of thrombi in a model of pulmonary embolism.

Amine-Regulated pri-SMTP Oxidation in SMTP Biosynthesis in Stachybotrys: Possible Implication in Nitrogen Acquisition.

SMTP (the name SMTP is derived from Stachybotrys microspora triprenyl phenol) is a family of triprenyl phenol secondary metabolites from a black mold, Stachybotrys microspora. Some SMTP congeners exhibit anti-inflammatory and profibrinolytic activities that, in combination, contribute to the treatment of ischemic stroke. The final step in the SMTP biosynthesis is a non-enzymatic amine conjugation with an o-phthalaldehyde moiety of the precursor pre-SMTP, which can form adducts with proteins and nucleic acids. Thus, pre-SMTP formation should be a precisely regulated, rate-limiting step in the SMTP biosynthesis. To address the mechanism backing this regulation, we purified a metabolite that rapidly disappeared following amine feeding, identifying a novel compound, pri-SMTP. Furthermore, an enzyme, designated as pri-SMTP oxidase, responsible for pri-SMTP conversion to pre-SMTP, was purified. The formation of pri-SMTP, which is regulated by nitrogen and carbon nutrients, occurred in particular septate mycelia. Although pri-SMTP oxidase was expressed constitutively, the consumption of pri-SMTP was accelerated only when a primary amine was fed. Thus, SMTP biosynthesis is regulated by at least three mechanisms: (i) pri-SMTP formation affected by nutrients, (ii) the compartmentalization of pri-SMTP formation/storage, and (iii) amine-regulated pri-SMTP oxidation. Amine-regulated SMTP formation (i.e., amine-capturing with pre-SMTP) may play a role in the nitrogen acquisition/assimilation strategy in S. microspora, since pri-SMTP synthesis occurs on non-preferred nitrogen.

Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion.

BACKGROUND AND PURPOSE: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. METHODS: The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. RESULTS: Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1ß, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 µg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. CONCLUSIONS: Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.