MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

PMPCB Silencing Sensitizes HCC Tumor Cells to Sorafenib Therapy.

Hepatocellular carcinoma (HCC) tumors invariably develop resistance to cytotoxic and targeted agents, resulting in failed treatment and tumor recurrence. Previous in vivo short hairpin RNA (shRNA) screening evidence revealed mitochondrial-processing peptidase (PMPC) as a leading gene contributing to tumor cell resistance against sorafenib, a multikinase inhibitor used to treat advanced HCC. Here, we investigated the contributory role of the ß subunit of PMPC (PMPCB) in sorafenib resistance. Silencing PMPCB increased HCC tumor cell susceptibility to sorafenib therapy, decreased liver tumor burden, and improved survival of tumor-bearing mice receiving sorafenib. Moreover, sorafenib + PMPCB shRNA combination therapy led to attenuated liver tumor burden and improved survival outcome for tumor-bearing mice, and it reduced colony formation in murine and human HCC cell lines in vitro. Additionally, PMPCB silencing enhanced PINK1-Parkin signaling and downregulated the anti-apoptotic protein MCL-1 in sorafenib-treated HCC cells, which is indicative of a healthier pro-apoptotic phenotype. Higher pre-treatment MCL-1 expression was associated with inferior survival outcomes in sorafenib-treated HCC patients. Elevated MCL-1 expression was present in sorafenib-resistant murine HCC cells, while MCL-1 knockdown sensitized these cells to sorafenib. In conclusion, our findings advocate combination regimens employing sorafenib with PMPCB knockdown or MCL-1 knockdown to circumvent sorafenib resistance in HCC patients.

Efficacy and safety of methionine aminopeptidase 2 inhibition in type 2 diabetes: a randomised, placebo-controlled clinical trial.

AIMS/HYPOTHESIS: This multicentre randomised double-blind placebo-controlled clinical trial assessed the efficacy and safety of a methionine aminopeptidase 2 (MetAP2) inhibitor, beloranib, in individuals with obesity (BMI ≥30 kg/m2) and type 2 diabetes (HbA1c 53-97 mmol/mol [7-11%] and fasting glucose <15.6 mmol/l). METHODS: Participants were randomised (via a centralised interactive web response system) to placebo, 1.2 or 1.8 mg beloranib s.c. twice weekly for 26 weeks. Participants, investigators and the sponsor were blinded to group assignment. The primary endpoint was the change in weight from baseline to week 26. The trial was terminated early when beloranib development was stopped because of an imbalance of venous thromboembolism events in beloranib-treated individuals vs placebo that became evident during late-stage development of the drug. RESULTS: In total, 153 participants were randomised, 51 to placebo, 52 to 1.2 mg beloranib and 50 to 1.8 mg beloranib. In participants who completed week 26, the least squares mean ± SE weight change (baseline 111 kg) was -3.1 ± 1.2% with placebo (n = 22) vs -13.5 ± 1.1% and -12.7 ± 1.3% with 1.2 and 1.8 mg beloranib, respectively (n = 25; n = 19; p < 0.0001). The change in HbA1c (baseline 67 mmol/mol [8.3%]) was -6.6 ± 2.2 mmol/mol (-0.6 ± 0.2%) with placebo vs -21.9 ± 2.2 mmol/mol (-2.0 ± 0.2%) or -21.9 ± 3.3 mmol/mol (-2.0 ± 0.3%) with 1.2 or 1.8 mg beloranib (p < 0.0001), respectively. The most common beloranib adverse events were sleep related. One beloranib-treated participant experienced a non-fatal pulmonary embolism. CONCLUSIONS/INTERPRETATION: MetAP2 inhibitors represent a novel mechanism for producing meaningful weight loss and improvement in HbA1c. TRIAL REGISTRATION: ClinicalTrials.gov NCT02324491 FUNDING: The study was funded by Zafgen, Inc.

Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

Blue light treatment of Pseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors.

Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.

Robust Reductions of Excess Weight and Hyperphagia by Beloranib in Rat Models of Genetic and Hypothalamic Obesity.

Hypothalamic lesions or deficient melanocortin (MC) signaling via MC4 receptor (MC4r) mutations often lead to hyperphagia and severe treatment-resistant obesity. We tested the methionine aminopeptidase 2-inhibitor beloranib (ZGN-440) in 2 male rat models of obesity, one modeling hypothalamic obesity with a combined medial hypothalamic lesion (CMHL) and the other modeling a monogenic form of obesity with MC4r mutations (MC4r knockout [MC4rKO]). In CMHL rats (age 3 months), postsurgery excess weight gain was significantly inhibited (ZGN-440, 0.2 ± 0.7 g/d; vehicle, 3.8 ± 0.6 g/d; P < 0.001) during 12 days of ZGN-440 treatment (0.1 mg/kg daily subcutaneously) together with a 30% reduction of daily food intake vs vehicle injection. In addition, ZGN-440 treatment improved glucose tolerance and reduced plasma insulin, and circulating levels of α-melanocyte stimulating hormone were increased. Serum lipid levels did not differ significantly in ZGN-440-treated vs vehicle-treated rats. Similar results were found in MC4rKO rats: ZGN-440 treatment (14-21 d) was associated with significant reductions of body weight gain (MC4rKO, -1.7 ± 0.6 vs 2.8 ± 0.4 g/d; lean wild-type controls, -0.7 ± 0.2 vs 1.7 ± 0.7 g/d; ZGN-440 vs vehicle, respectively), reduction of food intake (MC4rKO, -28%; lean controls, -7.5%), and insulin resistance, whereas circulating levels of interleukin-1ß did not change. In both obesity models, body temperature and locomotor activity were not affected by ZGN-440 treatment. In conclusion, the robust reduction of body weight in response to ZGN-440 observed in rats with severe obesity is related to a strong reduction of food intake that is likely related to changes in the central regulation of feeding.

In Silico Investigation of Traditional Chinese Medicine for Potential Lead Compounds as SPG7 Inhibitors against Coronary Artery Disease.

Coronary artery disease (CAD) is the most common cause of heart attack and the leading cause of mortality in the world. It is associated with mitochondrial dysfunction and increased level of reactive oxygen species production. According to the Ottawa Heart Genomics Study genome-wide association study, a recent research identified that Q688 spastic paraplegia 7 (SPG7) variant is associated with CAD as it bypasses the regulation of tyrosine phosphorylation of AFG3L2 and enhances the processing and maturation of SPG7 protein. This study aims to identify potential compounds isolated from Traditional Chinese Medicines (TCMs) as potential lead compounds for paraplegin (SPG7) inhibitors. For the crystallographic structure of paraplegin, the disordered disposition of key amino acids in the binding site was predicted using the PONDR-Fit protocol before virtual screening. The TCM compounds saussureamine C and 3-(2-carboxyphenyl)-4(3H)-quinazolinone, have potential binding affinities with stable H-bonds and hydrophobic contacts with key residues of paraplegin. A molecular dynamics simulation was performed to validate the stability of the interactions between each candidate and paraplegin under dynamic conditions. Hence, we propose these compounds as potential candidates as lead drug from the compounds isolated from TCM for further study in drug development process with paraplegin protein for coronary artery disease.

Efficacy and safety of beloranib for weight loss in obese adults: a randomized controlled trial.

AIM: To assess the efficacy, safety and tolerability of beloranib treatment for obesity. METHODS: This phase II, double-blind, randomized study investigated the effects of beloranib suspension (0.6, 1.2 and 2.4 mg) or placebo, administered subcutaneously, for 12 weeks in 147 participants (primarily white women) with obesity. No diet or exercise advice was administered. RESULTS: At week 12, beloranib resulted in dose-dependent progressive weight loss of -5.5 ± 0.5, -6.9 ± 0.6 and -10.9 ± 1.1 kg for the 0.6, 1.2 and 2.4 mg beloranib doses, respectively, compared with -0.4 ± 0.4 kg with placebo (all p < 0.0001 vs placebo). Weight loss with beloranib was associated with corresponding reductions in waist circumference and body fat mass, as well as improvements in lipids, high-sensitivity C-reactive protein and blood pressure. Sleep disturbance and gastrointestinal adverse events were more common with beloranib than with placebo; these were generally mild to moderate, transient and dose-related, and led to more early study withdrawals in participants in the group with the highest dose of beloranib. CONCLUSIONS: In this 12-week phase II study, beloranib produced clinically and statistically significant weight loss and corresponding improvements in cardiometabolic risk factors. Beloranib appeared safe, and the 0.6 and 1.2 mg doses were generally well tolerated. The 2.4 mg dose was associated with increased sleep latency and mild to moderate gastrointestinal adverse events over the first month of treatment. These findings represent a novel mechanism for producing clinically meaningful weight loss.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Effects of Tamarind (Tamarindus indicus Linn) seed extract on Russell's viper (Daboia russelli siamensis) venom.

Snake bite has been regarded as an important health problem in Myanmar since early 1960's. In the recent years, there has been growing interest in alternative therapies and therapeutic use of natural products, especially those derive from plants. In Myanmar and Indian traditional medicine, various plants have used as a remedy for treating snake bite. The present study was carried out to evaluate the effects of alcohol extract of Tamarind (Tamarindus indica Linn.) seed on some biologic properties of Russell's viper (Daboia russelli siamensis) venom (RVV). The Phospholipase A2 (PLA2) enzyme, coagulase enzyme and caseinolytic enzyme activities of Russell's viper venom (RVV) were reduced when mixed and incubated with the extract. When the RVV and the different amount of extracts were preincubated and injected intramuscularly into mice, all of them survived, but all the mice in the control group died. On the other hand, when RVV were injected first followed by the extract into mice, all of them died. If the extract was injected near the site where Russell's viper venom was injected, all the mice survived for more than 24 hours and the survival time prolonged but they all died within 96 hours. In conclusion, according to the results obtained, the extract neutralizes some biologic properties of the Russell's viper venom and prolonged the survival time if the extract was injected near the site where the Russell's viper venom was injected.

Anti-inflammatory and antinociceptive effects of cordymin, a peptide purified from the medicinal mushroom Cordyceps sinensis.

The anti-inflammatory and antinociceptive activities of cordymin, a peptide purified from the medicinal mushroom Cordyceps sinensis, were studied. The effects of cordymin on cytokine levels and total antioxidant activity were analysed. The antinociceptive effects of cordymin in vivo and in vitro were also determined. Cordymin treatment decreased the levels of tumour necrosis factor alpha, interleukin 1 beta and total antioxidant status. Cordymin inhibited the acetic acid-induced abdominal constrictions in mice in a dose-dependent manner. In the hot-plate test, results showed that cordymin significantly inhibited the reaction time to thermal stimuli at 30, 60 and 90 min. In neurolysin inhibition assay, cordymin showed strong activities against neurolysin (IC(50) = 0.1 µM). Our results show that cordymin is a potent anti-inflammatory and analgesic medicine.

The development of MetAP-2 inhibitors in cancer treatment.

Methionine aminopeptidases (MetAPs), which remove methionine residue from newly synthesized polypeptide chains, are a class of metalloproteases ubiquitously distributed in both eukaryotes and prokaryotes. MetAP-2 inhibition can induce G1 cell cycle arrest, cytostasis in tumor cells in vitro and inhibition of tumor growth in vivo. The discovery of fumagillin with potent antiangiogenic and antiproliferative activities promoted the development of fumagillin analogues as a novel class of anticancer agents. Early drug discovery efforts have focused on analogs of fumagillin, which irreversibly inhibit MetAP-2 through covalent modification of an epoxide. Several fumagillin analogs, like CKD-732, TNP-470 and PPI-2458, were found to be potent selective inhibitors of MetAP-2 (proteolytic activity) and endothelial cell proliferation. Further, they have entered in clinical trials for the treatment of different types of tumors. Recently, attention has been paid to reversible human MetAP-2 inhibitors, such as bengamides, 2-hydroxy-3-aminoamides, anthranilic acid sulfonamides and triazole analogs, which have demonstrated their potential to inhibit angiogenesis and tumor growth in vivo as well. This review article mainly discussed the development of MetAP-2 inhibitors in cancer therapy and also summarized their structure-activity relationships.

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors.

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.

Renoprotective effects of combined endothelin-converting enzyme/neutral endopeptidase inhibitor SLV338 in acute and chronic experimental renal damage.

BACKGROUND: Acute kidney injury (AKI) as well as chronic renal failure are associated with a huge mortality/morbidity. However, so far no drugs have been approved for the treatment of acute kidney failure and only a few for the treatment of chronic kidney disease (CKD). We analysed the effect of SLV338, a neutral endopeptidase (NEP)/endothelin converting enzyme (ECE)-inhibitor in animal models of acute kidney failure as well as chronic renal failure. METHODS: Acute renal failure was induced in male Wistar rats by uninephrectomy and clamping of the remaining kidney for 55 minutes. SLV338 (total dose: 4.9 mg/kg) or vehicle was continuously infused for 2 hours (starting 20 minutes prior to clamping). Sham operated animals served as controls. Plasma creatinine was measured at baseline and day 2 and 8 after renal ischemia-reperfusion. Hypertensive renal damage was induced in male Sprague Dawley rats by nitric oxide deficiency using L-NAME (50 mg/kg per day, added to drinking water for 4 weeks). One group was treated over the same time period with SLV338 (30 mg/kg per day, mixed with food). Systolic blood pressure was monitored weekly. At study end, urine and blood samples were collected and kidneys were harvested. RESULTS: Acute renal ischemia-reperfusion caused a 5-fold plasma creatinine elevation (day 2), which was significantly attenuated by more than 50% in animals treated with SLV338 (p < 0.05). Renal failure was accompanied by a 67% mortality in vehicle-treated rats, but only 20% after SLV338 treatment (p = 0.03 compared to sham controls). Chronic L-NAME administration caused hypertension, urinary albumin excretion, glomerulosclerosis, renal arterial remodelling, and renal interstitial fibrosis. Treatment with SLV338 did not significantly affect blood pressure, but abolished renal tissue damage (interstitial fibrosis, glomerulosclerosis, renal arterial remodelling (p < 0.05 versus L-NAME group in each case). CONCLUSIONS: The dual ECE/NEP inhibitor SLV338 preserves kidney function and reduces mortality in severe acute ischemic renal failure. Moreover, combined ECE/NEP inhibition prevents hypertensive renal tissue damage in a blood pressure independent manner in L-NAME-treated rats.

High-throughput screening of improved protease inhibitors using a yeast cell surface display system and a yeast cell chip.

Protease-targeted inhibitors have been promising pharmaceuticals. Here, we combined a yeast cell surface display system with a yeast cell chip for the high-throughput screening of protease inhibitors, and succeeded in improving the activity of a protease inhibitor.

Antihypertensive effects of Ocimum basilicum L. (OBL) on blood pressure in renovascular hypertensive rats.

Ocimum basilicum L. (OBL), sweet basil, is a medicinal herb used in traditional Chinese medicine to treat cardiovascular diseases including hypertension. The objective of the study was to investigate the possible antihypertensive effects of OBL extract in renovascular hypertensive rats. The two-kidney one-clip (2K1C) Goldblatt model of renovascular hypertension was used in Wistar rats. Rats were randomized into sham, untreated 2K1C, captopril- (30 mg kg(-1) per day orally) and OBL- (100, 200, 400 mg kg(-1) per day orally) (low (L)-, medium (M)-, high (H)-OBL) treated 2K1C groups (n=10-12 per group), followed up for 4 weeks. Blood pressure, heart weight/body weight, plasma angiotensin-II and endothelin (ET)-1 were studied. OBL reduced systolic and diastolic blood pressure by about 20 and 15 mm Hg, respectively, compared with 35 and 22 mm Hg for captopril, from the lowest dose tested with no dose dependency. Cardiac hypertrophy was reduced from 3.6+/-0.7 mg g(-1) for untreated 2K1C to 3.0+/-0.6, 2.9+/-0.6 and 2.4+/-0.4 mg g(-1) for L-, M- and H-OBL, respectively, compared with 2.6+/-0.5 for sham and 3.1+/-0.4 mg g(-1) for captopril (P<0.05). Renal function was improved with captopril. Angiotensin was reduced to a lesser extent than with captopril. ET was reduced to lower concentrations (78+/-15, 80+/-22, 82+/-15 pg ml(-1) for L-, M-, H-OBL, respectively) than in sham (116+/-31 pg ml(-1)), untreated 2K1C (174+/-72 pg ml(-1)) or captopril (117+/-72 pg ml(-1)) groups. The effects of OBL on blood pressure, cardiac hypertrophy and ET, are consistent with an effect on ET-converting enzyme, and warrant further exploration.

Phosphinic tripeptides as dual angiotensin-converting enzyme C-domain and endothelin-converting enzyme-1 inhibitors.

A new series of phosphinic inhibitors able to interact with both angiotensin-converting enzyme (ACE) C-domain and endothelin-converting enzyme-1 (ECE-1), while sparing neprilysin (NEP), has been developed. The most potent and selective inhibitor in this series (compound 8(F2)) displays K(i) values of 0.65 nM, 150 nM, 14 nM and 6.7 microM toward somatic ACE C-domain, ACE N-domain, ECE-1, and NEP, respectively. Remarkably, in this series, the inhibitor's ability to discriminate between ECE-1 and NEP was observed to depend on the stereochemistry of the residue present in the inhibitor's P(1)' position. After iv administration, compound 8(F2) (10 mg/kg) lowered mean arterial blood pressure by 24 +/- 2 mmHg in spontaneously hypertensive rats, as compared with controls. Mixed ACE/ECE-1 inhibitor may lead to a new generation of vasopeptide inhibitors that should reduce the levels of angiotensin-II and endothelin-1, without interfering with bradykinin cleavage.

A meprin inhibitor suppresses atherosclerotic plaque formation in ApoE-/- mice.

Meprin is a member of the astacin family of zinc metalloendopeptidases. It is widely distributed in the body, and hydrolyzes and inactivates several endogenous vasoactive peptides, some of which could alter various functions of cells in the arterial wall. We assessed the influence of chronic meprin inhibition by daily administration of actinonin (5mg/kg body weight per day; i.p.) on the development of atherosclerotic changes in ApoE(-/-) mice. Mice were fed a high-fat (21% fat), cholesterol-rich (1% cholesterol) Western-type diet for 16 weeks starting at 10 weeks of age. At 20 weeks of age, randomly selected ApoE(-/-) mice were treated with Western-type diet chow pellets supplemented with commercially available actinonin (meprin-I group) for 6 weeks; the diet of control ApoE(-/-) mice was supplemented with saline (placebo group). There was no difference in body weight, hemodynamic data and serum lipids between the two groups at the end of the dietary period. Meprin-I treatment was found to elevate levels of natriuretic peptides (NPs) in plasma and the vascular wall by radioimmunoassay. Meprin-I treatment also decreased plaque volume and suppressed lipid deposition in carotid arteries. Meprin-I treatment reduced production of reactive oxygen species (ROS) and apoptosis (which are associated with atherosclerosis) in the vascular wall. In in vitro experiments, meprin-I treatment increased NP function on cell apoptosis, proliferation, and intracellular ROS generation in the THP-1 cell line and primary vascular smooth muscle cells (VSMC). These results suggest that the meprin inhibitor actinonin may have a protective role in atherosclerosis, and that meprin inhibition may be therapeutically useful in atherosclerosis prevention. Suppression of degradation in the arteries of endogenously released NPs (particularly atrial natriuretic peptide and brain natriuretic peptide), or other kinins known to have anti-atherosclerotic actions, may at least partially contribute to the inhibitory effects of meprin-I on atherosclerotic changes.

News from an ancient world: two novel astacin metalloproteases from the horseshoe crab.

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase micro) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase micro. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase micro. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.