Rubiarbonol B induces RIPK1-dependent necroptosis via NOX1-derived ROS production.

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.

Pedicled frontal periosteal rescue flap via eyebrow incision for skull base reconstruction (SevEN-002).

PURPOSE: Cerebrospinal fluid (CSF) leakage is one of the major complications after endoscopic endonasal surgery. The reconstructive nasoseptal flap is widely used to repair CSF leakage. However, it could not be utilized in all cases; thus, there was a need for an alternative. We developed a pericranial rescue flap that could cover both sellar and anterior skull base defects via the endonasal approach. A modified surgical technique that did not violate the frontal sinus and cause cosmetic problems was designed using the pericranial rescue flap. METHODS: We performed 12 cadaveric dissections to investigate the applicability of the lateral pericranial rescue flap. An incision was made, extending from the middle to the lateral part of the eyebrow. The pericranium layer was dissected away from the galea layer, from the supraorbital region towards the frontoparietal region. With endoscopic assistance, the periosteal flap was raised, the flap base was the pericranium layer at the eyebrow incision. After a burr-hole was made in the supraorbital bone, the pericranial flap was inserted via the intradural or extradural pathway. RESULTS: The mean size of the pericranial flap was 11.5 cm × 3.2 cm. It was large enough to cross the midline and cover the dural defects of the anterior skull base, including the sellar region. CONCLUSION: We demonstrated a modified endoscopic technique to repair the anterior skull base defects. This minimally invasive pericranial flap may resolve neurosurgical complications, such as CSF leakage.

Diagnostic Accuracy of Ischemia-Modified Albumin for Acute Coronary Syndrome: A Systematic Review and Meta-Analysis.

The diagnostic usefulness of ischemia-modified albumin in acute coronary syndrome (ACS) has been questioned. The goal of this systematic review and meta-analysis was to see how accurate ischemia-modified albumin (IMA) was in diagnosing ACS in patients admitted to emergency departments (EDs). We searched for relevant literature in databases such as MEDLINE, EMBASE, and the Cochrane Library. Primary studies that reliably reported on patients with symptoms suggestive of ACS and evaluated IMA on admission to emergency departments were included. The QUADAS-2 tool was used to assess the risk of bias in the included research. A total of 4,761 patients from 19 studies were included in this systematic review. The sensitivity and specificity were 0.74 and 0.40, respectively, when the data were pooled. The area under the curve value for IMA for the diagnosis of ACS was 0.75, and the pooled diagnostic odds ratio value was 3.72. Furthermore, ACS patients with unstable angina had greater serum IMA levels than those with non-ischemic chest pain. In contrast to prior meta-analyses, our findings suggest that determining whether serum IMA levels are effective for diagnosing ACS in the emergency department is difficult. However, the accuracy of these findings cannot be ascertained due to high heterogeneity between studies.

Efficacy of Quantitative Pupillary Light Reflex for Predicting Neurological Outcomes in Patients Treated with Targeted Temperature Management after Cardiac Arrest: A Systematic Review and Meta-Analysis.

Background and objectives: This study aims to evaluate the usefulness of the quantitative pupillary light reflex as a prognostic tool for neurological outcomes in post-cardiac arrest patients treated with targeted temperature management (TTM). Material and Methods: We systematically searched MEDLINE, EMBASE, and the Cochrane Library (search date: 9 July 2021) for studies on post-cardiac arrest patients treated with TTM that had measured the percent constriction of pupillary light reflex (%PLR) with quantitative pupillometry as well as assessed the neurological outcome. For an assessment of the methodological quality of the included studies, two authors utilized the prognosis study tool independently. Results: A total of 618 patients from four studies were included in this study. Standardized mean differences (SMDs) were calculated to compare patients with good or poor neurological outcomes. A higher %PLR measured at 0-24 h after hospital admission was related to good neurological outcomes at 3 months in post-cardiac arrest patients treated with TTM (SMD 0.87; 95% confidence interval 0.70-1.05; I2 = 0%). A higher %PLR amplitude measured at 24-48 h after hospital admission was also associated with a good neurological outcome at 3 months in post-cardiac arrest patients treated with TTM, but with high heterogeneity (standardized mean difference 0.86; 95% confidence interval 0.40-1.32; I2 = 70%). The evidence supporting these findings was of poor quality. For poor neurological outcome, the prognosis accuracy of %PLR was 9.19 (pooled diagnostic odds ratio, I2 = 0%) and 0.75 (area under the curve). Conclusions: The present meta-analysis could not reveal that change of %PLR was an effective tool in predicting neurological outcomes for post-cardiac arrest patients treated with TTM owing to a paucity of included studies and the poor quality of the evidence.

3-O-acetylrubianol C (3AR-C) induces RIPK1-dependent programmed cell death by selective inhibition of IKKß.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKß. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKß phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKß inhibitor to overcome TNF resistance in cancer therpay.

Tulipiferamide A, an Alkamide from Liriodendron tulipifera, Exhibits an Anti-Inflammatory Effect via Targeting IKKß Phosphorylation.

Three new alkamides, tulipiferamides A-C (1-3, respectively), and 30 known compounds (4-33) were obtained from the roots of Liriodendron tulipifera (Magnoliaceae). Dehydrotemisin (4), an elemane sesquiterpene lactone, was isolated for the first time from nature. The structures were deduced by the interpretation of NMR spectroscopic and MS spectral data. The geometries of the double bonds in tulipiferamides A-C (1-3, respectively) were determined on the basis of 1H-1H coupling constants and 13C chemical shifts. The presence of the alkamide type in this plant is reported for the first time. An analysis of the inflammatory response revealed that seven compounds (1, 4, 7, 9, 14, 23, and 27) suppressed the nitric oxide production induced by LPS in RAW264.7 macrophages. Furthermore, tulipiferamide A (1) inhibits NF-κB activation by selectively targeting IKKß, an upstream kinase of NF-κB, resulting in the suppression of inflammatory mediators, including iNOS, COX-2, IL-1ß, TNFα, and IL-6. Our results provide a rationale for the further development of tulipiferamide A as a selective IKKß inhibitor to modulate inflammatory diseases.

Paratrimerins J-Y, Dimeric Coumarins Isolated from the Stems of Paramignya trimera.

Paratrimerins J-Y (1-13 and 16-18), new dimeric coumarins, were obtained from the EtOH(aq) extract of the stems of Paramignya trimera (Rutaceae) utilizing LC/MS guided isolation. The structures of the dimeric coumarins were elucidated based on 1D/2D NMR spectroscopic and HR-ESIMS data analyses. The absolute configurations of paratrimerins J-Y along with those of two known dimers paratrimerins A (14) and B (15) were established on the basis of the experimental and simulated ECD data. In addition, the absolute configurations of the sugar units of paratrimerins A, B, and J-V (1-15) were confirmed by LC/MS analysis on l-cysteine methyl ester and phenyl isothiocyanate derivatives. The variety of the absolute configurations of the dimeric diastereomers 1-15 highlighted a diversity in stereochemical outcomes following a Diels-Alder biosynthesis in P. trimera. With regard to P. trimera being a recently emerging medicinal resource for liver cancer, the dimers 1-18 were evaluated for cytotoxicity against a wide panel of human cancer cell lines. Paratrimerin W (16) was cytotoxic toward Huh7 hepatocellular carcinoma, HT1080 fibrosarcoma, and HT29 colorectal cancer cells with IC50 values of 14.9, 18.4, and 22.5 µM, respectively.

Configurational Assignment of a Flexible Benzo[g]isochromene Stereodiad from Rubia philippinensis and Inhibition of Soluble Epoxide Hydrolase Activity.

A new benzo[g]isochromene possessing a conformationally mobile moiety was identified from Rubia philippinensis. The 2D structure was established utilizing spectrometric and spectroscopic techniques with variable temperatures. The configurational investigation of the flexible moiety was investigated utilizing contemporary NMR-combined computational tools such as DP4, direct J-DP4, and DP4 Plus. The probabilities computed from DP4 Plus analysis, featuring inclusion of an additional geometry optimization process, demonstrated more conclusive probability scores among the analyses used. The configurational assignment was also supported by compositional and molecular orbital analyses. Compound 1 inhibited soluble epoxide hydrolase (IC50 = 0.6 ± 0.01 µM), an enzyme associated with cardiovascular disorders.

Isolation of Sesquiterpenoids and Steroids from the Soft Coral Sinularia brassica and Determination of Their Absolute Configuration.

Two undescribed rearranged cadinane-type sesquiterpenoids (1-2), named sinulaketol A-B, together with one new chlorinated steroid (3), one new gorgosterol (4), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7-8) were isolated from the soft coral Sinularia brassica. The structures of these compounds were established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. Their absolute configurations were also determined by the ECD calculations and DP4+ probability analysis. Antileishmanial activity of compounds 1-8 was evaluated in vitro against the amastigote forms of Leishmania donovani, in which compounds 3, 6, and 7 inhibited the growth of L. donovani by 58.7, 74.3, 54.7%, respectively, at a concentration of 50 µM. Antimicrobial effect of the isolated compounds were also evaluated against Candida albicans, Staphylococcus aureus, and Escherichia coli. Compound 6, a brominated diterpene, exhibited antimicrobial effect against S. aureus.

Ilimaquinone inhibits neovascular age-related macular degeneration through modulation of Wnt/ß-catenin and p53 pathways.

Neovascular age-related macular degeneration (nAMD) is a common cause of irreversible vision loss in the elderly. Anti-vascular endothelial growth factor has been effective in treating pathological ocular neovascularization, but it has limitations including the need for repeated intraocular injections for the maintenance of therapeutic effects in most patients and poor or non-response to this agent in some patients. in vitro cellular studies were conducted using retinal pigment epithelial cell lines (ARPE-19 and hTERT-RPE1), human umbilical vein endothelial cells (HUVECs), and human umbilical vein smooth muscle cells (HUVSMCs). in vivo efficacy of ilimaquinone (IQ) was tested in laser-induced choroidal neovascularization mouse and rabbit models. Tissue distribution study was performed in male C57BL6/J mice. IQ, 4,9-friedodrimane-type sesquiterpenoid isolated from the marine sponge, repressed the expression of angiogenic/inflammatory factors and restored the expression of E-cadherin in retinal pigment epithelial cells by inhibiting the Wnt/ß-catenin pathway. In addition, it selectively inhibited proliferation and tube formation of HUVECs by activating the p53 pathway. Topical and intraperitoneal administration of IQ significantly reduced choroidal neovascularization in rabbits and mice with laser-induced choroidal neovascularization. Notably, IQ by the oral route of exposure was highly permeable to the eyes and suppressed abnormal vascular leakage by downregulation of ß-catenin and stabilization of p53 in vivo. Our findings demonstrate that IQ functions through regulation of p53 and Wnt/ß-catenin pathways with conceivable advantages over existing cytokine-targeted anti-angiogenic therapies.

Cytotoxic Activity of Aplykurodin A Isolated From Aplysia kurodai against AXIN1-Mutated Hepatocellular Carcinoma Cells by Promoting Oncogenic ß-Catenin Degradation.

Dysregulation of the Wnt/ß-catenin signaling pathway is involved in the development of human hepatocellular carcinoma and has thus emerged as a therapeutic target for this malignant tumor. In this study, we employed sensitive cell-based assays to identify aplykurodin A isolated from Aplysia kurodai as an antagonist of Wnt/ß-catenin signaling. Aplykurodin A inhibited ß-catenin responsive transcription, which was stimulated by a Wnt3a-conditioned medium or a glycogen synthase kinase 3ß inhibitor by accelerating intracellular ß-catenin degradation. Aplykurodin A downregulated the level of oncogenic ß-catenin and decreased the expression of ß-catenin-dependent gene, leading to inhibition of human hepatoma Hep3B and SNU475 cell proliferation. Moreover, apoptosis and autophagy were elicited by aplykurodin A, as indicated by an increase the number of Annexin V-FITC-stained cells and the formation of microtubule-associated protein 1 light chain 3 puncta, respectively, in Hep3B and SNU475 cells. Our findings suggest that aplykurodin A provides a novel therapeutic strategy for human hepatocellular carcinoma via stimulation of oncogenic ß-catenin degradation.

Protective Effect of Tetrahydroquinolines from the Edible Insect Allomyrina dichotoma on LPS-Induced Vascular Inflammatory Responses.

The larva of Allomyrina dichotoma (family Scarabaeidae) is an edible insect that is registered in the Korean Food Standards Codex as a food resource. The chemical study on the larvae of A. dichotoma resulted in the isolation of three new tetrahydroquinolines, allomyrinaines A-C (1-3), one new dopamine derivative, allomyrinamide A (4), and four known compounds (5-8). The structures were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) and MS spectroscopic data analysis. Allomyrinaines A-C (1-3) possessed three stereogenic centers at C-2, C-3, and C-4, whose relative configurations were determined by analyses of the coupling constants and the nuclear Overhauser enhancement spectroscopy (NOESY) data, as well as DP4+ calculation. The anti-inflammatory effects of compounds 1-4 were evaluated in human endothelial cells. Allomyrinaines A-C (1-3) could stabilize vascular barrier integrity on lipopolysaccharide (LPS)-induced vascular inflammation via inhibition of the nuclear factor-κB (NF-κB) pathway. The physiologically relevant concentration was confirmed by Q-TOF-MS-based quantitative analysis on allomyrinaines A-C in crude extract. This study suggests that allomyrinaines A-C (1-3) are bioactive constituents of A. dichotoma to treat vascular inflammatory disorder.

Catechol-Type Flavonoids from the Branches of Elaeagnus glabra f. oxyphylla Exert Antioxidant Activity and an Inhibitory Effect on Amyloid-ß Aggregation.

Elaeagnus glabra f. oxyphylla (Elaeagnaceae) is a small evergreen tree with narrow lanceolate leaves that is native to Korea. In this work, we studied the chemical composition of E. glabra f. oxyphylla branches (EGFOB) for the first time. Additionally, we evaluated the effects of the ethanol extract of EGFOB and each of its chemical components on key mediators of Alzheimer's disease (AD), namely, amyloid-ß (Aß) aggregation and oxidative stress. The ethanol extract of EGFOB decreased Aß aggregation (IC50 = 32.01 µg/mL) and the levels of the oxidative free radicals 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50 = 11.35 and 12.32 µg/mL, respectively). Sixteen compounds were isolated from EGFOB. Among them, procyanidin B3 (8), procyanidin B4 (9), and helichrysoside (13) significantly inhibited Aß aggregation (IC50 = 14.59, 32.64, and 44.45 µM, respectively), indicating their potential as bioactive compounds to control Aß aggregation. Furthermore, these compounds markedly enhanced in vitro scavenging activity against ABTS (IC50 = 3.21-4.61 µM). In the DPPH test, they showed lower scavenging activity than in the ABTS test (IC50 ≥ 54.88 µM). Thus, these results suggest that EGFOB and specifically compounds 8, 9, and 13 may be beneficial in AD prevention and treatment through their antioxidant and anti-Aß aggregation activities.

Stereo-Selective Pharmacokinetics of Ilimaquinone Epimers Extracted from a Marine Sponge in Rats.

An ilimquinone (IQ) mixture isolated from Hippiospongia metachromia, consisting of IQ and epi-ilimaquinone (epi-IQ), exerts anti-HIV, anti-microbial, anti-inflammatory, and anti-cancer effects. An HPLC-MS/MS method was developed for simultaneous determination of the two epimers in rat plasma, separating them using a biphenyl column. Ascorbic acid is added during the sample preparation to ensure the stability of both isomers. The plasma concentrations of the isomers were monitored following intravenous and oral administration of the IQ mixture in rats as well as the individual epimers that were separately orally administered. Compare to IQ, epi-IQ was much more stable in rat plasma, likely due to its configurations of decalin. Both substances decayed in more than bi-exponential pattern, with an elimination rate constant of 1.2 h-1 for IQ and 1.7 h-1 for epi-IQ. The epi-IQ was distributed more widely than IQ by about two-fold. Consequently, the clearance of epi-IQ was greater than that of IQ by about three-fold. The oral absolute bioavailability for IQ was 38%, and, that for epi-IQ, was 13%. Although the systemic exposure of IQ was greater than that of epi-IQ by ~8.7-fold, the clearance of each isomer was similar when administered either orally or intravenously, when normalized for bioavailability. The stereo-specific behavior of the isomers appears to originate from differences in both their tissue distribution and gastrointestinal permeability.

Protective effects of Oxya chinensis sinuosa Mishchenko against ultraviolet B-induced photodamage in hairless mice.

BACKGROUND: Edible insects, including Oxya chinensis sinuosa Mishchenko (Oc), which is consumed as food in Asia, are considered as a human food shortage alternative, and also as a preventive measure against environmental destruction. Ultraviolet B (UVB) irradiation, which causes skin photodamage, is considered as an extrinsic skin aging factor. It reduces skin hydration, and increases wrinkle formation and reactive oxygen species (ROS) and inflammatory cytokine expression. Thus, the objective of this study was to investigate the anti-aging effects of an ethanol extract of Oc (Oc.Ex). METHODS: A UVB-irradiated hairless mouse model was used to examine relevant changes in skin hydration, wrinkle formation, and skin epidermal thickness. Also, antioxidant markers such as superoxide dismutase (SOD) and catalase (CAT) were analyzed, and Oc. Ex skin protective effects against UVB irradiation-induced photoaging were examined by determining the levels of skin hydration factors. RESULTS: Oc.Ex improved epidermal barrier dysfunctions such as increased transepidermal water loss (TEWL) and capacitance reduction in UVB-irradiated mice. It upregulated skin hydration-related markers, including hyaluronic acid (HA), transforming growth factor (TGF)-ß, and pro-collagen, in UVB-irradiated mice, compared with the vehicle control group. It also reduced UVB-induced wrinkle formation, collagen degradation, and epidermal thickness. Additionally, it remarkably suppressed the increased expression of matrix metalloproteinases (MMPs), and restored the activity of SOD and CAT in UVB-irradiated mice, compared with the vehicle control group. Furthermore, Oc. Ex treatment downregulated the production of inflammatory cytokines and phosphorylation of the mitogen-activated protein kinases (MAPKs) signaling pathway activated by UVB irradiation. CONCLUSION: This study revealed that Oc. Ex reduced skin thickness and the degradation of collagen fibers by increasing hydration markers and collagen-regulating factors in the skin of UVB-irradiated mice. It also inhibited UVB-induced antioxidant enzyme activity and inflammatory cytokine expression via MAPK signaling downregulation, suggesting that it prevents UVB-induced skin damage and photoaging, and has potential for clinical development in skin disease treatment.

Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

Ghost probiotics with a combined regimen: a novel therapeutic approach against the Zika virus, an emerging world threat.

The Zika virus (ZIKV) used to be an obscure flavivirus closely related to dengue virus (DENV). Transmission of this epidemic pathogen occurs mainly via mosquitoes, but it is also capable of placental and sexual transmission. Although the characteristics of these viruses are well defined, infections are unpredictable in terms of disease severity, unusual clinical manifestations, unexpected methods of transmission, long-term persistence, and the development of new strains. Recently, ZIKV has gained huge medical attention following the large-scale epidemics around the world, and reported cases of congenital abnormalities associated with Zika virus infections which have created a public health emergency of international concern. Despite continuous research on ZIKV, no specific treatment or vaccine has been developed, excepting a preventive strategy for congenital ZIKV infection. Probiotics, known as GRAS, are bacteria that confer various health beneficial effects, and have been shown to be effective at curing a number of viral diseases by modulating the immune system. Furthermore, probiotic preparations consisting of dead cells and cellular metabolites, so-called "Ghost probiotics", can also act as biological response modifiers. Here, we review available information on the epidemiology, transmission, and clinical features of ZIKV, and on treatment and prevention strategies. In addition, we emphasize the use of probiotics and plant-based natural remedies and describe their action mechanisms, and the green technologies for microbial conversion, which could contribute to the development of novel therapies that may reduce the pathogenicity of ZIKV. Accordingly, we draw attention to new findings, unanswered questions, unresolved issues, and controversies regarding ZIKV.

NMR-Based Investigation of Hydrogen Bonding in a Dihydroanthracen-1(4 H)one from Rubia philippinensis and Its Soluble Epoxide Hydrolase Inhibitory Potential.

Hydrogen bonding is a vital feature of a large ensemble of chemical structures. Soluble epoxide hydrolase (sEH) has been targeted for development of the treatment for inflammation-associated diseases. Compounds 1 and 2 were purified from Rubia philippinensis, and their structures were established via physical data analysis. Compound 1 possesses intramolecular hydrogen bonding, sufficiently robust to transfer heteronuclear magnetization via a nonbonded interaction. The bonding strength was assessed using the 1H NMR chemical shift temperature coefficients (-1.8 ppb/K), and the heteronuclear coupling constants were measured. The stereochemical details were investigated using interproton distance analysis and ECD. Purified compounds displayed moderate sEH-inhibitory activity.

Discovery of rubiarbonone C as a selective inhibitor of cytochrome P450 4F enzymes.

Cytochrome P450 (CYP) enzymes, particularly CYP4A/4F, are the major ω-hydroxylases of arachidonic acid (AA) that can produce 20-hydroxyeicosatetraenoic acid (20-HETE). Although there are dissimilarities in substrate specificity, tissue distribution, and gene regulation between CYP4A and CYP4F, selective CYP4A or 4F inhibitors are currently unavailable. Therefore, this study was designed to develop CYP4F selective inhibitors using a novel inhibitory assay of 20-HETE formation. The assay was established using pooled human kidney microsomes (HKMs) and human recombinant CYP4 enzymes incubated with 1,2,3,4,5-13C AA (13C5 AA) as a substrate to minimize interference by endogenous AA. The intrinsic clearance (Vmax/Km) values were 9.5 µL/min/mg for HKMs and 0.02, 0.9, and 10.1 µL/min/pmol for CYP4A11, CYP4F2, and CYP4F3B, respectively, which suggests a major role for CYP4F in ω-hydroxylation of AA. To validate the assay, we tested well-known pan-CYP4 inhibitor HET0016 along with 50 compounds derived from natural products. Of the screened compounds, rubiarbonone C showed the most potent inhibitory activity. The 50% inhibitory concentrations of rubiarbonone C against CYP4A11, CYP4F2, and 4F3B were > 50, 4.2, and 4.2 µM, respectively. Moreover, epoxyeicosatrienoic acid formation from 13C5 AA was not inhibited by up to 30 µM rubiarbonone C. Meanwhile, in pooled human liver microsomes, CYP1, 2, and 3 family enzymes involved in drug metabolism were not substantially inhibited by rubiarbonone C. Thus, rubiarbonone C is a selective inhibitor of CYP4F and can be used to discriminate among CYP4 family enzymes and evaluate their roles in physiological and pathophysiological conditions.

Sesterterpenoid and Steroid Metabolites from a Deep-Water Alaska Sponge Inhibit Wnt/ß-Catenin Signaling in Colon Cancer Cells.

The Wnt/ß-catenin signaling pathway is known to play critical roles in a wide range of cellular processes: cell proliferation, differentiation, migration and embryonic development. Importantly, dysregulation of this pathway is tightly associated with pathogenesis in most human cancers. Therefore, the Wnt/ß-catenin pathway has emerged as a promising target in anticancer drug screening programs. In the present study, we have isolated three previously unreported metabolites from an undescribed sponge, a species of Monanchora (Order Poecilosclerida, Family Crambidae), closely related to the northeastern Pacific species Monanchora pulchra, collected from deep waters off the Aleutian Islands of Alaska. Through an assortment of NMR, MS, ECD, computational chemical shifts calculation, and DP4, chemical structures of these metabolites have been characterized as spirocyclic ring-containing sesterterpenoid (1) and cholestane-type steroidal analogues (2 and 3). These compounds exhibited the inhibition of ß-catenin response transcription (CRT) through the promotion of ß-catenin degradation, which was in part implicated in the antiproliferative activity against two CRT-positive colon cancer cell lines.