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Biochemical Pharmacology (v.85, #10)
The circadian clock circuitry and the AHR signaling pathway in physiology and pathology by George Anderson; Timothy V. Beischlag; Manlio Vinciguerra; Gianluigi Mazzoccoli (pp. 1405-1416).
Life forms populating the Earth must face environmental challenges to assure individual and species survival. The strategies predisposed to maintain organismal homeostasis and grant selective advantage rely on anticipatory phenomena facing periodic modifications, and compensatory phenomena facing unpredictable changes. Biological processes bringing about these responses are respectively driven by the circadian timing system, a complex of biological oscillators entrained to the environmental light/dark cycle, and by regulatory and metabolic networks that precisely direct the body's adjustments to variations of external conditions and internal milieu. A critical role in organismal homeostatic functions is played by the aryl hydrocarbon receptor (AHR) complex, which senses environmental and endogenous compounds, influences metabolic responses controlling phase I/II gene expression, and modulates vital phenomena such as development, inflammation and adaptive immunity. A physiological cross-talk between circadian and AHR signaling pathways has been evidenced. The alteration of AHR signaling pathway deriving from genetic damage with polymorphisms or mutations, or produced by exogenous or endogenous AHR activation, and chronodisruption caused by mismatch between the body's internal clock and geophysical time/social schedules, are capable of triggering pathological mechanisms involved in metabolic, immune-related and neoplastic diseases. On the other hand, the molecular components of the circadian clock circuitry and AHR signaling pathway may represent useful tools for preventive interventions and valuable targets of therapeutic approaches.
Keywords: AHR; ARNT; Clock gene; Circadian rhythm
Current perspectives on parathyroid hormone (PTH) and PTH-related protein (PTHrP) as bone anabolic therapies by Pedro Esbrit; María José Alcaraz (pp. 1417-1423).
Display OmittedOsteoporosis is characterized by low bone mineral density and/or poor bone microarchitecture leading to an increased risk of fractures. The skeletal alterations in osteoporosis are a consequence of a relative deficit of bone formation compared to bone resorption. Osteoporosis therapies have mostly relied on antiresorptive drugs. An alternative therapeutic approach for osteoporosis is currently available, based on the intermittent administration of parathyroid hormone (PTH). Bone anabolism caused by PTH therapy is mainly accounted for by the ability of PTH to increase osteoblastogenesis and osteoblast survival. PTH and PTH-related protein (PTHrP)–an abundant local factor in bone- interact with the common PTH type 1 receptor with similar affinities in osteoblasts. Studies mainly in osteoporosis rodent models and limited data in postmenopausal women suggest that N-terminal PTHrP peptides might be considered a promising bone anabolic therapy. In addition, putative osteogenic actions of PTHrP might be ascribed not only to its N-terminal domain but also to its PTH-unrelated C-terminal region. In this review, we discuss the underlying cellular and molecular mechanisms of the anabolic actions of PTH and the similar potential of PTH-related protein (PTHrP) to increase bone mass and improve bone regeneration.
Keywords: PTH; PTHrP; Bone formation; Bone repair; Bone anabolic therapyAbbreviations; BMPs; bone morphogenetic proteins; BMU; basic multicellular unit; ERKs; extracellular signal-regulated kinases; FGF-2; basic fibroblast growth factor-2; FoxO; forkhead box O; IGFs; insulin-like growth factors; LRP; low density-receptor-like proteins; MAPK; mitogen-activated protein kinase; PI-3; K; phosphoinositide 3-kinase; PKA; protein kinase A; PKC; protein kinase C; PLC; phospholipase C; PPR; PTH/PTHrP type 1 receptor; PTH; parathyroid hormone; PTHrP; PTH-related protein; RANKL; receptor activator of NF-κB ligand; Runx2; runt-related transcription factor 2; TGF-β; transforming growth factor-β; VEGF; vascular endothelial growth factor
Antitumor efficacy of the heparanase inhibitor SST0001 alone and in combination with antiangiogenic agents in the treatment of human pediatric sarcoma models by G. Cassinelli; C. Lanzi; M. Tortoreto; D. Cominetti; G. Petrangolini; E. Favini; N. Zaffaroni; C. Pisano; S. Penco; I. Vlodavsky; F. Zunino (pp. 1424-1432).
The activity of heparanase is responsible for heparan sulfate cleavage, thus resulting in the release of heparan sulfate-bound growth factors. Since heparanase activity is upregulated in several tumor types and is implicated in the malignant behavior, the enzyme is regarded as a promising target for antitumor therapy. Based on previous evidence that the heparanase inhibitor SST0001, a non-anticoagulant N-acetylated glycol split heparin, is effective against an Ewing's sarcoma model, the present study was performed to extend the preclinical evaluation of SST0001 to a panel of pediatric sarcoma models, representative of various tumor histotypes (soft tissue and bone sarcomas) and to further elucidate its mode of action. SST0001 treatment downregulated several angiogenic factors in the conditioned media of sarcoma cells, inhibited the pro-invasive effect of heparin-binding factors (VEGF, bFGF, HGF, PDGF), and abrogated PDGF receptor tyrosine phosphorylation. Subcutaneous administration of SST0001 was very effective, resulting in a significant growth inhibition (range, 64–95%) of all tested tumor xenografts. The efficacy of SST0001 was enhanced in combination with antiangiogenic agents (bevacizumab, sunitinib) as documented by the high rate of complete response. The synergistic effect of SST0001 in combination with antiangiogenic agents is consistent with the heparanase mode of action and with the relevant role of heparin-binding proangiogenic/growth factors in the malignant behavior of sarcoma cells.
Keywords: Heparanase inhibitor; SST0001; Antiangiogenic agents; Pediatric sarcomas; Antitumor activity
Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect by Christopher F. Prada; Raquel Álvarez-Velilla; Rafael Balaña-Fouce; Carlos Prieto; Estefania Calvo-Álvarez; Jose Miguel Escudero-Martínez; José María Requena; César Ordóñez; Alessandro Desideri; Yolanda Pérez-Pertejo; Rosa M. Reguera (pp. 1433-1440).
In this article, we characterized the leishmanicidal profile of several camptothecin derivatives. Gimatecan was the most powerful DNA TopIB poison, killing infecting-amastigotes in the nanomolar range.The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA–protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.
Keywords: Abbreviations; Top; DNA topoisomerases; TopIB; DNA topoisomerase IB; LdTopIB; L; .; donovani; TopIB; LiTopIB; L. infantum; TopIB; IFP1.4; infrared fluorescent protein 1.4; SI; 48h; selectivity index at 48; h; MDR-1; multidrug resistant protein 1DNA topoisomerase IB; Gimatecan; Camptothecin; Splenic explants; Leishmania; spp.
Lipid raft modulation by Rp1 reverses multidrug resistance via inactivating MDR-1 and Src inhibition by Un-Jung Yun; Ji-Hye Lee; Kyung Hee Koo; Sang-Kyu Ye; Soo-Youl Kim; Chang-Hun Lee; Yong-Nyun Kim (pp. 1441-1453).
Rp1 synergistic effect with anti-cancer drugs via lipid rafts alteration, MDR-1 redistribution, downregulation, and inactivation, and thus intracellular accumulation of anti-cancer drug.Multidrug resistance (MDR) is a major obstacle to effective cancer therapy. The membrane transporter MDR-1 (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) transporter family, effluxes anti-cancer drugs from cancer cells. Increased activity of MDR-1 is known to be the main mechanism for multidrug resistance. MDR-1 is known to be localized in the cholesterol- and sphingolipid-enriched plasma membrane microdomains, known as lipid rafts. Disruption of lipid rafts by cholesterol depletion alters lipid raft functions, indicating that cholesterol is critical for raft function. Because ginsenosides are structurally similar to cholesterol, in this study, we investigated the effect of Rp1, a novel ginsenoside derivative, on drug resistance using drug-sensitive OVCAR-8 and drug-resistant NCI/ADR-RES and DXR cells. Rp1 treatment resulted in an accumulation of doxorubicin or rhodamine 123 by decreasing MDR-1 activity in doxorubicin-resistant cells. Rp1 synergistically induced cell death with actinomycin D in DXR cells. Rp1 appeared to redistribute lipid rafts and MDR-1 protein. Moreover, Rp1 reversed resistance to actinomycin D by decreasing MDR-1 protein levels and Src phosphorylation with modulation of lipid rafts. Addition of cholesterol attenuated Rp1-induced raft aggregation and MDR-1 redistribution. Rp1 and actinomycin D reduced Src activity, and overexpression of active Src decreased the synergistic effect of Rp1 with actinomycin D. Rp1-induced drug sensitization was also observed with several anti-cancer drugs, including doxorubicin. These data suggest that lipid raft-modulating agents can be used to inhibit MDR-1 activity and thus overcome drug resistance.
Keywords: MDR-1; Lipid rafts; Ginsenosides; Src; Actinomycin D
Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion by Yan Xie; Peter W. Abel; Joseph K. Kirui; Caishu Deng; Poonam Sharma; Dennis W. Wolff; Myron L. Toews; Yaping Tu (pp. 1454-1462).
Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis.
Keywords: PI3Kγ; Breast cancer; Metastasis; Migration and invasion; Lamellipodia
Endoplasmic reticulum stress induced by 2-deoxyglucose but not glucose starvation activates AMPK through CaMKKβ leading to autophagy by Haibin Xi; Julio C. Barredo; Jaime R. Merchan; Theodore J. Lampidis (pp. 1463-1477).
Autophagy, a well-conserved cellular self-eating process, has been shown to play a critical role in the pathophysiology of cancer. Previously, we reported that under normal O2 conditions (21% O2), the dual glucose metabolism inhibitor 2-deoxyglucose (2-DG) activates a cytoprotective autophagic response in cancer cells mainly through the induction of endoplasmic reticulum (ER) stress rather than ATP2 reduction. However, the pathway(s) by which this occurs was unknown. Here, we find that ER stress induced by 2-DG as well as tunicamycin activates AMPK via Ca2+-CaMKKβ leading to stimulation of autophagy. These results suggest a new role for AMPK as a sensor of ER stress. In contrast, we find that although physiologic glucose starvation (GS) leads to ER stress which contributes to autophagy activation, it does so by a different mechanism. In addition to ER stress, GS also stimulates autophagy through lowering ATP and activating the canonical LKB1–AMPK energy sensing pathway as well as through increasing reactive oxygen species resulting in the activation of ERK. Furthermore, under hypoxia we observe that both 2-DG and GS inhibit rather than activate autophagy. This inhibition correlates with dramatically depleted ATP levels, and occurs through reduction of the PI3K III-Beclin1 complex for autophagy initiation, blockage of the conjugation of ATG12 to ATG5 for autophagosome expansion, as well as inhibition of the functional lysosomal compartment for autophagic degradation. Taken together, our data support a model where under normoxia therapeutic (2-DG) and physiologic (GS) glucose restriction differentially activate autophagy, while under hypoxia they similarly inhibit it.
Keywords: Abbreviations; 2-DG; 2-deoxyglucose; 4-PBA; sodium 4-phenylbutyrate; ACC; acetyl-CoA carboxylase; AMPK; AMP-activated protein kinase; ATF6; activating transcription factor 6; ATG; autophagy-related gene; ATP; adenosine triphosphate; BAPTA; BAPTA-AM; Beclin1; coiled-coil, moesin-like BCL2 interacting protein; [Ca; 2+; ]; c; cytoplasmic Ca; 2+; concentration; [Ca; 2+; ]; ER; ER Ca; 2+; concentration; CaMKKβ; Ca; 2+; /calmodulin-dependent kinase kinase β; EGFP; enhanced green fluorescent protein; ER; endoplasmic reticulum; ERK; extracellular signal-regulated protein kinase; EST/Pep A; EST and pepstatin A; Grp78; glucose-regulated protein 78 KDa; GS; glucose starvation; Indo-1; Indo-1-AM; IRE1; inositol requiring enzyme 1; LC3B; microtubule-associated protein 1 light chain 3B; LKB1; liver kinase B1; LTG; LysoTracker Green; Man; mannose; MEF; mouse embryonic fibroblast; MAPK; mitogen-activated protein kinase; mtDNA; mitochondrial DNA; mTOR; mammalian target of rapamycin; NAC; N-acetyl-; l; -cysteine; OM; oligomycin; p53; tumor protein p53; p70S6K; 70; kDa ribosomal protein S6 kinase; PERK; PKR-like ER kinase; PI3K III; class III phosphatidylinositol 3′ kinase; PPP; pentose phosphate pathway; ROS; reactive oxygen species; STO; STO-609; TG; thapsigargin; TM; tunicamycin; TSC; tuberous sclerosis complex; UPR; unfolded protein response2-Deoxyglucose; Autophagy; Endoplasmic reticulum stress; Glucose starvation; Hypoxia
Hinokitiol inhibits platelet activation ex vivo and thrombus formation in vivo by Kuan H. Lin; Jinn R. Kuo; Wan J. Lu; Chi L. Chung; Duen S. Chou; Shih Y. Huang; Hsiu C. Lee; Joen R. Sheu (pp. 1478-1485).
Hinokitiol is a tropolone-related bioactive compound that has been used in hair tonics, cosmetics, and food as an antimicrobial agent. Recently, hinokitiol has attracted considerable interest because of its anticancer activities. Platelet activation plays a crucial role in atherothrombotic processes. We examined the effects of hinokitiol treatment on platelet activation using human platelets. In the present study, hinokitiol (1 and 2μM) inhibited the collagen-induced aggregation of human platelets, but did not inhibit the activation of platelets by other agonists, including thrombin, arachidonic acid, and ADP. Hinokitiol inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt in collagen-activated human platelets, and significantly reduced intracellular calcium mobilization and hydroxyl radical (OH) formation. Hinokitiol also reduced the PKC activation and platelet aggregation stimulated by PDBu. In addition, hinokitiol significantly prolonged thrombogenesis in mice. Hinokitiol did not influence the binding of a fluorescent triflavin probe to the αIIbβ3 integrin on platelet membrane, and neither ODQ nor SQ22536 significantly reversed the hinokitiol-mediated inhibition of platelet aggregation. In conclusion, hinokitiol may inhibit platelet activation by inhibiting the PLCγ2-PKC cascade and hydroxyl radical formation, followed by suppressing the activation of MAPKs and Akt. Our study suggests that hinokitiol may represent a potential therapeutic agent for the prevention or treatment of thromboembolic disorders.
Keywords: Abbreviations; BSA; bovine serum albumin; cPLA; 2; cytosolic phospholipase A; 2; CVDs; cardiovascular diseases; DAG; diacylglycerol; DTS; the dense tubular system; ERK; extracellular signal-regulated kinase; ESR; electron spin resonance; HO; hydroxyl radical; IP; 3; inositol 1,4,5-trisphosphate; JNK; c-Jun N-terminal kinase; NTG; nitroglycerin; PGE; 1; prostaglandin E; 1; PKC; protein kinase C; PRP; platelet-rich plasma; TxA; 2; thromboxane A; 2Hinokitiol; Hydroxyl radical; MAPK; Occlusion time; Platelet activation; PLCγ2
Angiotensin II upregulates KCa3.1 channels and stimulates cell proliferation in rat cardiac fibroblasts by Li-Ping Wang; Yan Wang; Li-Mei Zhao; Gui-Rong Li; Xiu-Ling Deng (pp. 1486-1494).
Ang II stimulates cell proliferation mediated by upregulating KCa3.1 channels via interacting with the AT1R and activating AP-1 complex through ERK1/2, p38-MAPK and PI3K/Akt signaling pathways in cultured rat cardiac fibroblasts.The proliferation of cardiac fibroblasts is implicated in the pathogenesis of myocardial remodeling and fibrosis. Intermediate-conductance calcium-activated K+ channels (KCa3.1 channels) have important roles in cell proliferation. However, it is unknown whether angiotensin II (Ang II), a potent profibrotic molecule, would regulate KCa3.1 channels in cardiac fibroblasts and participate in cell proliferation. In the present study, we investigated whether KCa3.1 channels were regulated by Ang II, and how the channel activity mediated cell proliferation in cultured adult rat cardiac fibroblasts using electrophysiology and biochemical approaches. It was found that mRNA, protein, and current density of KCa3.1 channels were greatly enhanced in cultured cardiac fibroblasts treated with 1μM Ang II, and the effects were countered by the angiotensin type 1 receptor (AT1R) blocker losartan, the p38-MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the PI3K/Akt inhibitor LY294002. Ang II stimulated cell proliferation and the effect was antagonized by the KCa3.1 blocker TRAM-34 and siRNA targeting KCa3.1. In addition, Ang II-induced increase of KCa3.1 expression was attenuated by transfection of activator protein-1 (AP-1) decoy oligodeoxynucleotides. These results demonstrate for the first time that Ang II stimulates cell proliferation mediated by upregulating KCa3.1 channels via interacting with the AT1R and activating AP-1 complex through ERK1/2, p38-MAPK and PI3K/Akt signaling pathways in cultured adult rat cardiac fibroblasts.
Keywords: Angiotensin II; Cardiac fibroblasts; Intermediate-conductance Ca; 2+; activated K; +; channels; Proliferation
Differential sensitivities of the vascular KATP channel to various PPAR activators by Yingji Wang; Lei Yu; Ningren Cui; Xin Jin; Daling Zhu; Chun Jiang (pp. 1495-1503).
Several agonists of the peroxisome proliferator-activated receptors (PPARs) are currently used for the treatment of metabolic disorders including diabetes. We have recently shown that one of them, Rosiglitazone, inhibits the vascular ATP-sensitive K+ (KATP) channel and compromises the coronary vasodilation by the β-adrenoceptor agonist. Here, we show evidence for the channel inhibition by various PPAR agonists, information that may be useful for finding new therapeutical agents with less cardiovascular side-effects and more selective KATP channel blockers targeting at the Kir6.1 subunit. Structural comparison of these PPAR agonists may shed insight into the critical chemical groups for the channel inhibition.Kir6.1/SUR2B channel was expressed in HEK293 cells and studied in whole-cell voltage clamp. The Kir6.1/SUR2B channel was strongly inhibited by several PPARγ agonists with potencies similar to, or higher than, that of Rosiglitazone, while other PPARγ agonists barely inhibited the channel. The Kir6.1/SUR2B channel was also inhibited by PPARα and PPARβ/δ agonists with intermediate potencies. The structure necessary for the channel inhibition appears to include the thiazole linked to an aromatic or furan ring. Additions of side groups such as small aliphatic chain increased the potency for channel inhibition, while additions of aromatic rings reduced it. These results indicate that the PPARγ agonists with weak KATP channel inhibition may be potential candidates as therapeutical agents, and those with strong channel inhibition may be used as selective KATP channel blockers. The structural information of the PPAR agonists may be useful for the development of new therapeutical modalities with less cardiovascular side-effects.
Keywords: Abbreviations; DM2; Type-2 diabetes mellitus; K; ATP; ATP-sensitive K; +; K; ir; inwardly rectifying potassium channel; PPAR; peroxisome proliferator-activated receptor; SUR; sulfonylurea receptor; TZDs; thiazolidinediones; VSM; vascular smooth musclesPPAR; Thiazolidinedione; Type-2 diabetes mellitus; Potassium channel; Vascular tones; Cardiovascular
A novel benzo[ d]imidazole derivate prevents the development of dextran sulfate sodium-induced murine experimental colitis via inhibition of NLRP3 inflammasome by Wen Liu; Wenjie Guo; Jing Wu; Qiong Luo; Feifei Tao; Yanhong Gu; Yan Shen; Jianxin Li; Renxiang Tan; Qiang Xu; Yang Sun (pp. 1504-1512).
Small molecular compound Fc11a-2 attenuates dextran sulfate sodium (DSS)-induced experimental colitis in mice through inhibiting NLRP3 inflammasome activation.NLRP3 inflammasome has been reported to be associated with various kinds of immunological diseases including colitis. However, there are few drug candidates targeting inflammasomes for the treatment of colitis. In the present study, we aimed at examining the effect of 1-ethyl-5-methyl-2-phenyl-1H-benzo[ d]imidazole, a synthetic small molecular compound also named Fc11a-2, for the treatment of dextran sulfate sodium (DSS)-induced experimental colitis in mice via targeting NLRP3 inflammasome. Treatment with Fc11a-2 dose-dependently attenuated the loss of body weight and shortening of colon length induced by DSS. In addition, the disease activity index, histopathologic scores and myeloperoxidase activity were also significantly reduced by Fc11a-2 treatment. Moreover, protein and mRNA levels of DSS-induced proinflammatory cytokines in colon, including TNF-α, IL-1β, IL-18, IL-17A and IFN-γ, were markedly suppressed by Fc11a-2. Furthermore, a decreased CD11c+ macrophage infiltration in colons and inactivation of caspase-1 in peritoneal macrophages were detected in Fc11a-2-treated mice. The mechanism of action of Fc11a-2 was related to the inhibition of the cleavage of pro-caspase-1, pro-IL-1β and pro-IL-18 which in turn suppressed the activation of NLRP3 inflammasome. Taken together, our results demonstrate the ability of Fc11a-2 to inhibit NLRP3 inflammasome activation and its potential use in the treatment of inflammatory bowel diseases.
Keywords: NLRP3 inflammasome; Experimental colitis; Inflammatory bowel diseases; Ulcerative colitis; IL-1β
Systematic evaluation of 640 FDA drugs for their effect on CD4+Foxp3+ regulatory T cells using a novel cell-based high throughput screening assay by Rui Mao; Wei Xiao; Haitao Liu; Bo Chen; Bing Yi; Piotr Kraj; Jin-Xiong She (pp. 1513-1524).
Regulatory T cells (Treg), which play a pivotal role in maintaining immune homeostasis by suppressing the proliferation of effector T cells, have great therapeutic potential for autoimmune diseases and transplantation. However, progress on their clinical application has been hampered by the lack of high throughput screening (HTS) strategies for the systematic and rapid evaluation of existing drugs and the identification of novel drug candidates. In this report, we present an innovative in vitro HTS assay using CD4+ T cells from Foxp3-GFP transgenic mice that specifically express the GFP signal in Foxp3+ Treg cells detectable by FACS analysis in a high throughput manner. Systematic evaluation of 640 FDA-approved drugs revealed that 70 drugs increased the number of Treg cells with suppression function only in the presence of TGFβ, 75 drugs increased Treg numbers even in the absence of TGFβ, and 32 drugs increased Treg numbers synergistically with TGFβ. The identified Treg-promoting drugs include those previously known to induce Treg (rapamycin and retinoic acid), statins, glucocorticoids and drugs in many other categories. Furthermore, Treg cells cultured with the identified drugs possess surface and intracellular markers characteristic of natural Treg cells and possess suppressive function. These results suggest that this Treg HTS assay can be used to screen compound libraries to identify novel chemical entities for Treg-based immune therapies.
Keywords: Immunosuppressive drugs; Regulatory T cells; Immune tolerance; High throughput screening; Foxp3Abbreviations; APC; antigen presenting cell; CSA; cyclosporine A; CTLA-4; Cytotoxic T-Lymphocyte Antigen 4; DMEM; Dulbecco's modified Eagle's medium; FACS; fluorescence activated cell sorting; FBS; fetal bovine serum; FDA; the Food and Drug Administration; Foxp3; forkhead box P3; GFP; Green fluorescent protein; GITR; glucocorticoid-induced TNFR-related protein; GVHD; graft; vs.; host disease; HTS; high throughput screening; IL; interleukin; NC; negative control; RA; Retinoic acid; rIL-2; recombinant interleukin 2; TGFβ1; transforming growth factor beta 1; Treg; regulatory T cells.
Molecular mechanisms underlying the anti-obesity potential of prunetin, an O-methylated isoflavone by Tae-Gue Ahn; Gabsik Yang; Heon-Myung Lee; Myung-Dong Kim; Ho-Young Choi; Kyoung-Sik Park; Sun-Dong Lee; Yoon-Bum Kook; Hyo-Jin An (pp. 1525-1533).
Prunetin is an O-methylated isoflavone, which is a type of flavonoid. There are a limited number of reports detailing the biological activities of prunetin. Although an anti-inflammatory effect of prunetin has been reported in vitro, to our knowledge, there have been no reports on anti-adipogenic effects of prunetin in obese animals. The aims of this study were to determine whether prunetin suppresses high-fat diet (HFD)-induced adipogenesis in the liver and visceral adipose tissues of mice, and to explore the underlying mechanisms mediating the actions of prunetin. To this end, mice were fed a HFD for 10 weeks to induce obesity, and prunetin (10μg/kg or 20μg/kg) was administered in the last 3 weeks. Compared to saline-treated mice, mice treated with prunetin showed significantly reduced body weight gain, visceral fat pad weights, and plasma glucose levels. We found that prunetin significantly inhibited the HFD-induced upregulation of the expression of important adipogenic genes (PPARγ, C/EBPα, SREBP, aP2, LPL adiponectin, and leptin), and suppressed HFD-mediated increase in expression of lipid metabolism-related genes (SREBP, PPARγ, LXR, and HMG-CoA) in the liver tissues. Furthermore, prunetin induced expression of adiponectin receptors 1 and 2 (adipoR1, adipoR2), as well as that of AMP-activated protein kinase (AMPK) in the liver and adipose tissue. These results suggest that prunetin mediates anti-obesity/adipogenesis effects by suppressing obesity-related transcription through a feedback mechanism that regulates the expression of adiponectin, adipoR1, adipoR2, and AMPK.
Keywords: Prunetin; High-fat diet (HFD); Mice; Adipogenic genes; Adiponectin receptors; AMP-activated protein kinase (AMPK)
α1-Adrenoceptor and serotonin 5-HT1A receptor affinity of homobivalent 4-aminoquinoline compounds: An investigation of the effect of linker length by Junli Chen; Ahsan K. Murad; Laurence P.G. Wakelin; William A. Denny; Renate Griffith; Angela M. Finch (pp. 1534-1541).
α1-adrenoceptor (α1-AR) subtype-selective ligands lacking off-target affinity for the 5-HT1A receptor (5-HT1A-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2–12 atoms (C2–C12) for α1A, α1B and α1D -ARs and the 5-HT1A-R. These ligands are α1A-AR antagonists with nanomolar affinity for α1A and α1B -ARs. They display linker-length dependent selectivity for α1A/B -ARs over α1D-AR and the 5-HT1A-R. The C2 diquinoline has the highest affinity for α1A-AR (p K i 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α1A-AR over α1D-AR and 5-HT1A-R respectively. A decrease in affinity for α1-ARs is observed as the linker length increases, reaching a nadir at 5 (α1A/1B-ARs) or 6 (α1D-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α1A/1B/1D-ARs) or 8 (5-HT1A-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α1D-AR and 5-HT1A-R affinity of these compounds makes them promising leads for future drug development of α1A-AR selective ligands without α1D-AR and the 5-HT1A-R off-target activity.
Keywords: GPCR; α; 1; -Adrenergic receptor; Subtype selectivity; Serotonin receptor; Diquinoline; Linker length
The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels by Niraj S. Bende; Eunji Kang; Volker Herzig; Frank Bosmans; Graham M. Nicholson; Mehdi Mobli; Glenn F. King (pp. 1542-1554).
One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.
Keywords: Voltage-gated sodium channel; Neurotoxin; Spider-venom peptide; Pore blocker; Gating modifier; Inhibitor cystine knot
Characterization of monomeric and multimeric snake neurotoxins and other bioactive proteins from the venom of the lethal Australian common copperhead ( Austrelaps superbus) by Francesca Marcon; Louise Purtell; Jerran Santos; Peter G. Hains; Pierre Escoubas; Andis Graudins; Graham M. Nicholson (pp. 1555-1573).
Envenomation by Australian copperheads results mainly in muscle paralysis largely attributed to the presence of postsynaptic α-neurotoxins. However, poorly reversible neurotoxic effects suggest that these venoms may contain snake presynaptic phospholipase A2 neurotoxins (SPANs) that irreversibly inhibit neurotransmitter release. Using size-exclusion liquid chromatography, the present study isolated the first multimeric SPAN complex from the venom of the Australian common copperhead, Austrelaps superbus. The multimeric SPAN P-elapitoxin-As1a (P-EPTX-As1a) along with two novel monomeric SPANs and a new postsynaptic α-neurotoxin were then pharmacologically characterized using the chick biventer cervicis nerve-muscle preparation. All SPANs inhibited nerve-evoked twitch contractions at the neuromuscular junction without inhibiting contractile responses to cholinergic agonists or KCl. These actions are consistent with a prejunctional action to inhibit neurotransmitter release, without direct myotoxicity. Furthermore, the multimeric P-EPTX-As1a caused tetanic ‘fade’ in muscle tension under high frequency nerve stimulation, and produced a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes such as taipoxin. Moreover, the neurotoxic α-subunit of P-EPTX-As1a shows high homology to taipoxin α-chain. Several other coagulopathic and myotoxic high mass proteins including a class PIII snake venom metalloproteinase, C-type lectin,l-amino acid oxidase, acetylcholinesterase and phospholipase B were also identified that may contribute to the overall toxicity of A. superbus venom. In conclusion, clinicians should be aware that early antivenom intervention might be necessary to prevent the onset of irreversible presynaptic neurotoxicity caused by multimeric and monomeric SPANs and that A. superbus venom is potentially capable of producing coagulopathic and myotoxic effects.
Keywords: Abbreviations; ACh; acetylcholine; AChE; acetylcholinesterase; BCA; bicinchoninic acid; 4-BPB; 4-bromophenacyl bromide; CBCNM; chick biventer cervicis nerve-muscle; CCh; carbachol; D/C; disintegrin-like/cysteine rich; EPP; end-plate potential; ESI-QTOF MS; electrospray ionization quadrupole time-of-flight mass spectrometry; LAAO; l; -amino acid oxidase; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; nAChR; nicotinic acetylcholine receptor; NSW; New South Wales; PDGF; platelet-derived growth factor; PLA; 2; phospholipase A; 2; PLB; phospholipase B; EPTX; elapitoxin; RP-HPLC; reversed-phase high-pressure liquid chromatography; SA; sinapinic acid; SPAN; snake presynaptic phospholipase A; 2; neurotoxin; sPLA; 2; secretory phospholipase A; 2; SVMP; snake venom metalloproteinase; t; 90; time to 90% neuromuscular blockade; THAP; 2,4,6-trihydroxyacetophenone; TFA; trifluoroacetic acid; TSAV; tiger snake antivenom; VDGF; venom-derived growth factor; V; e; elution volume; V; o; void volumeSnake toxin; Mass fragmentation; Snake presynaptic phospholipase A; 2; neurotoxin; Australian copperhead; Snake antivenom