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Biochemical Pharmacology (v.84, #11)

Editorial Board (pp. iii).

THIOCYANATE: A potentially useful therapeutic agent with host defense and antioxidant properties by Joshua D. Chandler; Brian J. Day (pp. 1381-1387).

Thiocyanate (SCN) functions in host defense as part of the secreted lactoperoxidase (LPO) microbicidal pathway. SCN is the preferred substrate for LPO-driven catalytic reduction of hydrogen peroxide (H₂O₂) forming hypothiocyanous acid (HOSCN). HOSCN is selectively generated by many peroxidase enzymes that can utilize SCN including: eosinophil peroxidase (EPO), gastric peroxidase (GPO), myeloperoxidase (MPO), salivary peroxidase (SPO), and thyroid peroxidase (TPO). These enzymes generate HOSCN through a two-electron halogenation reaction. HOSCN is a potent microbicidal agent that kills or nullifies invading pathogens but is better tolerated by host tissue. Some controversy exists as to whether physiologic levels of HOSCN are non-toxic to host tissue, but the disagreement appears to be based on results of enzymatic generation (yielding moderate steady-state exposure) versus direct high level acute exposure in mammalian cell lines. This apparent duality is also true of other endogenous oxidants such as hydrogen peroxide and relates to the difference between physiologically relevant oxidant production versus supra-physiologic bolus dosing approaches. SCN has antioxidant properties that include the ability to protect cells against oxidizing agents such as hypochlorous acid (HOCl) and repair protein chloramines. SCN is an important endogenous molecule that has the potential to interact in complex and elegant ways with its host environment and foreign organisms. SCN's diverse properties as both host defense and antioxidant agent make it a potentially useful therapeutic.

Keywords: Abbreviations; SCN; thiocyanate; LPO; lactoperoxidase; HOSCN; hypothiocyanous acid; EPO; eosinophil peroxidase; GPO; gastric peroxidase; MPO; myeloperoxidase; SPO; salivary peroxidase; TPO; thyroid peroxidase; HOCl; hypochlorous acid; ELF; epithelial lining fluid; NLF; nasal lining fluid; Pa; pseudomonas aeruginosa; BALF; bronchoalveolar lavage fluid; NIS; sodium-iodide symporter; CFTR; cystic fibrosis transmembrane conductance regulator; CaCC; calcium-dependent chloride channel; TCA; trichloroacetic aid; H; ₂; O; ₂; hydrogen peroxide; ^-; OSCN; hypothiocyanite; RS-SCN; sulfenyl thiocyanates; RSOH; sulfenic acids; RSSR; disulfides; GSH; glutathione; GSSG; glutathione disulfide; TBN; 2-nitro-5thiobenzoate; SCN; ₂; thiocyanaogen; GOX; glucose oxidase; IFN-γ; interferon gamma; Duox; dual oxidase; HOBr; hypobromous acid; HUVEC; human umbilical vein endothelial cells; AEC; alveolar epithelial cells; CF; cystic fibrosis; IL; interleukinCystic fibrosis; Hypothiocyanite; Hypochlorite; Lactoperoxidase; Myeloperoxidase

Clinical perspectives for irreversible tyrosine kinase inhibitors in cancer by Caterina Carmi; Marco Mor; Pier Giorgio Petronini; Roberta R. Alfieri (pp. 1388-1399).

Irreversible inhibitors provide potent and selective inhibition of tyrosine kinase enzymes. Use of such inhibitors has proved promising in overcoming the tumor resistance encountered with reversible tyrosine kinase inhibitors. Irreversible inhibitors inactivate their protein target through covalent interaction with a nucleophilic cysteine residue within the nucleotide binding pocket of the kinase domain.Different irreversible tyrosin kinase inhibitors directed against epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor tyrosine kinase (FGFR) have been developed and some of them have been employed clinically as anticancer agents. This review focuses on recent preclinical and clinical progress with currently available irreversible tyrosine kinase inhibitors. The chemical structures of the candidates, structure-activity relationships, biological activities and results of current clinical investigations are described.

Keywords: Abbreviations; AE; adverse event; BTK; Bruton's tyrosine kinase; CI; confidence interval; CLL; chronic lymphocytic leukemia; CR; complete response; DTL; dose limiting toxicity; EGFR; epidermal growth factor receptor; FGFR; fibroblast growth factor receptor; HR; hazard ratio; MTD; maximum tolerated dose; NSCLC; non small cell lung cancer; NHL; Non-Hodgkin's lymphoma; ORR; overall response rate; OS; overall survival; PD; progressive disease; PDGFR; platelet derived growth factor receptor; PFS; progression free survival; PR; partial response; SCCHN; squamous cell carcinoma of the head and neck; SD; stable disease; TK; tyrosine kinase; TKI; tyrosine kinase inhibitor; VEGFR; vascular endothelial growth factor receptorTyrosine kinase; Irreversible inhibitors; EGFR; BTK; Covalent interaction

The role of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate during viral replication by Leen Delang; Jan Paeshuyse; Johan Neyts (pp. 1400-1408).

Phosphoinositides (PI) are phospholipids that mediate signaling cascades in the cell by binding to effector proteins. Reversible phosphorylation of the inositol ring at positions 3, 4 and 5 results in the synthesis of seven different phosphoinositides. Each phosphoinositide has a unique subcellular distribution with a predominant localization in subsets of membranes. These lipids play a major role in recruiting and regulating the function of proteins at membrane interfaces [1]. Several bacteria and viruses modulate and exploit the host PI metabolism to ensure efficient replication and survival. Here, we focus on the roles of cellular phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4-kinases (PI4Ks) during the replication cycle of various viruses. It has been well documented that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ, EC 2.7.1.67) is indispensable for viral RNA replication of several picornaviruses. Two recruitment strategies were reported: (i) binding and modulation of GBF1/Arf1 to enhance recruitment of PI4KIIIβ and (ii) interaction with ACBD3 for recruitment of PI4KIIIβ. PI4KIII has also been demonstrated to be crucial for hepatitis C virus (HCV) replication. PI4KIII appears to be directly recruited and activated by HCV NS5A protein to the replication complexes. In contrast to picornaviruses, it is still debated whether the α or the β isoform is the most important. PI4KIII can be explored as a target for inhibition of viral replication. The challenge will be to develop highly selective inhibitors for PI4KIIIα and/or β and to avoid off-target toxicity.

Keywords: PI4KIIIα; PI4KIIIβ; PI4P; Viral replication; Antiviral target

Gli family transcription factors are drivers of patupilone resistance in ovarian cancer by Simona Mozzetti; Enrica Martinelli; Giuseppina Raspaglio; Silvia Prislei; Marta De Donato; Flavia Filippetti; Shohreh Shahabi; Giovanni Scambia; Cristiano Ferlini (pp. 1409-1418).

Outline of the Gli1 involvement in the process of patupilone-resistance.Epothilones constitute a novel class of antitubulin agents that are active in patients who relapse after treatment with other chemotherapeutics. This study investigated the molecular mechanisms leading to the onset of epothilone-B (patupilone) resistance in ovarian cancer. Results demonstrated that the Gli family of transcription factors was overexpressed in resistant cells and that treatment with a specific Gli1 inhibitor (GANT58) made cells more susceptible to treatment, partially reversing drug resistance. We also demonstrated that Gli1 knockdown halted growth in resistant cells that were exposed to patupilone, confirming that Gli1 is capable of directly mediating epothilone-B resistance. Another observation from our research was that patupilone-resistant cells produced HGF and acquired characteristics of a mesenchymal phenotype. However, HGF silencing alone was not capable of converting the drug-resistant phenotype to a susceptible one, and in this case we demonstrated that Gli1 overexpression led to an increase in HGF, establishing a functional link between Gli1 and HGF. These results demonstrated that Gli1 played a key role in driving resistance to patupilone, suggesting that the combination of epothilones and Gli1-targeted agents could be exploited to improve outcomes in ovarian cancer patients resistant to standard treatments.

Keywords: Epothilones; Patupilone; Gli1; Gli2; Gli3; Gli-inhibitors; Drug resistance

1,3-Bis(3,5-dichlorophenyl) urea compound ‘COH-SR4’ inhibits proliferation and activates apoptosis in melanoma by Sharad S. Singhal; James Figarola; Jyotsana Singhal; Kathryn Leake; Lokesh Nagaprashantha; Christopher Lincoln; B. Gabriel Gugiu; David Horne; Richard Jove; Sanjay Awasthi; Samuel Rahbar (pp. 1419-1427).

SR4 induces growth inhibition and apoptosis in melanoma. Apoptotic cells showed green fluorescence.The current clinical interventions in malignant melanomas are met with poor response to therapy due to dynamic regulation of multiple melanoma signaling pathways consequent to administration of single target agents. In this context of limited response to single target agents, novel candidate molecules capable of effectively inducing tumor inhibition along with targeting multiple critical nodes of melanoma signaling assume translational significance. In this regard, we investigated the anti-cancer effects of a novel dichlorophenyl urea compound called COH-SR4 in melanoma. The SR4 treatment decreased the survival and inhibited the clonogenic potential of melanomas along with inducing apoptosis in vitro cultures. SR4 treatments lead to inhibition of GST activity along with causing G2/M phase cell cycle arrest. Oral administration of 4mg/kg SR4 leads to effective inhibition of tumor burdens in both syngeneic and nude mouse models of melanoma. The SR4 treatment was well tolerated and no overt toxicity was observed. The histopathological examination of resected tumor sections revealed decreased blood vessels, decrease in the levels of angiogenesis marker, CD31, and proliferation marker, Ki67, along with an increase in pAMPK levels. Western blot analyses of resected tumor lysates revealed increased PARP cleavage, Bim, pAMPK along with decreased pAkt, vimentin, fibronectin, CDK4 and cyclin B1. Thus, SR4 represents a novel candidate for the further development of mono and combinatorial therapies to effectively target aggressive and therapeutically refractory melanomas.

Keywords: Melanoma; SR4; Syngeneic model; Tumor xenograftsAbbreviation; COH-SR4; 1,3-bis(3,5-dichlorophenyl)urea “City of Hope compound”

Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity by Vania Vidimar; Xiangjun Meng; Marcelina Klajner; Cynthia Licona; Ludivine Fetzer; Sébastien Harlepp; Pascal Hébraud; Marjorie Sidhoum; Claude Sirlin; Jean-Philippe Loeffler; Georg Mellitzer; Gianni Sava; Michel Pfeffer; Christian Gaiddon (pp. 1428-1436).

Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation.

Keywords: Abbreviations; CHOP; C/EBP homologous protein; DDIT3; DNA damage inducible transcript 3; FACS; fluorescence activated cell sorter; FRET; Förster resonance energy transfer; NAC; n-acetylcysteineOrganometallic; Anticancer; Caspase 8; ROS; Ruthenium

Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase by Tran Thi Hien; Won Keun Oh; Bui Thu Quyen; Trong Tuan Dao; Jung-Hoon Yoon; Sei Young Yun; Keon Wook Kang (pp. 1437-1450).

Endothelial nitric oxide synthase (eNOS) has important regulatory functions in vascular tone, and impaired endothelium-dependent vasodilatation is a key event in diabetes and atherosclerosis. Vitis amurensis grapes containing resveratrol oligomers are consumed as wine and fruit and have antioxidative and neuroprotective effects. In this study, our goal was identify the most potent eNOS-activating compound among six stilbenes and oligostilbenes found in V. amurensis and to clarify its molecular mechanism. Among the six tested compounds, amurensin G most potently relaxed endothelium-intact aortic rings and increased eNOS phosphorylation and nitric oxide (NO) production. Amurensin G increased both estrogen receptor (ER) phosphorylation and ER-dependent gene transcription, and ERα or ERβ inhibition suppressed amurensin G-mediated eNOS phosphorylation. Amurensin G enhanced the activities of phosphatidylinositol 3-kinase (PI3K) and Src and their chemical inhibitors suppressed amurensin G-stimulated eNOS phosphorylation. Moreover, amurensin G activated AMP-activated protein kinase (AMPK), and amurensin G-stimulated eNOS phosphorylation and PI3K activation were reversed by AMPK inhibition. ER inhibition reversed AMPK-dependent PI3K activation in response to amurensin G. Amurensin G-mediated endothelium-dependent relaxation was blocked by inhibition of AMPK, ER, Src, or PI3K. These results suggest that amurensin G enhances NO production via eNOS phosphorylation in endothelial cells, and ER-dependent AMPK/PI3K pathways are required. Amurensin G would be applicable to prevent atherosclerosis.

Keywords: Abbreviations; ACC; acetyl-CoA carboxylase; ACh; acetylcholine; AICAR; 5-aminoimidazole-4-carboxamide-1-β-; d; -ribofuranoside; AMPK; adenosine 5′-monophosphate-activated protein kinase; CA-AMPK; constitutive active AMPK; CaMK II; calmodulin-dependent protein kinase II; DAF-2; DA (4,5-diaminofluorescein diacetate); DN-AMPK; dominant negative mutant of AMPK; eNOS; endothelial nitric oxide synthase; ER; estrogen receptor; ERK; extracellular signal-regulated kinase; GR; glucocorticoid receptor; JNK; c-Jun activated protein kinase; LKB1; liver kinase B1; MAPK; mitogen-activated protein kinase; l; -NAME; N; ^G; -nitro-; l; -arginine methyl ester; MPP; Methyl-piperidino-pyrazole; NO; nitric oxide; NOS; nitric oxide synthase; PBS; phosphate buffered saline; PI3K; phosphatidylinositol 3-kinase; PP2; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; THC; tetrahydrochryseneAmurensin G; AMPK; eNOS; Estrogen receptor; PI3-kinase/Akt

Pharmacodynamic and pharmacokinetic analysis of apoE4 [L261A, W264A, F265A, L268A, V269A], a recombinant apolipoprotein E variant with improved biological properties by Angeliki Lampropoulou; Vassilis I. Zannis; Kyriakos E. Kypreos (pp. 1451-1458).

Physiological levels of wild-type (wt) apolipoprotein E (apoE) in plasma mediate the clearance of cholesterol-rich atherogenic lipoprotein remnants while higher than normal plasma apoE concentrations fail to do so and trigger hypertriglyceridemia. This property of wt apoE reduces significantly its therapeutic value as a lead biological for the treatment of dyslipidemia. Recently, we reported the generation of a recombinant apoE variant, apoE4 [L261A, W264A, F265A, L268A, V269A] (apoE4mut1) with improved biological functions. Specifically, in apoE-deficient (apoE^−/−) mice this variant can normalize high plasma cholesterol levels without triggering hypertriglyceridemia, even at supraphysiological levels of expression. In the present study we performed pharmacodynamic and pharmacokinetic analysis of apoE4mut1 in experimental mice. Using adenovirus-mediated gene transfer in LDL receptor deficient (LDLr^−/−) mice, we show that the cholesterol lowering potential of apoE4mut1 is dependent on the expression of a functional classical LDLr. Bolus infusion of apoE4mut1-containing proteoliposomes in apoE^−/− mice fed western-type diet for 6 weeks indicated that exogenously synthesized apoE4mut1 maintains intact its ability to normalize the high cholesterol levels of these mice with a maximum pharmacological effect obtained at 10h post-treatment. Interestingly, plasma cholesterol levels remained significantly reduced up to 24h following intravenous administration of apoE4mut1 proteoliposomes. Measurements of plasma apoE levels indicated that apoE4mut1 in the form of proteoliposomes used in the study has a half-life of 15.8h. Our data suggest that purified apoE4mut1 may be an attractive new candidate for the acute correction of hypercholesterolemia in subjects expressing functional LDL receptor.

Keywords: Abbreviations; ApoE; apolipoprotein E; ApoE2; apolipoprotein E2; ApoE3; apolipoprotein E3; ApoE4; apolipoprotein E4; ApoE; ^−/−; apoE-deficient; apoA-I; apolipoprotein A-I; Ad; adenovirus; ELISA; ELISA-linked immunoassay; GFP; green fluorescence protein; HDL; high density lipoprotein; PCR; polymerase chain reaction; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; VLDL; very low density lipoprotein; wt; wild-typeApolipoproteins E; Pharmacodynamics; Pharmacokinetics; Hypercholesterolemia; LDL-receptor; LRP-1

Evaluation of chemical mediators and cellular response during acute and chronic gut inflammatory response induced by dextran sodium sulfate in mice by Allisson Freire Bento; Daniela Ferraz Pereira Leite; Rodrigo Marcon; Rafaela Franco Claudino; Rafael Cypriano Dutra; Maíra Cola; Alessandra Cadete Martini; João B. Calixto (pp. 1459-1469).

Inflammatory events during experimental colitis induced by DSS.Inflammatory bowel disease (IBD) affects millions of people worldwide but its pathophysiology remains unclear. Therefore, experimental models of colitis have contributed crucially for the understanding of IBD, and also in the investigations for effective therapies. Herein we investigated the kinetics of inflammatory mediator production and cell infiltration during acute and chronic dextran sodium sulfate (DSS)-induced colitis. The induction phases with DSS were characterized by severe disease activity with massive colonic polymorphonuclear infiltration and increased levels of tumor necrosis factor-α (TNF-α), keratinocyte-derived chemokine (CXCL1/KC), interleukin (IL)-17 and vascular adhesion molecule-1 (VCAM-1). Interestingly, in the recovery periods, we found marked increase of anti-inflammatory mediators IL-10, IL-4, transforming growth factor-β (TGF-β) and cyclooxygenase 2 (COX-2) that seems be essential for the resolution of intestinal inflammation. Furthermore, nuclear factor κB (NFκB) and regulatory T cell marker forkhead box P3 (FoxP3) were increased gradually during experimental colitis, demonstrating a discrepant profile response and evident immune disbalance in the chronic phase of intestinal mucosal inflammation. Taken together, these results provide valuable information for studies on DSS-induced colitis and especially for the identification of biomarkers that predict disease course and possible therapeutic interventions.

Keywords: Dextran sodium sulfate; Colitis; Inflammatory mediators; Cell influx; Chronic inflammation

Identification of a unique nsaid, fluoro-loxoprofen with gastroprotective activity by Shintaro Suemasu; Naoki Yamakawa; Tomoaki Ishihara; Teita Asano; Kayoko Tahara; Ken-ichiro Tanaka; Hirofumi Matsui; Yoshinari Okamoto; Masami Otsuka; Koji Takeuchi; Hidekazu Suzuki; Tohru Mizushima (pp. 1470-1481).

We previously proposed that direct cytotoxicity of NSAIDs due to their membrane permeabilization activity, together with their ability to decrease gastric prostaglandin E₂, contributes to production of gastric lesions. Compared to loxoprofen (LOX), fluoro-loxoprofen (F-LOX) has much lower membrane permeabilization and gastric ulcerogenic activities but similar anti-inflammatory activity. In this study, we examined the mechanism for this low ulcerogenic activity in rats. Compared to LOX, the level of gastric mucosal cell death was lower following administration of F-LOX. However, the gastric level of prostaglandin E₂ was similar in response to treatment with the two NSAIDs. Oral pre-administration of F-LOX conferred protection against the formation of gastric lesions produced by subsequent administration of LOX and orally administered F-LOX resulted in a higher gastric pH value and mucus content. In the presence of a stimulant of gastric acid secretion, the difference in the ulcerogenic activity of F-LOX and LOX was less apparent. Furthermore, an increase in the mucus was observed in gastric cells cultured in the presence of F-LOX in a manner dependent of increase in the cellular level of cAMP. These results suggest that low ulcerogenic activity of F-LOX involves its both low direct cytotoxicity and protective effect against the development of gastric lesions. This protective effect seems to be mediated through an increase in a protective factor (mucus) and a decrease in an aggressive factor (acid).

Keywords: Abbreviations; ABTS; 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-2NH; ₄; BAPTA-AM; 1,2-bis(2-aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; COX; cyclooxygenase; DAPI; 4′,6-diamidino-2-phenylindole dihydrochloride; EIA; enzyme immunoassay; ELLA; enzyme-linked lectin-binding assay; F-LOX; fluoro-loxoprofen; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; H & E; hematoxylin and eosin; IBMX; 3-isobutyl-1-methylxanthine; LOX; loxoprofen; NSAID; non-steroidal anti-inflammatory drug; PG; prostaglandin; RGM1; rat normal gastric epithelial cell line; SD; Sprague-Dawley; SBA; soybean agglutinin; TCA; trichloroacetic acid; TUNEL; terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelingNSAIDs; Gastrointestinal complications; Mucus

Japonicone A antagonizes the activity of TNF-α by directly targeting this cytokine and selectively disrupting its interaction with TNF receptor-1 by Zhenlin Hu; Jiangjiang Qin; Huahua Zhang; Dan Wang; Yaping Hua; Jieping Ding; Lei Shan; Huizi Jin; Junping Zhang; Weidong Zhang (pp. 1482-1491).

Anti-TNF biologics are effective therapies for various inflammatory diseases. Unfortunately, their clinical use is associated with an increased risk of infections. Selectively inhibiting TNF receptor-1 (TNFR1)-mediated signaling while preserving TNFR2 signaling may reduce inflammation yet maintain host immune response to pathogens. However, few small molecules that selectively target the TNF/TNFR system have been discovered. In the present study, we identified Japonicone A (Jap A), a nature compound derived from Inula japonica Thunb, as a novel TNF-α antagonist, as it reduced the TNF-α-mediated cytotoxicity on L929 cells and inhibited the binding of¹²⁵I-labeled TNF-α to L929 cell surface. Furthermore, Jap A could directly bind to TNF-α rather than TNFR1 as determined by surface plasmon resonance. More importantly, Jap A could effectively inhibit the binding of TNF-α to TNFR1, while displaying only marginal inhibitory effects on that to TNFR2. Jap A also could block TNFR1-mediated signaling as it inhibited TNF-α-induced NF-κB activation in 293 cells. In addition, Jap A suppressed TNF-α-induced expressions of adhesion molecules (ICAM-1, VCAM-1) and chemokine (MCP-1) in the endothelial cells by blocking TNF-α-triggered multiple signaling pathways. Data from in vivo experiments demonstrated that Jap A protected mice from acute hepatitis induced by TNF-α/d-galactosamine, but did not compromise host antiviral immunity in adenovirus-infected mice. These results indicate that Jap A can directly target TNF-α, selectively disrupt its interaction with TNFR1, and antagonize its pro-inflammatory activities without compromising host defense against virus, thus emphasizing the potential of Jap A as an interesting lead compound for development of new anti-inflammatory drugs.

Keywords: Tumor necrosis factor-α; Tumor necrosis factor receptor-1; Japonicone A; Antagonist; Inflammation

Tanshinone IIA sodium sulfonate facilitates endocytic HMGB1 uptake by Yusong Zhang; Wei Li; Shu Zhu; Arvin Jundoria; Jianhua Li; Huan Yang; Saijun Fan; Ping Wang; Kevin J. Tracey; Andrew E. Sama; Haichao Wang (pp. 1492-1500).

TSN-SS facilitates clathrin- or caveolin-dependent endocytosis of exogenous HMGB1, triggering a cellular degradation process, autophagy. Subsequently, endosomes merge with autophagosomes to form amphisomes, which then fuse with lysosomes to degrade the amphisome contents.Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.

Keywords: Abbreviations; HMGB1; high mobility group box 1; LC3; microtubule-associated protein1 light chain 3; TSN-SS; tanshinone IIA sodium sulfonateHMGB1; Macrophage; Endocytosis; Tanshinone; Autophagy

Piperine, an LXRα antagonist, protects against hepatic steatosis and improves insulin signaling in mice fed a high-fat diet by Hyejeong Jwa; Youngshim Choi; Ui-Hyun Park; Soo-Jong Um; Seung Kew Yoon; Taesun Park (pp. 1501-1510).

This study investigated the role of piperine in the transcriptional regulation of liver X receptor α (LXRα) and the effects of dietary piperine on high-fat diet (HFD)-induced hepatic steatosis and insulin resistance in mice. Furthermore, we explored the potential molecular mechanisms through which the protective effects of piperine may work. In the present study, piperine significantly reduced ligand-induced LXRα activity in a dose-dependent manner and gradually disrupted the interaction between ligand-bound LXRα and GST–CBP. In mice, an HFD supplemented with 0.05% piperine (PSD) significantly decreased body and liver weight as well as plasma and hepatic lipid levels. In agreement with our in vitro study, in mice fed an HFD, dietary piperine markedly decreased LXRα mRNA expression and its lipogenic target genes (i.e., SREBP1c, ChREBPα, FAS, and CD36). Piperine also significantly decreased plasma insulin and glucose concentrations, while increasing insulin sensitivity in mice fed an HFD. In addition, piperine downregulated the expression of genes involved in ER stress, including GRP78, activating transcription factor 6, and eukaryotic translation initiation factor 2α, and upregulated GLUT2 translocation from the cytosol to the plasma membrane in the livers of PSD mice. Piperine antagonized LXRα transcriptional activity by abolishing the interaction of ligand-bound LXRα with the co-activator CBP. The effects of piperine on hepatic lipid accumulation were likely regulated via alterations in LXRα-mediated lipogenesis in mice fed an HFD. Dietary piperine also led to reduced ER stress and increased insulin sensitivity and prevented hepatic insulin resistance in mice fed the HFD.

Keywords: Abbreviations; AMPK; AMP-activated protein kinase; LXRα; liver X receptor α; CD36; CD36 antigen; SREBP1c; sterol regulatory element binding protein 1c; ChREBPα; carbohydrate response element binding protein α; ER; endoplasmic reticulum; HFD; high fat diet; BIP; binding immunoglobulin protein; PERK; protein kinase-like ER kinase; Insig-1; insulin-induced gene 1; JNK; C-jun N-terminal kinase; IRS-1; insulin receptor substrate 1; GLUT2; glucose transporter 2; CBP; CREB-binding protein; ND; normal diet; PSD 0.05%; piperine-supplemented diet; ALT; aminotransferase; AST; aspartate aminotransferase; ATF6; activating transcription factor 6; sXBP1; spliced XBP1; eIF2α; phosphorylated eukaryotic translation initiation factor 2α; AKT; protein kinase B; G6Pase; glucose 6-phosphatase; PEPCK; phosphoenolpyruvate carboxykinase; HOMA-IR; homeostasis model assessment of basal insulin resistanceFatty liver; Insulin resistance; Hyperlipidemia; GLUT2; ER stress

Effects of amino acid substitutions at positions 33 and 37 on UDP-glucuronosyltransferase 1A9 (UGT1A9) activity and substrate selectivity by Porntipa Korprasertthaworn; Andrew Rowland; Benjamin C. Lewis; Peter I. Mackenzie; Krongtong Yoovathaworn; John O. Miners (pp. 1511-1521).

UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure–function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), sulfinpyrazone (SFZ), frusemide (FSM), ( S)-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K_m values were generally higher and V_max and CL_int values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL_int values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.

Keywords: UDP-glucuronosyltransferase; UGT1A9; Glucuronidation; Structure–function; Genetic polymorphism

N-Acetylcysteine ethyl ester (NACET): A novel lipophilic cell-permeable cysteine derivative with an unusual pharmacokinetic feature and remarkable antioxidant potential by Daniela Giustarini; Aldo Milzani; Isabella Dalle-Donne; Dimitrios Tsikas; Ranieri Rossi (pp. 1522-1533).

Recent large clinical trials failed to confirm the supposed beneficial effects of N-acetylcysteine (NAC) in preventing oxidative stress-related diseases. This may be due to its low bioavailability. We thought that esterification of the carboxyl group of NAC to produce N-acetylcysteine ethyl ester (NACET) would drastically increase the lipophilicity of NAC, thus greatly improving its pharmacokinetics.In the present work, we report on representative chemical, pharmacological and anti-oxidant properties of NACET, especially in direct comparison with its congener NAC. We found that NACET is rapidly absorbed in rats after oral administration but reaches very low concentrations in plasma. This is due to a unique feature of NACET: it rapidly enters the cells where it is trapped being transformed into NAC and cysteine. After oral treatment, NACET (but not NAC) was able to increase significantly the glutathione content of most tissues examined, brain included, and to protect from paracetamol intoxication in the rat. NACET has also the unique feature to accumulate in human erythrocytes where it behaves as a potent protector against hydroperoxide-induced oxidative damage. Our study shows that being able to enter cells and to produce NAC and cysteine, NACET increases circulating hydrogen sulfide (H₂S), thus representing a good candidate for the oral use as an H₂S producer, with clear advantages over NAC.NACET has the potential to substitute NAC as a mucolytic agent, as a paracetamol antidote and as a GSH-related antioxidant.

Keywords: Glutathione; Cysteine; N; -Acetylcysteine; Paracetamol; Metabolism

Pharmacodynamics, tissue distribution, toxicity studies and antitumor efficacy of the vascular targeting fusion toxin VEGF₁₂₁/rGel by Khalid A. Mohamedali; Gang Niu; Troy A. Luster; Philip E. Thorpe; Haokao Gao; Xiaoyuan Chen; Michael G. Rosenblum (pp. 1534-1540).

As a part of an ongoing assessment of its mechanism of action, we evaluated the in vivo pharmacokinetics, tissue distribution, toxicity and antitumor efficacy of VEGF₁₂₁/rGel, a novel fusion protein. Pharmacokinetic studies showed that VEGF₁₂₁/rGel cleared from the circulation in a biphasic manner with calculated half-lives of 0.3 and 6h for the alpha and beta phases, respectively. Pharmacokinetic evaluation of⁶⁴Cu–DOTA–VEGF₁₂₁/rGel showed relatively high blood retention 30min after injection (26.6±1.73% ID/g), dropping to 11.8±2.83% and 0.82±0.11% ID/g at 60 and 240min post injection, respectively. Tissue uptake studies showed that kidneys, liver and tumor had the highest drug concentrations 48h after administration. The maximum tolerated dose (MTD), based on a QOD×5 i.v. administration schedule, was found to be 18mg/kg with an LD₅₀ of 25mg/kg. Treatment of BALB/c mice with VEGF₁₂₁/rGel at doses up to the MTD caused no alterations in hematologic parameters. However, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) parameters increased in a dose-related manner. The no-observable-adverse-effect-level (NOAEL) was determined to be 20% of the MTD (3.6mg/kg). VEGF₁₂₁/rGel treatment of mice bearing orthotopically-placed MDA-MB-231 breast tumors caused increased vascular permeability of tumor tissue by 53% compared to saline-treated controls. Immunohistochemical analysis showed significant tumor hypoxia and necrosis as a consequence of vascular damage. In summary, VEGF₁₂₁/rGel appears to be an effective therapeutic agent causing focused damage to tumor vasculature with minimal toxic effects to normal organs. This agent appears to be an excellent candidate for further clinical development.

Keywords: Angiogenesis; Necrosis; Pharmacokinetics; Toxicology; VEGF; Vascular permeability

Corrigendum to “Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology” [Biochem. Pharmacol. 84 (2012) 341–351] by Christian Seeger; Tony Christopeit; Karoline Fuchs; Katharina Grote; Werner Sieghart; U. Helena Danielson (pp. 1541-1541).

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Biochemical Pharmacology (v.84, #11)

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Biochemical Pharmacology (v.84, #11)