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Biochemical Pharmacology (v.83, #4)

Editorial Board (pp. iii).

Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter by Huailing Zhong; Connie Sánchez; Marc G. Caron (pp. 435-442).

The serotonin transporter (SERT) functions to transport serotonin (5-HT) from the extracellular space into neurons to maintain homeostatic control of 5-HT. It is the molecular target for selective serotonin reuptake inhibitor (SSRI) antidepressants. Preclinical research has shown that some SERT inhibitors can bind to two distinct binding sites on the SERT, a primary high affinity binding site and a low affinity allosteric binding site. Mutational studies of the SERT and computational modeling methods with escitalopram resulted in the identification of key amino acid residues important for the function of the allosteric binding site. While this allosteric binding site appears to influence the clinical efficacy of escitalopram under physiological conditions, the molecular mechanism of this effect is still poorly understood and may involve a large network of protein–protein interactions with the SERT. Dynamic interfaces between the SERT and the SERT interacting proteins (SIPs) potentially influence not only the SERT on its uptake function, its regulation, and trafficking, but also on known as well as yet to be identified non-canonical signaling pathways through SIPs. In this commentary, we outline approaches in the areas of selective small-molecule allosteric compound discovery, biochemistry, in vivo genetic knock-in mouse models, as well as computational and structural biology. These studies of the intra-molecular allosteric modulation of the SERT in the context of the myriad of potential inter-molecular signaling interactions with SIPs may help uncover unknown physiological functions of the SERT.

Keywords: Escitalopram (S-citalopram); Serotonin (5-HT); Serotonin transporter (SERT); Selective serotonin reuptake inhibitor (SSRI); SERT interacting proteins (SIPs); Allosteric

Thymoquinone: Potential cure for inflammatory disorders and cancer by Chern Chiuh Woo; Alan Prem Kumar; Gautam Sethi; Kwong Huat Benny Tan (pp. 443-451).

Thymoquinone is an active ingredient isolated from Nigella sativa and has been investigated for its anti-oxidant, anti-inflammatory and anticancer activities in both in vitro and in vivo models since its first extraction in 1960s. Its anti-oxidant/anti-inflammatory effect has been reported in various disease models, including encephalomyelitis, diabetes, asthma and carcinogenesis. Moreover, thymoquinone could act as a free radical and superoxide radical scavenger, as well as preserving the activity of various anti-oxidant enzymes such as catalase, glutathione peroxidase and glutathione-S-transferase. The anticancer effect(s) of thymoquinone are mediated through different modes of action, including anti-proliferation, apoptosis induction, cell cycle arrest, ROS generation and anti-metastasis/anti-angiogenesis. In addition, this quinone was found to exhibit anticancer activity through the modulation of multiple molecular targets, including p53, p73, PTEN, STAT3, PPAR-γ, activation of caspases and generation of ROS. The anti-tumor effects of thymoquinone have also been investigated in tumor xenograft mice models for colon, prostate, pancreatic and lung cancer. The combination of thymoquinone and conventional chemotherapeutic drugs could produce greater therapeutic effect as well as reduce the toxicity of the latter. In this review, we summarize the anti-oxidant/anti-inflammatory and anticancer effects of thymoquinone with a focus on its molecular targets, and its possible role in the treatment of inflammatory diseases and cancer.

Keywords: Abbreviations; PTEN; phosphatase and tensin homolog; STAT3; signal transducer and activator of transcription 3; PPAR-γ; peroxisome proliferator-activated receptors gamma; ROS; reactive oxygen species; NO; nitric oxide; TNF-α; tumor necrosis factor; IL; interleukin; MAPK; mitogen activated protein kinase; NF-κB; nuclear factor-κB; IκB; inhibitory subunit of NF-κB; LPS; lipopolysaccharides; ERK; extracellular-signal-regulated kinase; JAK-2; Janus kinase 2; Mcl-1; myeloid cell leukemia sequence 1; VEGF; vascular endothelial growth factor; MMP; matrix metalloproteinase; COX; cyclooxygenase; cdk; cyclin-dependent kinase; AKT; serine threonine protein kinase B; PARP; poly-ADP ribose polymerase; IAP; inhibitor of apoptosis; XIAP; X-linked inhibitor of apoptosis; DNA-PKcs; DNA-dependent protein kinase; JNK; c-Jun N-terminal kinase; UHRF1; ubiquitin-like, containing PHD and RING finger domains, 1; PDE1A; phosphodiesterase 1AThymoquinone; Inflammatory disorders; Cancer; Apoptosis; Drug toxicity

Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to Aurora kinase inhibitors by Lifang Xie; Michelle Kassner; Ruben M. Munoz; Qiang Q. Que; Jeff Kiefer; Yu Zhao; Spyro Mousses; Hongwei H. Yin; Daniel D. Von Hoff; Haiyong Han (pp. 452-461).

PDGFR inhibitors (e.g. imatinib) sensitize pancreatic cancer cells to the treatment of Aurora kinase inhibitors (e.g. PHA-739358).Aurora kinases are a family of mitotic kinases that play important roles in the tumorigenesis of a variety of cancers including pancreatic cancer. A number of Aurora kinase inhibitors (AKIs) are currently being tested in preclinical and clinical settings as anti-cancer therapies. However, the antitumor activity of AKIs in clinical trials has been modest. In order to improve the antitumor activity of AKIs in pancreatic cancer, we utilized a kinome focused RNAi screen to identify genes that, when silenced, would sensitize pancreatic cancer cells to AKI treatment. A total of 17 kinase genes were identified and confirmed as positive hits. One of the hits was the platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), which has been shown to be overexpressed in pancreatic cancer cells and tumor tissues. Imatinib, a PDGFR inhibitor, significantly enhanced the anti-proliferative effect of ZM447439, an Aurora B specific inhibitor, and PHA-739358, a pan-Aurora kinase inhibitor. Further studies showed that imatinib augmented the induction of G2/M cell cycle arrest and apoptosis by PHA-739358. These findings indicate that PDGFRA is a potential mediator of AKI sensitivity in pancreatic cancer cells.

Keywords: Pancreatic cancer; Aurora kinase; siRNA screening; PDGFR; Imatinib

Inactivation of a class A and a class C β-lactamase by 6β-(hydroxymethyl)penicillanic acid sulfone by Krisztina M. Papp-Wallace; Christopher R. Bethel; Thomas D. Gootz; Wenchi Shang; Justin Stroh; William Lau; Dale McLeod; Loren Price; Anthony Marfat; Anne Distler; Sarah M. Drawz; Hansong Chen; Emily Harry; Micheal Nottingham; Paul R. Carey; John D. Buynak; Robert A. Bonomo (pp. 462-471).

β-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the β-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6β-(hydroxymethyl)penicillanic acid sulfone (6β-HM-sulfone) was tested against isolates expressing the class A TEM-1 β-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6β-HM-sulfone inhibitor to ampicillin was highly effective. 6β-HM-sulfone inhibited TEM-1 with an IC₅₀ of 12±2nM and PDC-3 with an IC₅₀ of 180±36nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios ( k_cat/ k_inact) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20h, 6β-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6β-HM-sulfone was found to form a major adduct of +247±5Da with TEM-1 and +245±5Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (HOH). Minor adducts of +88±5Da with TEM-1 and +85±5Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6β-HM-sulfone is an effective and versatile β-lactamase inhibitor of representative class A and C enzymes.

Keywords: β-Lactamase inhibitor; PDC-3; TEM-1; β-Lactamase; Sulfone; β-Lactam

The molecular pharmacology of AMD11070: An orally bioavailable CXCR4 HIV entry inhibitor by Renee M. Mosi; Virginia Anastassova; Jennifer Cox; Marilyn C. Darkes; Stefan R. Idzan; Jean Labrecque; Gloria Lau; Kim L. Nelson; Ketan Patel; Zefferino Santucci; Rebecca S.Y. Wong; Renato T. Skerlj; Gary J. Bridger; Dana Huskens; Dominique Schols; Simon P. Fricker (pp. 472-479).

AMD11070 is a selective, orally bioavailable inhibitor of CXCR4 and of X4 HIV-1 replication. Mechanistic studies indicate AMD11070 is allosteric inhibitor acting via the extracellular region of CXCR4.In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1α ligand binding (IC₅₀=12.5±1.3nM), inhibits SDF-1 mediated calcium flux (IC₅₀=9.0±2.0nM) and SDF-1α mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC₅₀=39.8±2.5nM) or a [³⁵S]-GTPγS binding assay (IC₅₀=19.0±4.1nM), and inhibits SDF-1α stimulated chemotaxis (IC₅₀=19.0±4.0nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT₁, demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1β isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4.

Keywords: AMD11070; HIV; Chemokine receptor; CXCR4; Allosteric

Akt and p53 are potential mediators of reduced mammary tumor growth by Chloroquine and the mTOR inhibitor RAD001 by Christian R. Loehberg; Pamela L. Strissel; Ralf Dittrich; Reiner Strick; Juergen Dittmer; Angela Dittmer; Ben Fabry; Willi A. Kalender; Thorsten Koch; David L. Wachter; Nicole Groh; Astrid Polier; Ina Brandt; Laura Lotz; Inge Hoffmann; Florentine Koppitz; Sonja Oeser; Andreas Mueller; Peter A. Fasching; Michael P. Lux; Matthias W. Beckmann; Michael G. Schrauder (pp. 480-488).

Chloroquine overriding RAD001-induced Akt-phosphorylation and its direct anti-tumor activity in murine mammary gland tumors present this anti-malarial as a promising combination partner of the mTOR-inhibitor RAD001.PI3K/Akt/mTOR and p53 signaling pathways are frequently deregulated in tumors. The anticancer drug RAD001 (everolimus) is a known mTOR-inhibitor, but mTOR-inhibition leads to phosphorylation of Akt inducing resistance against RAD001 treatment. There is growing evidence that conflicting signals transduced by the oncogene Akt and the tumorsuppressor p53 are integrated via negative feedback between the two pathways. We previously showed that the anti-malarial Chloroquine, a 4-alkylamino substituted quinoline, is a p53 activator and reduced the incidence of breast tumors in animal models. Additionally, Chloroquine is an effective chemosensitizer when used in combination with PI3K/Akt inhibitors but the mechanism is unknown. Therefore, our aim was to test, if Chloroquine could inhibit tumor growth and prevent RAD001-induced Akt activation.Chloroquine and RAD001 caused G1 cell cycle arrest in luminal MCF7 but not in mesenchymal MDA-MB-231 breast cancer cells, they significantly reduced MCF7 cell proliferation on a collagen matrix and mammospheroid formation. In a murine MCF7 xenograft model, combined treatment of Chloroquine and RAD001 significantly reduced mammary tumor growth by 4.6-fold ( p=0.0002) compared to controls. Chloroquine and RAD001 inhibited phosphorylation of mTOR and its downstream target, S6K1. Furthermore, Chloroquine was able to block the RAD001-induced phosphorylation of Akt serine 473.The Chloroquine effect of overcoming the RAD001-induced activation of the oncogene Akt, as well as the promising antitumor activity in our mammary tumor animal model present Chloroquine as an interesting combination partner for the mTOR-inhibitor RAD001.

Keywords: RAD001; Chloroquine; mTOR; p53; Breast cancer

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax by M. Espona-Fiedler; V. Soto-Cerrato; A. Hosseini; J.M. Lizcano; V. Guallar; R. Quesada; T. Gao; R. Pérez-Tomás (pp. 489-496).

Here we describe mTOR as a new molecular target of the anticancerous molecules prodigiosin and obatoclax. These small molecules bind to the active site of mTOR inhibiting both mTOR complexes leading to cancer cell death.The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

Keywords: Abbreviations; PG; prodigiosin; OBX; obatoclax; Rap; rapamycin; mTOR; mammalian target of rapamycin; PI3K; phosphoinositide 3-kinase; AKT/PKB; protein kinase BProdigiosin; Obatoclax; Melanoma; PI3K/AKT; mTOR inhibitors; mTOR complexes

Carvedilol blocks the cloned cardiac Kv1.5 channels in a β-adrenergic receptor-independent manner by Imju Jeong; Bok Hee Choi; Shin Hee Yoon; Sang June Hahn (pp. 497-505).

Carvedilol, a non-selective β-adrenergic blocker, is widely used for the treatment of angina pectoris and hypertension. We examined the action of carvedilol on cloned Kv1.5 expressed in CHO cells, using the whole-cell patch clamp technique. Carvedilol reduced the peak amplitude of Kv1.5 and accelerated the inactivation rate in a concentration-dependent manner with an IC₅₀ of 2.56μM. Using a first-order kinetics analysis, we calculated k₊₁=19.68μM⁻¹s⁻¹ for the association rate constant, and k₋₁=44.89s⁻¹ for the dissociation rate constant. The apparent K_D ( k −₁/ k +₁) was 2.28μM, which is similar to the IC₅₀ value. Other β-adrenergic blockers (alprenolol, oxprenolol and carteolol) had little or no effect on Kv1.5 currents. Carvedilol slowed the deactivation time course, resulting in a tail crossover phenomenon. Carvedilol-induced block was voltage-dependent in the voltage range for channel activation, but voltage-independent in the voltage range for full activation. The voltage dependences for both steady-state activation and inactivation were unchanged by carvedilol. Carvedilol affected Kv1.5 in a use-dependent manner. When stimulation frequencies were increased to quantify a use-dependent block, however, the block by carvedilol was slightly increased with IC₅₀ values of 2.56μM at 0.1Hz, 2.38μM at 1Hz and 2.03μM at 2Hz. Carvedilol also slowed the time course of recovery from inactivation of Kv1.5. These results indicate that carvedilol blocks Kv1.5 in a reversible, concentration-, voltage-, time-, and use-dependent manner, but only at concentrations slightly higher than therapeutic plasma concentrations in humans. These effects are probably relevant to an understanding of the ionic mechanism underlying the antiarrhythmic property of carvedilol.

Keywords: Abbreviations; Kv; voltage-gated K; ⁺; channel; CHO; Chinese hamster ovary; HERG; human ether-a-go-go related geneKv1.5; Carvedilol; Antiarrhythmic drug; Open channel block; Use-dependent block

Ozone induces synthesis of systemic prostacyclin by cyclooxygenase-2 dependent mechanism in vivo by Siegfried Schulz; Simone Ninke; Bernhard Watzer; Rolf Michael Nüsing (pp. 506-513).

Under certain pathological conditions, e.g., infectious or neoplastic diseases, application of ozone exerts therapeutic effects. However, pharmacological mechanisms are not understood. Since an interaction with the arachidonic acid metabolism is suggested we investigated the effect of intraperitoneal insufflation of ozone on prostanoid system in vivo. Upon ozone application (4mg/kg) to rats we observed an approximate 3-fold increase in excretion rate of 6-keto-prostaglandin (PG) F_1α and of 2,3-dinor-6-keto-PGF_1α, the measurable stable products of prostacyclin. In plasma and vessel tissue 6-keto-PGF_1α concentration was also significantly increased. In contrast, excretion rates for PGE₂ and thromboxane (TX) B₂ did not change. F2-isoprostanes, regarded as endogenous indicators of oxidative stress, were also unaffected by ozone application. Oxygen insufflation used as control was without any effect on prostanoid levels. Ozone caused increase in 6-keto-PGF_1α by arterial but not by venous vessel tissues with peak activity 6–9h following insufflation. The increase in PGI₂ synthesis was dependent on cyclooxygenase (COX)-2 activity, demonstrated by its sensitivity towards COX-2 inhibition, and by enhanced COX-2 mRNA and protein expression in vessels. Ozone exerted no rise in excretion rate of prostacyclin metabolites in COX-2^−/− but in COX-1^−/− mice. Enzymatic activity and mRNA expression of vascular PGI₂ synthase (PGIS) was unaffected by ozone treatment. In summary our study shows for the first time that ozone insufflation causes enhanced expression of COX-2 in the vessel system leading to exclusive elevation of systemic PGI₂ levels. We assume that PGI₂ stimulation may contribute to the beneficial effects of ozone treatment.

Keywords: Abbreviations; BW; body weight; COX; cyclooxygenase; PG; prostaglandin; PGIS; prostacyclin synthase; TX; thromboxane; ROS; reactive oxygen speciesOzone; Insufflation; Cyclooxygenase; Isoprostanes; Prostanoids; Oxidative stress

Paradoxical effects of polyphenolic compounds from Clusiaceae on angiogenesis by Alexis Lavaud; Raffaella Soleti; Anne-Emmanuelle Hay; Pascal Richomme; David Guilet; Ramaroson Andriantsitohaina (pp. 514-523).

Among six tested polyphenolic compounds from Clusiaceae, the two endothelium-dependent vasodilatators isocalolongic acid (IA) and 2-deprenylrheediaxanthone (DRX) showed paradoxical effects on in vitro angiogenesis, IA being pro-angiogenic and DRX anti-angiogenic.Clusiaceae plants display high contents of xanthones and coumarins, the effects of which on endothelium, more particularly on angiogenesis, have not been assessed yet. We screened the capacity of six molecules from Clusiaceae – belonging to xanthones, coumarins and acid chromanes classes – to induce endothelium-dependent relaxation on mice aortic rings. Endothelial nitric oxide (NO) production was assessed in endothelial cell line using electron paramagnetic resonance technique. Then, the capacity of these molecules to induce capillary-like structures of endothelial cells was assessed. Cellular processes implicated in angiogenesis (adhesion, migration and proliferation) and Western blot analyses were then investigated. Among the tested molecules, isocalolongic acid (IA) and 2-deprenylrheediaxanthone (DRX) induced an endothelium-dependent relaxation of the aorta associated with an increase of NO production in endothelial cells. Using in vitro and ex vivo angiogenesis assays, it was shown that IA treatment promoted the formation of capillary-like network. In contrast, DRX prevented the ability of vascular endothelial growth factor (VEGF) to increase the formation of capillary-like network. IA increased endothelial cell proliferation while DRX decreased all cellular processes of angiogenesis. Western blot analysis showed that IA increased VEGF expression whereas DRX decreased ICAM-1 expression. Altogether, these data allowed identifying isolated molecules from Clusiaceae that exhibit a potential activity towards the modulation of endothelium-dependent relaxation involving NO release. Interestingly, they also highlighted paradoxical effects of the two compounds on cellular angiogenic processes, IA being pro-angiogenic and DRX anti-angiogenic.

Keywords: Endothelium; Angiogenesis; Clusiaceae; Xanthone; Nitric oxide

Bismuth ions inhibit the biological activity of non-amidated gastrins in vivo by Suzana Kovac; Su-Wen Loh; Shamilah Lachal; Arthur Shulkes; Graham S. Baldwin (pp. 524-530).

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. Bi³⁺ ions also bind to glycine-extended gastrin17 (Ggly), but inhibit Ggly-induced cell proliferation and migration in gastrointestinal cell lines in vitro. The aims of the present study were firstly, to establish the mechanism by which Bi³⁺ ions inhibit the binding of Fe³⁺ ions to Ggly, and secondly, to test the effect of Bi³⁺ ions on the activity of non-amidated gastrins in vivo. The interaction between Bi³⁺ ions, Fe³⁺ ions and Ggly was investigated by ultraviolet spectroscopy. The effect of Bi³⁺ ions on colorectal mucosal proliferation was measured in three animal models. In vitro in the presence of Bi³⁺ ions the affinity of Fe³⁺ ions for Ggly was substantially reduced; the data was better fitted by a mixed, rather than a competitive, inhibition model. In rats treated with Ggly alone proliferation in the rectal mucosa was increased by 318%, but was reduced to control values ( p<0.001) in animals receiving oral bismuth plus Ggly. Proliferation in the colonic mucosa of mice overexpressing Ggly or progastrin was significantly greater than in wild-type mice, but was no greater than control ( p<0.01) in animals receiving oral bismuth. Thus a reduction in the binding of Fe³⁺ ions to Ggly and progastrin in the presence of Bi³⁺ ions is a likely explanation for the ability of oral bismuth to block the biological activity of non-amidated gastrins in vivo.

Keywords: Abbreviations; CCK2R; cholecystokinin-2 receptor; CRC; colorectal carcinoma; Ggly; glycine-extended gastrin; PBS; phosphate-buffered salineBismuth ions; Colorectal mucosa; Ferric ions; Glycine-extended gastrin; Proliferation; Progastrin

Euphol prevents experimental autoimmune encephalomyelitis in mice: Evidence for the underlying mechanisms by Rafael Cypriano Dutra; Paula Roberta de Cezaro de Souza; Allisson Freire Bento; Rodrigo Marcon; Maíra Assunção Bicca; Luiz Francisco Pianowski; João B. Calixto (pp. 531-542).

Euphol effectively ameliorates both the clinical and pathological parameters of EAE, mainly by inhibiting the expression of pro-inflammatory mediators in vitro and in vivo.Multiple sclerosis (MS) is a severe chronic T cell-mediated autoimmune inflammatory disease of the central nervous system (CNS), the existing therapy of which is only partially effective and is associated with undesirable side effects. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. However there are no reports about the effects and mechanisms of euphol in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here we report the effects and the underlying mechanisms of action of euphol in EAE. Euphol (1–10mg/kg) was administered orally at different time-points of EAE. Immunological and inflammatory responses were evaluated by real-time PCR, Western blot and flow cytometry assays. We provide evidence that euphol significantly attenuates neurological signs of EAE. These beneficial effects of euphol seem to be associated with the down-regulation of mRNA and protein expression of some pro-inflammatory mediators such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the CNS. Furthermore, in vitro, euphol consistently inhibited the T cell-mediated immune response including the production of T_H1 and T_H17 cytokines in spleen cells of untreated EAE animals. Likewise, oral euphol treatment inhibited the infiltration of T_H17 myelin-specific cells into the CNS through the adhesion molecule, lymphocyte function-associated antigen 1 (LFA-1). Our findings reveal that oral administration of euphol consistently reduces and limits the severity and development of EAE. Therefore, euphol might represent a potential molecule of interest for the treatment of MS and other T_H17 cell-mediated inflammatory diseases.

Keywords: Abbreviations; CNS; central nervous system; CFA; complete Freund's adjuvant; EAE; experimental autoimmune encephalomyelitis; MS; multiple sclerosis; MOG; myelin oligodendrocyte glycoprotein; PBS; phosphate buffered saline; T; _H; T helper cells; IFN-γ; interferon gamma; IL; interleukin; ICAM; intercellular adhesion molecule; VCAM; vascular cell adhesion molecule; PECAM; platelet endothelial cell adhesion molecule; LFA-1; lymphocyte function-associated antigen-1; TNF-α; tumor necrosis factor alpha; TGF-β; transforming growth factor beta; RT-PCR; real-time PCR; NO; nitric oxide; iNOS; inducible nitric oxide synthase; COX-2; cyclooxygenase-2; PGs; prostaglandins; ANOVA; analysis of variance; SEM; standard error meanMultiple sclerosis; Experimental autoimmune encephalomyelitis; Euphol; Adhesion molecules

Structurally distinct nicotine immunogens elicit antibodies with non-overlapping specificities by M. Pravetoni; D.E. Keyler; R.R. Pidaparthi; F.I. Carroll; S.P. Runyon; M.P. Murtaugh; C.A. Earley; P.R. Pentel (pp. 543-550).

Nicotine conjugate vaccine efficacy is limited by the concentration of nicotine-specific antibodies that can be reliably generated in serum. Previous studies suggest that the concurrent use of 2 structurally distinct nicotine immunogens in rats can generate additive antibody responses by stimulating distinct B cell populations. In the current study we investigated whether it is possible to identify a third immunologically distinct nicotine immunogen. The new 1′-SNic immunogen (2 S)- N, N′-(disulfanediyldiethane-2,1-diyl)bis[4-(2-pyridin-3-ylpyrrolidin-1-yl)butanamide] conjugated to keyhole limpet hemocyanin (KLH) differed from the existing immunogens 3′-AmNic–rEPA and 6-CMUNic–BSA in linker position, linker composition, conjugation chemistry, and carrier protein. Vaccination of rats with 1′-SNic–KLH elicited high concentrations of high affinity nicotine-specific antibodies. The antibodies produced in response to 1′-SNic–KLH did not appreciably cross-react in ELISA with either 3′-AmNic–rEPA or 6-CMUNic–BSA or vice versa, showing that the B cell populations activated by each of these nicotine immunogens were non-overlapping and distinct. Nicotine retention in serum was increased and nicotine distribution to brain substantially reduced in rats vaccinated with 1′-SNic–KLH compared to controls. Effects of 1′-SNic–KLH on nicotine distribution were comparable to those of 3′-AmNic–rEPA which has progressed to late stage clinical trials as an adjunct to smoking cessation. These data show that it is possible to design multiple immunogens from a small molecule such as nicotine which elicit independent immune responses. This approach could be applicable to other addiction vaccines or small molecule targets as well.

Keywords: Nicotine; Immunotherapy; Addiction; Vaccine; Immunogenicity

In vitro drug metabolism by C-terminally truncated human flavin-containing monooxygenase 3 by Gianluca Catucci; Gianfranco Gilardi; Lars Jeuken; Sheila J. Sadeghi (pp. 551-558).

Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K_m values of 45±8μM and 25±4μM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme.The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.

Keywords: Flavin-containing monooxygenase; Molecular docking; In vitro; expression; Drug metabolism

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Biochemical Pharmacology (v.83, #4)