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Biochemical Pharmacology (v.82, #6)

Editorial Advisory Board (pp. iii).

Asthma translational medicine: Report card by Kevin Mullane (pp. 567-585).

Research has developed a standardized, unifying mechanism of asthma and identified key targets for therapeutic intervention. Unfortunately, for 39 compounds and biologics targeted at 23 different sites in this immune cascade, so far the clinical results have uniformly failed to match expectations.Over the last 30 years, scientific research into asthma has focused almost exclusively on one component of the disorder – airway inflammation – as being the key underlying feature. These studies have provided a remarkably detailed and comprehensive picture of the events following antigen challenge that lead to an influx of T cells and eosinophils in the airways. Indeed, in basic research, even the term “asthma” has become synonymous with a T helper 2 cell-mediated disorder. From this cascade of cellular activation processes and mediators that have been identified it has been possible to pinpoint critical junctures for therapeutic intervention, leading experimentalists to produce therapies that are very effective in decreasing airway inflammation in animal models. Many of these compounds have now completed early Phase 2 “proof-of-concept” clinical trials so the translational success of the basic research model can be evaluated. This commentary discusses clinical results from 39 compounds and biologics acting at 23 different targets, and while 6 of these drugs can be regarded as a qualified success, none benefit the bulk of asthma sufferers. Despite this disappointing rate of success, the same immune paradigm and basic research models, with a few embellishments to incorporate newly identified cells and mediators, continue to drive target identification and drug discovery efforts. It is time to re-evaluate the focus of these efforts.

Keywords: Abbreviations; ACQ; asthma control questionnaire; AHR; airway hyper-responsiveness; AP-1; activator protein 1; APC; antigen-presenting cell; AQLQ; asthma quality-of-life questionnaire; BALF; bronchoalveolar lavage fluid; CTLA-4; cytotoxic T lymphocyte-associated antigen 4; cysLT; cysteinyl leukotrienes; DC; dendritic cell; EAR; early airway response; FEV1; forced expiratory volume in 1 second; FLAP; 5-lipoxygenase activating protein; GM-CSF; granulocyte-macrophage colony-stimulating factor; ICAM-1; intercellular adhesion molecule 1; ICOS; inducible costimulator; ICS; inhaled corticosteroid; IFN; interferon; IgE; immunoglobulin E; IL; interleukin; LAR; late asthmatic response; LPS; lipopolysaccharide; LT; leukotriene; mAb; monoclonal antibody; Maf-1; musculoaponeurotic fibrosarcoma 1; MHC; major histocompatibility complex; mRNA; messenger ribonucleic acid; NF-IL-6; nuclear factor interleukin-6; NKT; natural killer T cell; PD-1; programmed death 1; PEFR; peak expiratory flow rate; PG; prostaglandin; SCID; severe combined immunodeficiency; STAT; signal transducer and activator of transcription factor; TCR; T cell receptor; TGFβ; transforming growth factor β; Th; T helper cell; TLR; toll-like receptor; TNF; tumor necrosis factor; Treg; regulatory T cell; TSLP; thymic stromal lymphopoietin; VCAM-1; vascular cell adhesion protein 1; VLA-4; very late antigen 4Cytokines; Chemokines; T helper 2 cells; Asthma therapeutics; Eosinophils; Animal models

The increasing challenge of discovering asthma drugs by Kevin Mullane (pp. 586-599).

The prevalence of asthma continues to rise. Current drugs provide symptomatic relief to some, but not all, patients. Despite the need for new therapeutics, and a huge research effort, only four novel agents from two classes of drugs – the antileukotrienes and an anti-IgE antibody – have been approved in the last 30 years. This review highlights three particular issues that contribute to the challenge of identifying new therapeutics. First is an over-reliance on animal models of allergy to define targets and expectations of efficacy that has met with poor translation to the clinical setting. While sensitivity to particular aeroallergens is one key risk factor for asthma, atopy and asthma are not synonymous, and while about half of adult asthmatics are atopic the incidence of allergic asthma is probably <50%. The second issue is a fundamental disconnect between the directions of basic research and clinical research. Basic research has developed a detailed, reductive, unifying mechanism of antigen-induced, T helper type 2 cell-mediated airway inflammation as the root cause of asthma. In contrast, clinical research has started to identify multiple asthma phenotypes with differing cellular components, mediators and sensitivities to asthma drugs, and probably varying underlying factors including susceptibility genes. Finally, different features of asthma – bronchoconstriction, symptoms, and exacerbations – respond diversely to treatment; effects that are not captured in animal models which do not develop asthma per se, but utilize unvalidated surrogate markers. Basic research needs to better integrate and utilize the clinical research findings to improve its relevance to drug discovery efforts.

Keywords: Abbreviations; ACQ; asthma control questionnaire; AHR; airway hyper-responsiveness; AQLQ; asthma quality-of-life questionnaire; BALF; bronchoalveolar lavage fluid; bn; billion; cysLT; cysteinyl leukotrienes; DZ; dizygotic; EAR; early airway response; FDA; Food and Drug Administration; FEV1; forced expiratory volume in 1 second; GPCR; G protein-coupled receptor; ICS; inhaled corticosteroid; IgE; immunoglobulin E; IL; interleukin; LABA; long acting β-agonist; LAR; late asthmatic response; MM; million; mRNA; messenger ribonucleic acid; MZ; monozygotic; PEFR; peak expiratory flow rate; SABA; short acting β-agonist; SMART; Salmeterol Asthma Research Trial; SNP; single nucleotide polymorphism; SRS-A; slow reacting substance of anaphylaxis; TGFβ; transforming growth factor β; Th2; T helper cell type 2; TLR; toll-like receptor; TNF; tumor necrosis factorAsthma phenotypes; Asthma models; Atopy; Asthma drugs; Asthma clinical trials; Asthma genetics

Novel proteasome-inhibitory syrbactin analogs inducing endoplasmic reticulum stress and apoptosis in hematological tumor cell lines by Ashish Anshu; Simmy Thomas; Puneet Agarwal; Tannya R. Ibarra-Rivera; Michael C. Pirrung; Axel H. Schönthal (pp. 600-609).

Novel synthetic syrbactin analogs trigger endoplasmic reticulum stress (induction of CHOP, ATF-3), downregulate anti-apoptotic proteins (survivin, Mcl-1), and cause apoptosis (activation of caspase 3, cleavage of PARP).The proteasome has been recognized as a druggable target in cancer cells, and this has led to searches for pharmacologic agents that target this cellular organelle for cancer therapeutic purposes. Syrbactins are a group of microbial metabolites consisting of two related families, the glidobactins and the syringolins. Some members of this group have revealed cytotoxic efficacy in tumor cells, and more recently it was discovered that they exert proteasome-inhibitory function. Based on this therapeutic promise and to gain further understanding of their molecular modes of action, we chemically synthesized de-novo three novel syrbactin analogs and characterized their proteasome-inhibitory and in vitro anti-neoplastic activity in human cell lines representing multiple myeloma, Waldenström's macroglobulinemia, and lymphocytic leukemia. Our results show that two of these novel compounds are able to inhibit proteasome activity in the nanomolar range, reduce the expression of anti-apoptotic proteins survivin and Mcl-1, and cause severe endoplasmic reticulum (ER) stress, resulting in pronounced tumor cell death. These anticancer effects can be synergistically enhanced when the agents are combined with thapsigargin, which further aggravates ER stress by a different mechanism. Taken together, our findings support the notion that syrbactin analogs may provide a structural platform for the development of novel cancer therapeutics, and that their efficacy may be further increased when complemented with other agents that trigger ER stress.

Keywords: Abbreviations; ER; endoplasmic reticulum; GlbA; glidobactin A; SylA; syringolin A; SylB; syringolin BSyrbactin; Proteasome inhibitor; Thapsigargin; Hematological tumors; Bortezomib

Heteromerization of human cytomegalovirus encoded chemokine receptors by Pia Tschische; Kenjiro Tadagaki; Maud Kamal; Ralf Jockers; Maria Waldhoer (pp. 610-619).

Human cytomegalovirus (HCMV) is a widespread pathogen that infects up to 80% of the human population and causes severe complications in immunocompromised patients. HCMV expresses four seven transmembrane (7TM) spanning/G protein-coupled receptors (GPCRs) – US28, US27, UL33 and UL78 – that show close homology to human chemokine receptors. While US28 was shown to bind several chemokines and to constitutively activate multiple signaling cascades, the function(s) of US27, UL33 and UL78 in the viral life cycle have not yet been identified. Here we investigated the possible interaction/heteromerization of US27, UL33 and UL78 with US28 and the functional consequences thereof. We provide evidence that these receptors not only co-localize, but also heteromerize with US28 in vitro. While the constitutive activation of the US28-mediated Gαq/phospholipase C pathway was not affected by receptor heteromerization, UL33 and UL78 were able to silence US28-mediated activation of the transcription factor NF-κB. Summarized, we provide evidence that these orphan viral receptors have an important regulatory capacity on the function of US28 and as a consequence, may ultimately impact on the viral life cycle of HCMV.

Keywords: Human cytomegalovirus; Chemokine receptors; Signaling; Post-endocytic sorting; Heteromerization; G-protein coupled receptor

The aromatic ketone 4′-hydroxychalcone inhibits TNFα-induced NF-κB activation via proteasome inhibition by Barbora Orlikova; Deniz Tasdemir; Frantisek Golais; Mario Dicato; Marc Diederich (pp. 620-631).

Chalcones are aromatic ketones, known to exhibit anti-microbial, anti-inflammatory and anti-cancer activities. The aim of this study was to investigate the anti-inflammatory and anti-cancer activity of 4′-hydroxychalcone. Here, we report that 4′-hydroxychalcone inhibits TNFα-induced NF-κB pathway activation in a dose-dependent manner. To investigate the underlying molecular mechanisms we demonstrate that 4′-hydroxychalcone inhibits proteasome activity in a dose-dependent manner but has no effect on IKK activity. Results show that 4′-hydroxychalcone inhibits TNFα-dependent degradation of IκBα and subsequently prevents p50/p65 nuclear translocation leading to 4′-hydroxychalcone-inhibited expression of NF-κB target genes. Most importantly, inhibition of NF-κB activation by 4′-hydroxychalcone is not leukemia cell-type specific and has no significant effect on non-transformed cell viability, thus highlighting the compound's potential in both prevention and treatment.

Keywords: Polyphenol; Chalcone; NF-κB; Inflammation; Cancer

Suppression of human CD4+ T cell activation by 3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is mediated by CXCL9 and CXCL10 by Anne Hertenstein; Theresa Schumacher; Ulrike Litzenburger; Christiane A. Opitz; Christine S. Falk; Tito Serafini; Wolfgang Wick; Michael Platten (pp. 632-641).

3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is an orally available anti-allergic drug with structural and functional homologies to immunosuppressive catabolites of the essential amino acid tryptophan and broad anti-inflammatory properties. It has recently been shown to be effective in animal models of multiple sclerosis and rheumatoid arthritis, two autoimmune diseases that are mediated by auto-aggressive Th1-polarized CD4+ T lymphocytes. Here we demonstrate potent suppressive effects of tranilast on the function of naïve human CD4+ T cells. Tranilast inhibited inhibits activation and proliferation of purified CD4+ T cells stimulated through the T cell receptor with an EC50 of less than 10μM, a concentration that is well below plasma levels achieved after oral administration of approved doses of 200–600mg in humans. The antiproliferative effects were less potent on naïve CD8+ T cells. Suppression of CD4+ and CD8+ T cell proliferation was associated with an inhibition of T cell activation. Cytokine analyses of naïve CD4+ T cells revealed that tranilast interferes with the production of cyto- and chemokines driven by signal transducer and activator of transcription 1 (STAT1), notably chemokine (C-X-C motif) ligands (CXCL) 9 and 10. Tranilast limited STAT1 phosphorylation in activated T cells and supplementation of CXCL9 or CXCL10 reversed the anti-proliferative effects of tranilast. These data imply CXCL9 and CXCL10 as novel therapeutic targets of tranilast in Th1-mediated autoimmune diseases and identify phospho-STAT1 and its target chemokines CXCL9 and CXCL10 as potential markers for monitoring the bioactivity of tranilast in humans.

Keywords: Multiple sclerosis; Rheumatoid arthritis; T cell; Anti-allergic drug; Tryptophan catabolism

Dimethoxycurcumin, a metabolically stable analogue of curcumin, exhibits anti-inflammatory activities in murine and human lymphocytes by Raghavendra S. Patwardhan; Rahul Checker; Deepak Sharma; Vineet Kohli; K.I. Priyadarsini; Santosh K. Sandur (pp. 642-657).

The aim of this study was to investigate whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue having higher metabolic stability over curcumin, could exhibit anti-inflammatory activity in murine and human lymphocytes. Both curcumin and DiMC suppressed mitogen as well as antigen driven proliferation of murine splenic lymphocytes. Further, mitogen and antigen-stimulated cytokine (IL-2, IL-4, IL-6 and IFN-γ) secretion by T cells was also abrogated by curcumin and DiMC. Interestingly, curcumin and DiMC suppressed B cell proliferation induced by lipopolysaccharide. Curcumin and DiMC also inhibited Con A-induced activation of early and late T cell activation markers. They scavenged basal reactive oxygen species and depleted GSH levels in lymphocytes. The suppression of mitogen-induced T cell proliferation and cytokine secretion by curcumin and DiMC was significantly abrogated by thiol containing antioxidants suggesting a role for redox in their anti-inflammatory activity. Further, the possibility of curcumin and DiMC directly interacting with thiol-containing antioxidant GSH was monitored by changes in absorbance. Both curcumin and DiMC inhibited Con A induced activation of NF-κB and MAPK. More importantly, curcumin and DiMC inhibited phytohaemagglutinin induced proliferation and cytokine secretion by human peripheral blood mononuclear cells. To explore their therapeutic efficacy, they were added to lymphocytes post-Con A stimulation and we observed a significant suppression of IL-2, IL-6 and IFN-γ. The present study for the first time demonstrates the potent anti-inflammatory activity of DiMC. Further, DiMC could find application as an alternative to curcumin, which is currently used in several clinical studies, due to its superior bioavailability and comparable efficacy.

Keywords: T cells; Cytokines; GSH; Reactive oxygen species; NF-κB

Functionally biased modulation of A₃ adenosine receptor agonist efficacy and potency by imidazoquinolinamine allosteric enhancers by Zhan-Guo Gao; Dennis Verzijl; Annelien Zweemer; Kai Ye; Anikó Göblyös; Adriaan P. IJzerman; Kenneth A. Jacobson (pp. 658-668).

Allosteric modulators for the G_i-coupled A₃ adenosine receptor (AR) are of considerable interest as therapeutic agents and as pharmacological tools to probe various signaling pathways. In this study, we initially characterized the effects of several imidazoquinolinamine allosteric modulators (LUF5999, LUF6000 and LUF6001) on the human A₃ AR stably expressed in CHO cells using a cyclic AMP functional assay. These modulators were found to affect efficacy and potency of the agonist Cl-IB-MECA differently. LUF5999 (2-cyclobutyl derivative) enhanced efficacy but decreased potency. LUF6000 (2-cyclohexyl derivative) enhanced efficacy without affecting potency. LUF6001 (2-H derivative) decreased both efficacy and potency. We further compared the agonist enhancing effects of LUF6000 in several other A₃ AR-mediated events. It was shown that although LUF6000 behaved somewhat differently in various signaling pathways, it was more effective in enhancing the effects of low-efficacy than of high-efficacy agonists. In an assay of cyclic AMP accumulation, LUF6000 enhanced the efficacy of all agonists examined, but in the membrane hyperpolarization assay, it only enhanced the efficacy of partial agonists. In calcium mobilization, LUF6000 did not affect the efficacy of the full agonist NECA but was able to switch the nucleoside antagonist MRS542 into a partial agonist. In translocation of β-arrestin2, the agonist-enhancing effect LUF6000 was not pronounced. In an assay of ERK1/2 phosphorylation LUF6000 did not show any effect on the efficacy of Cl-IB-MECA. The differential effects of LUF6000 on the efficacy and potency of the agonist Cl-IB-MECA in various signaling pathway were interpreted quantitatively using a mathematical model.

Keywords: Abbreviations; CCPA; 2-chloro-; N; ⁶; -cyclopentyladenosine; Cl-IB-MECA; 2-chloro-; N; ⁶; -(3-iodobenzyl)-adenosine-5′-; N; -methyluronamide; ERK; extracellular signal-regulated kinase; GPCR; G protein-coupled receptor; LUF5833; 2-aminophenyl-6-(1; H; -imidazol-2-ylmethylsulfanyl)-pyridine-3,5-dicarbonitrile; LUF6000; (; N; -(3,4-dichloro-phenyl)-2-cyclohexyl-1; H; -imidazo[4,5-; c; ]quinolin-4-amine); LUF5999; (; N; -(3,4-dichloro-phenyl)-2-cyclobutyl-1; H; -imidazo[4,5-; c; ]quinolin-4-amine); LUF6001; (; N; -(3,4-dichloro-phenyl)-1; H; -imidazo[4,5-; c; ]quinolin-4-amine); MRS541; N; ⁶; -(3-iodobenzyl)adenosine; MRS542; 2-chloro-N6-(3-iodobenzyl)adenosine; NECA; adenosine-5′-N-ethyluronamide; PKA; protein kinase AG protein-coupled receptors; Purines; Positive allosteric modulator; Second messenger; Nucleoside

Cytochrome b₅ shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy by Věra Kotrbová; Barbora Mrázová; Michaela Moserová; Václav Martínek; Petr Hodek; Jiří Hudeček; Eva Frei; Marie Stiborová (pp. 669-680).

Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b₅ alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine–DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b₅ enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine–DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine–DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b₅ might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b₅ in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b₅ and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.

Keywords: Abbreviations; COX; cyclooxygenase; CYP; cytochrome P450; HRN; hepatic cytochrome P450 reductase null; GAPDH; glyceraldehyde phosphate dehydrogenase; HPLC; high-performance liquid chromatography; i.p.; intra-peritoneal; LPO; lactoperoxidase; MPO; myeloperoxidase; PEI-cellulose; polyethylenimine-cellulose; RAL; relative adduct labeling; r.t.; retention time; TLC; thin layer chromatographyCYP1A1/2; Cytochrome b; ₅; Ellipticine; Modulation; Activation

Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5 by Rüdiger Kaspera; Suresh B. Naraharisetti; Eric A. Evangelista; Kristin D. Marciante; Bruce M. Psaty; Rheem A. Totah (pp. 681-691).

Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k_cat seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR ( K_D and K_m,red) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k_cat. In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k_cat between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.

Keywords: Abbreviations; CYP; cytochrome P450; CPR; cytochrome P450 reductase; CER; cerivastatin; AQ; amodiaquine; PAC; paclitaxel; RG; rosiglitazone; DEAQ; N; -de ethyl amodiaquine; DLPC; L-α-dilauryl-sn-glycero-3-phosphocholine; HLMs; human liver microsomes; SIR EI; ⁺; selective ionization reaction positive ionization; MRM EI; ⁺; multiple reaction mode positive ionizationPaclitaxel; Amodiaquine; Rosiglitazone; Cerivastatin; Pharmacogenetics; Ligand binding

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Biochemical Pharmacology (v.82, #6)