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Biochemical Pharmacology (v.82, #4)

Editorial Advisory Board (pp. iii).

Targeting Siglecs—A novel pharmacological strategy for immuno- and glycotherapy by Camilla Jandus; Hans-Uwe Simon; Stephan von Gunten (pp. 323-332).

The immune system must be tightly held in check to avoid bystander tissue damage as well as autoreactivity caused by overwhelming immune reactions. A novel family of immunoregulatory, carbohydrate-binding receptors, the Siglecs (sialic acid binding immunoglobulin-like lectins), has received particular attention in light of their capacity to mediate cell death, anti-proliferative effects and to regulate a variety of cellular activities. Siglec receptors are mainly expressed on leukocytes in a cell type-specific and differentiation-dependent manner. Siglecs might potentially be exploited as targets of novel immune- and glycotherapeutics for cell-directed therapies in autoimmune and allergic diseases, as well as in haematologic malignancies. Here we present novel insights on structural and functional characteristics, expression patterns and evolutionary aspects of Siglecs and their ligands. Pharmacological strategies using Siglec agonistic cross-linking therapeutics, such as monoclonal or engineered antibodies, intravenous immunoglobulin (IVIG), or glycomimetics are discussed. Modulation of immune responses by targeting Siglecs using agonistic or antagonistic therapeutics may have important clinical implications and may pave the way for novel pharmacological avenues for the treatment of autoimmune and allergic diseases or for tumor immunotherapy.

Keywords: Siglecs; Lectins; Immunotherapy; Cell-directed therapy; Glycotargeting

CD3⁺CD4⁻CD8⁻ (double negative) T cells: Saviours or villains of the immune response? by Fulvio D’Acquisto; Tessa Crompton (pp. 333-340).

Recent studies have shown that T cells are not just the latecomers in inflammation but might also play a key role in the early phase of this response. In this context, a number of T cell subsets including NKT cells, mucosal-associated invariant T cells and γ/δ T cells have been shown, together with classical innate immune cells, to contribute significantly to the development and establishment of acute and chronic inflammatory diseases. In this commentary we will focus our attention on a somewhat neglected class of T cells called CD3⁺CD4⁻CD8⁻ double negative T cells and on their role in inflammation and autoimmunity. We will summarize the most recent views on their origin at the thymic and peripheral levels as well as their tissue localization in immune and non-lymphoid organs. We will then outline their potential pathogenic role in autoimmunity as well as their homeostatic role in suppressing excessive immune responses deleterious to the host. Finally, we will discuss the potential therapeutic benefits or disadvantages of targeting CD3⁺CD4⁻CD8⁻ double negative T cells for the treatment of autoimmune disease. We hope that this overview will shed some light on the function of these immune cells and attract the interest of investigators aiming at the design of novel therapeutic approaches for the treatment of autoimmune and inflammatory conditions.

Keywords: Thymocyte positive and negative selection; CD4; ⁻; CD8; ⁻; DN T cells; Unconventional T cells; Inflammation; Autoimmune and infectious diseases

Protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity by Vaibhav Kapuria; Luke F. Peterson; H.D. Hollis Showalter; Paul D. Kirchhoff; Moshe Talpaz; Nicholas J. Donato (pp. 341-349).

Ubiquitin-activating enzyme 1 (UBE1) is a critical regulator of the ubiquitination cycle and its targeted inhibition may be an appropriate therapeutic strategy as tumor cells are reported to have increased dependence on protein ubiquitination. PYR-41 is a small molecule with previously described UBE1 inhibitory activity. PYR-41 blocks ubiquitination reactions but paradoxically leads to the accumulation of high MW ubiquitinated proteins. Detailed evaluation of PYR-41 activity demonstrated that PYR-41 inhibited UBE1 activity but also had equal or greater inhibitory activity against several deubiquitinases (DUBs) in intact cells and purified USP5 in vitro. Both UBE1 and DUB inhibition were mediated through PYR-41-induced covalent protein cross-linking which paralleled the inhibition of the target proteins enzymatic activity. PYR-41 also mediated cross-linking of specific protein kinases (Bcr-Abl, Jak2) to inhibit their signaling activity. Chemical reactivity modeling provided some insight into the cross-linking potential and partial target selectivity of PYR-41. Overall, our results suggest a broader range of targets and a novel mechanism of action for this UBE1 inhibitor. In addition, since PYR-41-related compounds have demonstrated anti-tumor activity in animal studies, partially selective protein cross-linking may represent an alternate approach to affect signal transduction modules and ubiquitin cycle-regulatory proteins for cancer therapy.

Keywords: Ubiquitin-activating enzyme; Deubiquitinase; Inhibitor; Cross-linking

Inhibition of human Na_v1.5 sodium channels by strychnine and its analogs by Chunhua Yuan; Lirong Sun; Meng Zhang; Shuji Li; Xuemin Wang; Tianming Gao; Xinhong Zhu (pp. 350-357).

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na_v1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na_v1.5 channel with the half-maximum inhibition (IC₅₀) values of 83.1μM and 104.6μM, respectively. Structure–function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na_v1.5 channel. Strychnine and icajine may bind to the channel by cation–π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na_v1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na_v1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na_v1.5 channel would be potential therapeutic advantages in certain arrhythmias.

Keywords: Na; _v; 1.5 channel; Alkaloid; Strychnine; Icajine; Arrhythmias

Structure–activity relationships for the interactions of 2′- and 3′-( O)-( N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases by Cibele Pinto; Gerald H. Lushington; Mark Richter; Andreas Gille; Jens Geduhn; Burkhard König; Tung-Chung Mou; Stephen R. Sprang; Roland Seifert (pp. 358-370).

We characterize interactions of 21 (M)ANT-nucleotides with mammalian adenylyl cyclases. Through combination of enzymological, fluorescence spectroscopy and molecular modelling techniques we provide evidence for ligand-specific conformational landscapes in ACs.Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2′,3′-( O)-( N-methylanthraniloyl) (MANT)-substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure–activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5. (M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases hypoxanthine>uracil>cytosine>adenine∼guanine≫xanthine. Omission of a hydroxyl group at the 2′ or 3′-position reduced inhibitor potency as did introduction of a γ-thiophosphate group or omission of the γ-phosphate group. Substitution of the MANT-group by an ANT-group had little effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1 homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure–activity relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity with no preference for the “cognate” base adenine as verified by enzyme inhibition, fluorescence spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate development of non-nucleotide inhibitors.

Keywords: Abbreviations; AC; membranous adenylyl cyclase; ANT; anthraniloyl-; FRET; fluorescence resonance energy transfer; FS; forskolin; GTPγS; guanosine 5′-[γ-thio]triphosphate; MANT; methylanthraniloyl-; NDP; nucleoside 5′-diphosphate; NTP; nucleoside 5′-triphosphate; MANT-GTP; specific fluorescent nucleotides studied were: 2′,3′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-triphosphate; 2′-d-3′-MANT-GTP; 2′-deoxy-3′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-triphosphate; 3′-d-2′-MANT-GTP; 3′-deoxy-2′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-triphosphate; MANT-GTPγS; 2′,3′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-[γ-thio]triphosphate; MANT-ATP; 2′,3′-; O; -(; N; -methylanthraniloyl)-adenosine 5′-triphosphate; 2′-d-3′-MANT-ATP; 2′-deoxy-3′-; O; -(; N; -methylanthraniloyl)-adenosine 5′-triphosphate; 3′-d-2′-MANT-ATP; 3′-deoxy-2′-; O; -(; N; -methylanthraniloyl)-adenosine 5′-triphosphate; MANT-UTP; 2′,3′-; O; -(; N; -methylanthraniloyl)-uridine 5′-triphosphate; MANT-CTP; 2′(3′)-; O; -(; N; -methylanthraniloyl)-cytidine 5′-triphosphate; MANT-ITP; 2′,3′-; O; -(; N; -methylanthraniloyl)-inosine 5′-triphosphate; MANT-ITPγS; 2′,3′-; O; -(; N; -methylanthraniloyl)-inosine 5′-[γ-thio]triphosphate; MANT-XTP; 2′,3′-; O; -(; N; -methylanthraniloyl)-xanthosine 5′-triphosphate; ANT-GTP; 2′,3′-; O; -anthraniloyl-guanosine 5′-triphosphate; ANT-ATP; 2′,3′-; O; -anthraniloyl-adenosine 5′-triphosphate; ANT-ADP; 2′,3′-; O; -anthraniloyl-adenosine 5′-diphosphate; MANT-GDP; 2′,3′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-diphosphate; MANT-ADP; 2′,3′-; O; -(; N; -methylanthraniloyl)-adenosine 5′-diphosphate; MANT-IDP; 2′,3′-; O; -(; N; -methylanthraniloyl)-inosine 5′-diphosphate; MANT-UDP; 2′,3′-; O; -(; N; -methylanthraniloyl)-uridine 5′-diphosphate; MANT-CDP; 2′,3′-; O; -(; N; -methylanthraniloyl)-cytidine 5′-diphosphate; MANT-IMP; 2′,3′-; O; -(; N; -methylanthraniloyl)-inosine 5′-monophosphateAdenylyl Cyclase; MANT-nucleotides; Fluorescence spectroscopy; Molecular modelling; Conformational landscape

Compound C stimulates heme oxygenase-1 gene expression via the Nrf2-ARE pathway to preserve human endothelial cell survival by Xiao-Ming Liu; Kelly J. Peyton; Ahmad R. Shebib; Hong Wang; William Durante (pp. 371-379).

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor 2 (Nrf2). Compound C also stimulated Nrf2 expression this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-l-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions.

Keywords: Compound C; Heme oxygenase-1; Oxidative stress; Endothelial cells

Endocannabinoids inhibit release of nerve growth factor by inflammation-activated mast cells by Giuseppina Cantarella; Mimmo Scollo; Laurence Lempereur; Gloria Saccani-Jotti; Francesco Basile; Renato Bernardini (pp. 380-388).

Nerve growth factor (NGF) is a pleiotropic member of the neurotrophin family. Beside its neuronal effects, NGF plays a role in various processes, including angiogenesis. Mast cells release NGF and are among elements contributing to angiogenesis, a process regulated by arrays of factors, including the inhibitory cannabinoids. The possible inhibitory role of cannabinoids on mast cell-related NGF mitogenic effect on endothelial cells was then investigated. Human mastocytic cells HMC-1, challenged with PMA to yield release of NGF, were preincubated with the endocannabinoid PEA. Then, conditioned media were added to HUVEC cultures. PMA-activated HMC-1 cells released substantial amounts of NGF, whereas PEA inhibited PMA-induced NGF release. HUVEC proliferation increased after treatment with media from activated HMC-1 cells, while was reduced with media from HMC-1 cells treated with PEA. To characterize receptors mediating such effects of PEA, RT-PCR and western blot analysis were performed on HMC-1 cells. None of the two cannabinoid CB1 and CB2 receptors was expressed by HMC-1 cells, which on the other hand expressed the orphan receptor GPR55. PEA was ineffective in inhibiting NGF release from HMC-1 cells treated with PMA and transfected with positive GPR55 RNAi, whereas it induced significant reduction of NGF in cells transfected with the corresponding negative control RNAi. Results indicate that NGF released from inflammatory mast cells induces angiogenesis. Cannabinoids attenuate such pro-angiogenic effects of NGF. Finally, cannabinoids could be considered for antiangiogenic treatment in disorders characterized by prominent inflammation.

Keywords: Endocannabinoids; NGF-related inflammation; Receptor; Antagonist

The four cysteine residues in the second extracellular loop of the human adenosine A_2B receptor: Role in ligand binding and receptor function by Anke C. Schiedel; Sonja Hinz; Dominik Thimm; Farag Sherbiny; Thomas Borrmann; Astrid Maaß; Christa E. Müller (pp. 389-399).

Adenosine A_2B receptors form only one disulfide bond between the conserved cysteine residues C78^3.25 and C171^45.50; the other three cysteines in the loop are not involved in disulfide bond formation.The adenosine A_2B receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A_2B receptor with dithiothreitol led to a 74-fold increase in the EC₅₀ value of the agonist NECA in cyclic AMP accumulation. In the C78^3.25S and the C171^45.50S mutant high-affinity binding of the A_2B antagonist radioligand [³H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25–C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A_2B receptors. In contrast, the C166^45.45S and the C167^45.46S mutant as well as the C166^45.45S–C167^45.46S double mutant behaved like the wild-type receptor, while in the C154^45.33S mutant significant, although more subtle effects on cAMP accumulation were observed – decrease (BAY60-6583) or increase (NECA) – depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A_2A receptor, which showed four disulfide bonds, the present data indicate that in the A_2B receptor only the C3.25–C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A_2B receptor not involved in stabilization of the receptor structure may have other functions.

Keywords: Adenosine A; _2B; receptor; Disulfide bonds; Extracellular loop 2; Mutagenesis; PSB-603

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca²⁺ channel subunits Orai1 and TRPC1 by Carmen Galán; Natalia Dionisio; Tarik Smani; Ginés M. Salido; Juan A. Rosado (pp. 400-410).

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca²⁺ sensor STIM1 and Ca²⁺ permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca²⁺ permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca²⁺ channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.

Keywords: Actin filaments; Microtubules; Calmodulin; TRPC1; STIM1; Orai1

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique by Wei Zhang; Shenglan Tan; Elijah Paintsil; Ginger E. Dutschman; Elizabeth A. Gullen; Edward Chu; Yung-Chi Cheng (pp. 411-417).

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Keywords: Ribonucleotides; Deoxyribonucleotides; LC/MS/MS; Hypoxia; Hydroxyurea; Aphidicolin

Activation of the P2Y₁ receptor induces apoptosis and inhibits proliferation of prostate cancer cells by Qiang Wei; Stefano Costanzi; Qiu-Zhen Liu; Zhan-Guo Gao; Kenneth A. Jacobson (pp. 418-425).

G protein-coupled receptors, the largest cell surface receptor family, have emerged as critical players in cell death and survival. High gene expression level of the G_q-coupled P2Y₁ nucleotide receptor in PC-3 prostate cancer cells was demonstrated using real-time quantitative PCR and confirmed by Western blotting and confocal laser scanning microscopy. A selective P2Y₁ receptor agonist, the ADP analogue MRS2365, concentration-dependently induced intracellular calcium mobilization (EC₅₀ 5.28nM), which was diminished by P2Y₁ receptor-selective antagonist MRS2500. P2Y₁ receptor activation by MRS2365 induced apoptosis in assays of Caspase-3, LDH release, and annexin-V staining. The pro-apoptotic effect of MRS2365 was blocked by MRS2500, P2Y₁ siRNA, and an inhibitor of the MAP kinase pathway PD98059. MRS2365 significantly inhibited the proliferation of PC-3 cells, examined using a MTT assay. Thus, activation of the P2Y₁ receptor induced cell death and inhibited growth of human prostatic carcinoma PC-3 cells. Activation of the P2Y₁ receptor should be a novel and promising therapeutic strategy for prostate cancer.

Keywords: Abbreviations; 7-AAD; 7-aminoactinomycin D; CHX; cycloheximide; ERK; extracellular receptor-activated kinase; FBS; fetal bovine serum; FITC; fluorescein isothiocyanate; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; LDH; lactate dehydrogenase; MAP kinase; mitogen-activated protein kinase; MRS2179; N; ⁶; -methyl-2′-deoxyadenosine-3′,5′-bisphosphate; MRS2365; (N)-methanocarba-2′-deoxy-2-methylthio-adenosine-5′-diphosphate; MRS2500; 2-iodo-; N; ⁶; -methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; 2-MeSADP; 2-methylthioadenosine 5′-diphosphate; MTT; 3-(4,5-dimethylethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate-buffered saline; TNF-α; tumor necrosis factor-alphaProstate cancer; P2Y; ₁; receptor; Apoptosis; Nucleotide; GPCR

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Biochemical Pharmacology (v.82, #4)