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Biochemical Pharmacology (v.70, #1)

Editorial Advisory Board (pp. iii).

Drugs targeting mitochondrial functions to control tumor cell growth by Nathalie Dias; Christian Bailly (pp. 1-12).

Mitochondria, the power houses of the cell, are at the cross-road of many cellular pathways. They play a central role in energy metabolism, regulate calcium flux and are implicated in apoptosis. Mitochondrial dysfunctions have been associated with various physiopathological disorders, especially neurodegenerative diseases and cancer. Structurally diverse pharmacological agents have shown direct effects on mitochondria ultra-structures and functions, either at the DNA level or upon targeting proteins located in the inner or outer mitochondrial membranes. The brief review deals with the molecular targets and mechanisms of action of chemically diverse small molecules acting on specific mitochondrial loci, such as the respiratory chain, DNA biogenesis, potassium channels, the Bcl-2 protein and the permeability transition pores (PTP). Drugs, which specifically compromise the structural and functional integrity of mitochondria, may provide novel opportunities to combat cancer cell proliferation, providing that these molecules can be selectively delivered to tumor sites. Different examples reported here show that mitochondrial insult or failure can rapidly lead to inhibition of cell survival and proliferation. Mitochondrial impairment may be a successful anti-cancer strategy.

Keywords: Abbreviations; ANT; adenine nucelotide transporter; KCO; potassium channel openers; MCR; mitochondrial respiratory chain; MMP; mitochondrial membrane permeability; PBR; peripheral benzodiazepine receptor; PTP; permeability transition pore; ROS; reactive oxygen species; SOD; superoxide dismutase; VDAC; voltage-dependent ion channelMitochondria; Cancer chemotherapy; Apoptosis; Respiratory chain; Permeability transition pore

Thymidine phosphorylase suppresses apoptosis induced by microtubule-interfering agents by Hei-Cheul Jeung; Xiao-Fang Che; Misako Haraguchi; Tatsuhiko Furukawa; Chun-Lei Zheng; Tomoyuki Sumizawa; Sun-Young Rha; Jae Kyung Roh; Shin-ichi Akiyama (pp. 13-21).

We investigated the ability of thymidine phosphorylase (TP) to confer cancer cells resistance to MIA (microtubule-interfering agents)-induced apoptosis. Jurkat cells were stably transfected with TP cDNA (Jurkat/TP) and the sensitivity to MIAs were examined. Jurkat/TP cells were more resistant to apoptosis induced by nocodazole, vincristine, vinblastine, paclitaxel and 2-methoxyestradiol than mock-trasfected Jurkat/CV cells. TP enzymatic activity was not required for this effect of TP.Jurkat/TP cells showed weak phosphorylation of Bcl-2, and kinase inhibitors staurosporine and genistein attenuated not only MIA-induced Bcl-2 phosphorylation but also cytotoxicity of MIA in Jurkat/CV, but not in Jurkat/TP. MIAs diminished expression of FasL in Jurkat/TP but not in Jurkat/CV, and neutralization of FasL by anti-FasL antibody considerably attenuated the cytotoxic effect of the MIAs in Jurkat/CV, but the effect of the antibody was marginal in Jurkat/TP cells. Our study provides further evidence that TP functions in conferring resistance on cancer cells to the stress induced by MIAs. In addition, we show that TP-induced inhibition of Bcl-2 phosphorylation and suppression of FasL may contribute to the protective function of TP in cancer cells.

Keywords: Thymidine phosphorylase; Microtubule; Apoptosis; Experimental therapeutics

Inhibition of dipeptidyl peptidase IV by novel inhibitors with pyrazolidine scaffold by Hyae Gyeong Cheon; Sung-Soo Kim; Kwang-Rok Kim; Sang-Dal Rhee; Sung-Don Yang; Jin Hee Ahn; Sung-Dae Park; Jae Mok Lee; Won Hoon Jung; Hye Sook Lee; Hee Youn Kim (pp. 22-29).

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity has been reported to improve nutrient-stimulated insulin secretion through the stabilization of glucagon-like peptide (GLP-1). In the present study, we identified novel DPP-IV inhibitors of pyrazolidine derivatives (Compounds1 and2) and characterized their biological effects in vitro and in vivo. Compound1, an isoleucine pyrazolidide with a phenyl urea group, inhibited rat plasma DPP-IV, porcine kidney DPP-IV, as well as human Caco-2 DPP-IV with IC₅₀ values of 1.70, 2.26, and 2.02μM, respectively. Because of the poor pharmacokinetic properties of Compound1, further optimization was carried out, leading to the discovery of Compound2, which had similar in vitro activities. Compound2 acted as a selective and competitive inhibitor of DPP-IV. MALDI-TOF mass spectrometric analysis proved that the compound (20μM) effectively blocked the degradation of active GLP-1 peptide by 61%. Although similar in in vitro potency, marked improvement of in vivo efficacy and pharmacokinetic properties was seen with Compound2. Oral administration of Compound2 resulted in potent and rapid inhibition of circulating DPP-IV in C57BL/6J mice, with ED₅₀ values of 26mg/kg (s.c.) and 42mg/kg (p.o.). In addition, this compound improved glucose tolerance in ob/ob mice, as determined by an oral glucose tolerance test (OGTT). These results indicate that Compound2 is a potent and selective DPP-IV inhibitor with oral anti-hyperglycemic activity in vivo.

Keywords: Dipeptidyl peptidase IV; GLP-1; Oral glucose tolerance test; ob/ob mice; Insulin secretion; Pharmacokinetics

Licofelone, a novel 5-LOX/COX-inhibitor, attenuates leukocyte rolling and adhesion on endothelium under flow by H. Ulbrich; O. Soehnlein; X. Xie; E.E. Eriksson; L. Lindbom; W. Albrecht; S. Laufer; G. Dannhardt (pp. 30-36).

The main mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of cycloxygenases COX-1 and COX-2. During recent years, combined 5-LOX/COX-inhibition, interfering with the biosynthesis of both prostaglandins and leukotrienes (LTs), has emerged as a possibility to avoid side effects related to COX-inhibition. The aim of the present study was to investigate if there is a contribution of mechanisms other than the reduction of inflammatory prostaglandins and leukotrienes to the anti-inflammatory effect of the LOX/COX inhibitor licofelone. In a flow chamber assay, licofelone (10–30μM) dose-dependently decreased both the rolling and adhesion of leukocytes on endothelial cells (EC). In contrast, no effects were found after treatment of EC with the unselective COX-1/COX-2 inhibitor indomethacin (30μM), the potent and selective 5-LOX inhibitor, ZD-2138 (30μM), the mainly COX-2 inhibitor aceclofenac (30μM), the selective COX-2 inhibitor celecoxib (30μM) and the combination of ZD-2138 with the selective COX-2 inhibitor celecoxib (30μM). In the presence of licofelone (30μM) the expression of E-selectin mRNA in cytokine-stimulated EC was attenuated, whereas no NSAID (30μM) tested showed any effect on E-selectin expression. Moreover, licofelone treatment (30μM) attenuated expression of VCAM-1 and ICAM-1 on inflammatory EC. The effect of licofelone on leukocyte recruitment was also evaluated in vivo. Using a mouse peritonitis model it was found that leukocyte accumulation was markedly reduced in licofelone treated animals (100mg/kg) compared to untreated mice. Thus, the novel 5-LOX/COX inhibitor licofelone possesses anti-inflammatory activity that, in addition to COX/LOX inhibition, involves effects on leukocyte-endothelial interactions.

Keywords: Cyclooxygenase; 5-Lipoxygenase; 5-LOX/COX inhibition; Leukocyte; Selectin; Adhesion; Rolling; Endothelium

Identification of a drug target motif: An anti-tumor drug NK109 interacts with a PNxxxxP by Kengo Morohashi; Ayako Yoshino; Atsushi Yoshimori; Seiichi Saito; Seiichi Tanuma; Kengo Sakaguchi; Fumio Sugawara (pp. 37-46).

The synthetic compound NK109 shows anti-tumor effects against a number of human cancer cell lines. The mechanism of action is thought to involve the inhibition of DNA topoisomerase II. However, NK109 also exhibits potent anti-tumor activities against doxorubicin-, cisplatin- and etoposide-resistant human cell lines. This paper describes target validation of NK109 using biotinylated NK109 and a T7 phage library screening procedure. Phage particles displaying an affinity for NK109 were isolated and the DNA sequence determined. The amino acid sequences of selected peptides, and the results of mutation experiments by alanine scanning, confirmed that the binding target motif of NK109 is PNxxxxP. In silico analysis of the interaction between NK109 and the peptide, by docking simulation and molecular dynamics, supported this conclusion. The PNxxxxP motif exists in the C2 domain of protein kinase Cα. NK109 was confirmed to bind the C2 domain from surface plasmon resonance analysis. Furthermore, NK109 moderately inhibited protein kinase C activity in vitro. Our results show that the anti-tumor activity of NK109 stems from interactions with multiple protein targets.

Keywords: Abbreviations; PKCα; protein kinase Cα; SPR; surface plasmon resonanceNK109; Benzo[; c; ]phenanthridine; Anti-cancer; Phage display; Molecular dynamics; Drug binding motif

Tamoxifen alters gating of the BK α subunit and mediates enhanced interactions with the avian β subunit by R.K. Duncan (pp. 47-58).

Mammalian BK channels are modulated by estrogen and non-steroidal estrogen-like compounds (i.e. xenoestrogens), but the effects are dependent on channel composition. (Xeno)estrogens preferentially activate BK channels through accessory β subunits, but reduce single-channel conductance by interaction with α subunits. In this report, the xenoestrogen tamoxifen was applied to chicken BK channels, in order to asses the mechanism behind drug interaction and to determine the extent to which (xeno)estrogen interaction is extended to avian BK homologs. As with mammalian isoforms, the properties of chicken BK channels were modulated by tamoxifen in a subunit-dependent manner. Tamoxifen reduced single-channel conductance through interaction with the α subunit. However, if the expression construct included the β subunit, tamoxifen increased the channel's open probability and shifted the voltage-activation range to more negative potentials. This effect on channel gating was concentration-dependent, with an EC₅₀ of about 0.2μM. Tamoxifen-mediated reductions in gating charge and in the intrinsic energetics that govern channel equilibrium. The relative contribution of these two effects on channel gating was altered by β co-expression. Modulation by (xeno)estrogens may be an evolutionarily conserved mechanism for non-genomic hormonal actions, and the limited conservation between avian and mammalian β subunits may suggest potential binding motifs. Alternatively, the data are consistent with a tamoxifen-mediated conformation change in the α subunit that alters the way α and β subunits interact, resulting in enhanced gating without direct binding to β.

Keywords: Calcium-activated potassium channel; Hormone; Estrogen; Xenoestrogen; Chicken

Low cytotoxicity of ecteinascidin 743 in yeast lacking the major endonucleolytic enzymes of base and nucleotide excision repair pathways by Daniele Grazziotin Soares; Nadine Paese Poletto; Diego Bonatto; Mirian Salvador; Gilberto Schwartsmann; Jo�o Antonio P�gas Henriques (pp. 59-69).

Ecteinascidin 743 (ET-743) is a promising antitumoral drug for the treatment of soft tissues sarcomas, becoming a good candidate for clinical trials. However, the molecular mechanism of how ET-743 induces cells death is poorly understood. The chemical structure of ET-743 suggests that it can form cytotoxic cross-links with proteins and DNA. Experiments with Escherichia coli and mammalian cells indicate that the nucleotide excision repair (NER) pathway promotes ET-743 cytotoxicity. We therefore analyzed cytotoxicity and tolerance to ET-743 in the yeast Saccharomyces cerevisiae, defective for NER and/or base excision repair (BER), either in single mutants or in combination with mutant alleles of genes encoding proteins involved in DNA translesion synthesis (TLS) and homologous recombination (HR). Treatment of haploid and diploid S. cerevisiae strains with ET-743 led to induced mutagenesis, mitotic gene conversion, and crossing-over. The results indicated that yeast strains lacking endonucleases of the NER and BER pathways are especially resistant for ET-743. The mutagenesis data points to a weak mutagenic activity of ET-743 in both WT and strains lacking BER/NER endonuclease, and that a mutant blocked in both BER and TLS totally lacks induced mutagenesis. The diploid strain shows an increase in the frequencies of crossing-over and mitotic recombination. These data lead us to propose a model for ET-743 action in eukaryotic cells, where the presence of BER and NER endonucleases results in cell death. However, ET-743 damage can be tolerated in BER and/or NER mutants by TLS (error-prone) or in combination with HR (error-free).

Keywords: Abbreviations; BER; base excision repair; ET-743; ecteinascidin 743; LOG; exponential phase; HR; homologous recombination; NER; nucleotide excision repair; SSBs; single-strand breaks; STSs; soft tissue sarcomas; SC; synthetic complete medium; TC-NER; transcription-coupled NER; TLS; translesion synthesis; WT; wild typeET-743; Base excision repair; Nucleotide excision repair; Saccharomyces cerevisiae; Apn1p; Rad1p

Functional expression of particular isoforms of excitatory amino acid transporters by rodent cartilage by Eiichi Hinoi; Liyang Wang; Akihiro Takemori; Yukio Yoneda (pp. 70-81).

In the present study, we have attempted to demonstrate functional expression by the rodent cartilage of particular isoforms of excitatory amino acid transporters (EAATs) essentially required for central glutamatergic signal termination. Constitutive expression of mRNA was shown for the first time with the neuronal EAAT subtype excitatory amino acid carrier-1 (EAAC1), in addition to glial subtypes such as glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), in rat costal chondrocytes cultured for 7–21 days on reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis confirmed the expression of corresponding proteins for both GLAST and GLT-1 in cultured chondrocytes. The accumulation of [³H]glutamate (Glu) occurred in a temperature- and sodium-dependent manner with biochemical and pharmacological profiles similar to those seen for brain EAATs in chondrocytes cultured for 7 days, while [³H]Glu accumulation consisted of a single component with a K_m of 39.1±2.3μM and a V_max of 1320±120pmol/mg protein/min, respectively. In organotypic cultured metatarsals isolated before vascularization from embryonic mice, where cells underwent maturational development from resting to proliferating, prehypertrophic, hypertrophic and calcified chondrocytes in a progressive order of cellular differentiation, moreover, mRNA expression was seen for GLAST, GLT-1 and EAAT4 but not for EAAC1 subtypes. Immunohistochemical analysis revealed distribution profiles different from each other with GLAST, GLT-1 and EAAT4 isoforms in sections of cultured metatarsals and isolated tibiae. These results suggest that extracellular Glu could be cleared up into intracellular locations through particular glial and/or neuronal EAAT isoforms functionally expressed by the rodent cartilage.

Keywords: Abbreviations; ALP; alkaline phosphatase; CCG-III; (2; S; ,3; S; ,4; R; )-2-(carboxycyclopropyl)glycine; CNS; central nervous system; DHK; dihydrokainate; DMEM; Dulbecco's modified Eagle's medium; EAAT; excitatory amino acid transporter; EAAC1; excitatory amino acid carrier-1; EAAT; excitatory amino acid transporter; FBS; fetal bovine serum; GLAST; glutamate aspartate transporter; GLT1; glutamate transporter-1; Glu; glutamate; HKR; HEPES Krebs-Ringer; l; -THA; l; -; threo; -β-hydroxyaspartate; NMDA; N; -methyl-; d; -aspartate; PB; phosphate buffer; PBS; phosphate-buffered saline; PTH1R; parathyroid hormone type 1 receptors; RT-PCR; reverse transcription polymerase chain reaction; SDS; sodium dodecylsulfate; T3MG; (±)-; threo; -3-methylglutamate; VGLUT; vesicular glutamate transporterGlutamate; EAATs; Chondrocytes; [; ³; H]Glutamate accumulation; Metatarsals; ATDC5

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene by Mar�a V. Castelli; Anabella F. Lodeyro; Angela Malheiros; Susana A.S. Zacchino; Oscar A. Roveri (pp. 82-89).

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC₅₀=2.4±0.1μM) which marginally affected electron transport and ATPase activity (IC₅₀=97±4μM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC₅₀=20±4μM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC₅₀=2.2±0.4μM) induced by succinate oxidation in submitochondrial particles but did not collapse the ΔpH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Δ ψ) and/or the localized transmembrane pH difference (ΔpH^S) without affecting the ΔpH between the two bulk aqueous phases (ΔpH^B). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

Keywords: Abbreviations; SMP; phosphorylating submitochondrial Mg; ⁺; -ATP particles; RLM; rat liver mitochondria; ANS; 8-anilino-1-naphtalene sulfonate; 9-AA; 9-aminoacridine; ACMA; 9-amino-6-chloro-2-methoxyacridine; FCCP; carbonyl cyanide; p; -trifluoromethoxyphenyl hydrazonePolygodial; ATP synthesis; Mitochondria; Uncoupler; Antifungal; Mechanism

Site specific alterations of adipose tissue mitochondria in 3′-azido-3′-deoxythymidine (AZT)-treated rats: An early stage in lipodystrophy? by Catherine Deveaud; Bertrand Beauvoit; St�phanie Hagry; Anne Galinier; Audrey Carri�re; B�n�dicte Salin; Jacques Schaeffer; Sylvie Caspar-Bauguil; Yvette Fernandez; Jean-Baptiste Gordien; Dominique Breilh; Luc Penicaud; Louis Casteilla; Michel Rigoulet (pp. 90-101).

Although it is well accepted that treatment with nucleoside reverse transcriptase inhibitors (NRTIs) modifies fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examines the effects of chronic oral administration of 3′-azido-3′-deoxythymidine (AZT) on the mitochondrial metabolism and the redox status management of rat white adipose tissues originating from two anatomical sites, as well as of the rat liver. Results showed that AZT treatment induced differential effects on the mitochondrial functions depending on the anatomical localisation. Indeed, in inguinal adipose tissue, a significant decrease in the cytochrome c oxidase activity and in the mitochondrial DNA (mtDNA) content was observed, whereas the activity of citrate synthase, a mitochondrial protein exclusively encoded by the nucleus, was not affected. In contrast, no significant change in these parameters could be detected for epididymal tissue and for liver. In parallel, no oxidative stress could be detected after treatment, for both white adipose tissues and for liver, even though treated liver exhibited several modifications in redox management. Taken together, these data demonstrate differential mitochondrial effects of AZT on subcutaneous versus visceral white adipose tissue. Moreover, the decrease in mitochondrial oxidative capacity of inguinal adipocyte consecutive to AZT treatment is not primarily due to an oxidative stress per se, but rather to a depletion of the mtDNA content per cell.

Keywords: Abbreviations; AUC; area under the curve; AZT; 3′–azido-3′–deoxythymidine; CCCP; carbonyl cyanide m-chlorophenylhydrazone; GPx; glutathione peroxidase; HAART; highly active anti retroviral therapy; MDA; malondialdehyde; mt DNA; mitochondrial DNA; nDNA; nuclear DNA; NRTI; nucleoside reverse transcriptase inhibitor; ROS; reactive oxygen specie; SOD; superoxide dismutaseAdipocyte; AZT; Lipodystrophy; MtDNA; Respiratory complexes; Reactive oxygen species (ROS)

Cell apoptosis induced by a synthetic carbazole compound LCY-2-CHO is mediated through activation of caspase and mitochondrial pathways by Ming-Jen Hsu; Yee Chao; Ying-Hsin Chang; Feng-Ming Ho; Li-Jiau Huang; Ya-Ling Huang; Tien-Yau Luh; Chih-Ping Chen; Wan-Wan Lin (pp. 102-112).

The mechanisms involved in the apoptotic effect of LCY-2-CHO [9-(2-chlorobenzyl)-9 H-carbazole-3-carbaldehyde], a synthetic carbazole derivative identified as an anti-inflammatory compound, were studied. Cell cycle analysis by propidium iodide staining in human THP-1 monocytic leukemia cells showed the ability of LCY-2-CHO to increase cell population in sub-G1 stage with time- and concentration-dependent manners. LCY-2-CHO-mediated cell death was also demonstrated by DNA laddering and was not related to the release of lactate dehydrogenase. Apoptosis in THP-1 cells induced by LCY-2-CHO was accompanied by the Bid cleavage, collapse of mitochondrial transmembrane potential, the release of cytochrome c and the activation of caspase-3. The apoptotic effect of LCY-2-CHO was diminished by the presence of zVEID-fmk (caspase-6 inhibitor), zIETD-fmk (caspase-8 inhibitor), and zVAD-fmk (non-selective caspase inhibitor), but was not altered by several antioxidants, and cathepsin inhibitor. The Bid cleavage and loss of mitochondrial transmembrane potential, but not the cytochrome c release, were reversed by zIETD-fmk. Comparing the cell selectivity of LCY-2-CHO, we found T-cell acute lymphoblastic CEM leukemia cells were sensitive to 1μM LCY-2-CHO, acute myeloid leukemia HL-60 cells underwent apoptosis at 10μM, while adherent cancer cells, such as PC3, HT29 and MCF-7, were resistant to 30μM LCY-2-CHO within 24-h incubation. Taken together in the present study, we demonstrated LCY-2-CHO might be apoptotic for malignant hematopoietic cells but not anchorage-dependent cells. This action is mediated by an intrinsic caspase-dependent apoptotic event involving mitochondria.

Keywords: Abbreviations; GSH; glutathione; LDH; lactate dehydrogenase; NAC; N; -acetyl cysteine; ROS; reactive oxygen species; zVEID-fmk; Z-Val-Glu-Ile-Asp-fluoromethylketone; zIETD-fmk; Z-Ile-Glu-Thr-Asp-fluoromethylketone; zVAD-fmk; Z-Val-Ala-Asp-fluoromethylketone; zYVAD-fmk; Z-Tyr-Val-Ala-Asp-fluoromethylketone; zVDVAD-fmk; Z-Val-Asp-Val-Ala-Asp-fluoromethylketoneLCY-2-CHO; Carbazole; Apoptosis; Caspases; Mitochondria

Gα_q-coupled receptor signaling enhances adenylate cyclase type 6 activation by Michael A. Beazely; Val J. Watts (pp. 113-120).

Calcium signaling robustly inhibits AC6 activity in membrane preparations and in intact cells via capacitative calcium entry (CCE). However, the release of intracellular calcium has not been demonstrated to robustly alter AC6 signaling and activation of Gα_q-coupled receptors in tissues that express AC6 enhances cyclic AMP accumulation. To specifically examine the ability of Gα_q-coupled receptors to modulate AC6 signaling in intact cells, we used stably transfected HEK-AC6 cells. We demonstrate that AC6 activation is potentiated by activation of endogenous muscarinic receptors expressed in HEK293 cells. Muscarinic receptor activation failed to potentiate the activation of the closely related AC5 isoform. Expression of recombinant Gα_q-coupled muscarinic or serotonin receptors, or constitutively active Gα_q, also potentiated drug-stimulated cyclic AMP accumulation in HEK-AC6 cells. Muscarinic receptor-mediated potentiation of AC6 activation was not due to activation of PKC or modulation of Gα_i/o-mediated inhibition of AC6. We demonstrate that calcium chelation or inhibition of calmodulin attenuates the effect of carbachol on AC6 activation. These data support the hypothesis that Gα_q-coupled receptor-mediated calcium signaling potentiates AC6 activation in intact cells.

Keywords: Abbreviations; AC; adenylate cyclase; BAPTA/AM; 1,2-bis(; o; -aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid tetra(acetoxymethyl) ester; CaMK; calmodulin-dependent kinase; CCE; capacitative calcium entry; HEK; human embryonic kidney; PKC; protein kinase C; W7; 1,2-bis(; o; -aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid tetra(acetoxymethyl) esterAdenylate cyclase; G protein; Calcium; PKC; Muscarinic receptor

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells by Tsipi Ben-Kasus; Zvi Ben-Zvi; Victor E. Marquez; James A. Kelley; Riad Agbaria (pp. 121-133).

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[¹⁴C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5′-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/ nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[¹⁴C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2′-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.

Keywords: Abbreviations; 5-Aza-C; 5-azacytidine; 5-Aza-dC; 5-aza-2′-deoxycytidine; Zeb; zebularine; Zeb-MP; zebularine-5′-monophosphate; Zeb-DP; zebularine-5′-diphosphate; Zeb-TP; zebularine-5′-triphosphate; Zeb-DP-EA; zebularine-5′-diphosphoethanolamine; Zeb-DP-Chol; zebularine-5′-diphosphocholine; 2′-dZeb-TP; 2′-deoxyzebularine-5′-triphosphate; CPEU; cyclopentenyl uridine; ODN; oligodeoxynucleotide; DMEM; Dulbecco's modified Eagle's medium; IC; ₅₀; drug concentration resulting in 50% inhibition of growth; CDA; cytidine deaminase; DNMT; DNA methyltransferase; PDE-1; snake venom phosphodiesterase-1; AP; alkaline phosphatase; UCK; uridine/cytidine kinaseZebularine; 2(1H)-Pyrimidinone riboside; T24 bladder carcinoma; Phosphorylation; DNA and RNA incorporation; DNA methyltransferase

Inhibition of dipeptidyl-peptidase IV catalyzed peptide truncation by Vildagliptin ((2S)-{[(3-hydroxyadamantan-1-yl)amino]acetyl}-pyrrolidine-2-carbonitrile) by Inger Brandt; Jurgen Joossens; Xin Chen; Marie-Berthe Maes; Simon Scharp�; Ingrid De Meester; Anne-Marie Lambeir (pp. 134-143).

Vildagliptin (NVP-LAF237/(2S)-{[(3-hydroxyadamantan-1-yl)amino]acetyl}-pyrrolidine-2-carbonitrile) was described as a potent, selective and orally bio-available dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5) inhibitor [Villhauer EB, Brinkman JA, Naderi GB, Burkey BF, Dunning BE, Prasad K, et al.1-[[(3-Hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem 2003;46:2774–89]. Phase III clinical trials for the use of this compound in the treatment of Type 2 diabetes were started in the first quarter of 2004. In this paper, we report on (1) the kinetics of binding, (2) the type of inhibition, (3) the selectivity with respect to other peptidases, and (4) the inhibitory potency on the DPP IV catalyzed degradation of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and substance P. Vildagliptin behaved as a slow-binding DPP IV inhibitor with an association rate constant of 1.4×10⁵M⁻¹s⁻¹ and a K_i of 17nM. It is a micromolar inhibitor for dipeptidyl-peptidase 8 and does not significantly inhibit dipeptidyl-peptidase II (EC 3.4.11.2), prolyl oligopeptidase (EC 3.4.21.26), aminopeptidase P (EC 3.4.11.9) or aminopeptidase M (EC 3.4.11.2). There was no evidence for substrate specific inhibition of DPP IV by Vildagliptin or for important allosteric factors affecting the inhibition constant in presence of GIP and GLP-1.

Keywords: Abbreviations; DPP; dipeptidyl-peptidase; GLP-1; glucagon-like peptide-1; GIP; glucose-dependent insulinotropic polypeptide; PO; prolyl oligopeptidase; p; NA; para-nitroaniline; DTT; dithiothreitolDipeptidyl-peptidase IV; LAF237; GLP-1; GIP; Substance P; Protease inhibitor

Limited protective role of V-PYRRO/NO against cholestasis produced by alpha-naphthylisothiocyanate in mice by Jie Liu; Yu-Ying He; Colin F. Chignell; James Clark; Page Myers; Joseph E. Saavedra; Michael P. Waalkes (pp. 144-151).

O²-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide donor that has been shown to protect against hepatotoxic effects of endotoxin, acetaminophen and cadmium. This study examined the effects of V-PYRRO/NO on alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in mice. Mice were given V-PYRRO/NO via osmotic pumps (5.4mg/ml; 0.5μl/h) starting 24h before receiving a hepatotoxic dose of ANIT (150mg/kg in olive oil, i.g.), and continuing for additional 48h (3-day pumps). V-PYRRO/NO administration partially ameliorated ANIT-induced hepatotoxicity, as evidenced by reduced serum alanine aminotransferase and alkaline phosphatase, markers of liver cell death, and by improved liver pathology. However, V-PYRRO/NO had no effect on ANIT-induced cholestasis, as ANIT-increased serum bilirubin levels and gamma-glutamyl transpeptidase activity were not ameliorated. Microarray and real time RT-PCR analysis revealed that ANIT intoxication altered expression of various genes, including genes encoding metabolic enzymes, transporter proteins, acute phase proteins, inflammation- and, apoptosis-related genes, as well as other genes related to liver injury. V-PYRRO/NO treatment attenuated ANIT-induced elevations in certain inflammation- and apoptosis-related genes, but had no effect on ANIT-induced disturbance on the expression of genes related to metabolism, transport, and acute phase proteins. Thus, the liver-selective NO donor, V-PYRRO/NO, was partially protective against ANIT-induced liver injury, without affecting ANIT-induced cholestasis and cholestasis-related gene expression.

Keywords: V-PYRRO/NO; ANIT hepatotoxicity; Cholestasis; Inflammation; Gene expression

Chronic Δ⁹-tetrahydrocannabinol treatment produces antinociceptive tolerance in mice without altering Protein Kinase A activity in mouse brain and spinal cord by George D. Dalton; Forrest L. Smith; Paul A. Smith; William L. Dewey (pp. 152-160).

The present study investigated the effect of different levels of Δ-9-tetrahydrocannabinol (Δ⁹-THC) antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain and spinal cord. To strengthen this investigation, a positive control was developed to demonstrate the assay utilized in this study was sensitive enough to detect an increase in PKA activity in the anatomical regions utilized in this study. The membrane-permeant and phosphodiesterase-resistant cAMP analog 8-Bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS) was utilized for the development of this positive control and this compound produced an increase in PKA activity in several mouse brain regions (i.c.v.) and lumbar spinal cord (i.t.) following its administration. Models were then developed in which mice expressed either a 13-fold or 49-fold level of Δ⁹-THC antinociceptive tolerance following chronic treatment with 10mg/kg Δ⁹-THC or 80mg/kg Δ⁹-THC for 6.5 days. Basal and total cytosolic and particulate PKA activities were measured directly in homogenates from the striatum, hippocampus, cerebellum, cortex and lumbar spinal cord. Results from this study indicate that chronic exposure to Δ⁹-THC does not produce an increase in PKA activity in these mouse brain regions or spinal cord. Future work is needed to determine the role of PKA in cannabinoid tolerance in mice.

Keywords: Cannabinoid; THC; Antinociception; Tolerance; Protein Kinase A

Transport of anthracyclines and mitoxantrone across membranes by a flip-flop mechanism by Ronit Regev; Daniella Yeheskely-Hayon; Hagar Katzir; Gera D. Eytan (pp. 161-169).

The objectives of the present work are to characterize the transport of mitoxantrone and three anthracyclines in terms of binding to the membrane surface, flip-flop across the lipid core of the membrane, and release into the medium. Mitoxantrone and anthracyclines are positively charged amphipathic molecules, and as such are located at the surface of membranes among the headgroups of the phospholipids. Therefore, their transport across membranes occurs by a flip-flop mechanism, rather than by diffusion down a continuous concentration gradient located in the lipid core of the membrane. Flip-flop rates have been estimated with liposomes labeled at their surface with 7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) moiety attached to the headgroup of phosphatidylethanolamine. Flip-flop of mitoxantrone, doxorubicin, daunorubicin, and idarubicin occurred with half-lives of 6, 0.7, 0.15, and 0.1min, respectively. Partition of the drugs into the membrane occurred with lipid phase/aqueous medium coefficients of 230,000, 8600, 23,000, and 40,000 for mitoxantrone, doxorubicin, daunorubicin, and idarubicin, respectively, which are much higher than their corresponding octanol/aqueous medium values. There was no direct correlation between the lipophilicity of the drugs and their lipid phase/aqueous medium partition coefficient or their flip-flop rate. Mitoxantrone exhibited the highest affinity toward liposome membranes, but the slowest flip-flop across the lipid core of the membranes. Simulation of drug uptake into liposomes revealed that transmembrane movement of the mitoxantrone and anthracyclines is determined by their flip-flop rate and affinity toward membranes.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphoglycerol; DOPC; 1,2-dioleyl-; sn; -glycero-3-phosphocholine; DOPG; 1,2-dioleyl-; sn; -glycero-3-phosphoglycerol; MDR; multidrug resistance; NBD-PE; 7-nitrobenzo-2-oxa-1,3-diazol-4-yl-phosphatidylethanolamineMembrane transport; Anthracyclines; Doxorubicin; Mitoxantrone; Multidrug resistance; P-glycoprotein

Z-analysis: A new approach to analyze stimulation curves with intrinsic basal stimulation by Peter B. Hedlund; Gabriel von Euler (pp. 170-175).

In the study of receptor biology it is of considerable importance to describe the stimulatory properties of an agonist according to mathematically defined models. However, the presently used models are insufficient if the experimental preparation contains an intrinsic basal stimulation. We have developed a novel approach, tentatively named Z-analysis. In this approach, the concentration of endogenous agonist is calculated by extending the stimulation curve to zero effect. The concentration of endogenous agonist is then combined with the concentration of added agonist to estimate the true EC₅₀ value. We developed a new model, the Z-model, specifically for this purpose, but in addition, we describe how Z-analysis can be applied to the traditional E₀-model. Models were applied to computer-generated curves with different Hill coefficients, using iterative curve fitting procedures. In addition to applying the models to ideal cases, we also used Monte Carlo-simulated data. Specific transformations were used to enable comparisons between parameters determined from these models. Both models were able to provide estimates of all eight parameters analyzed, both using ideal data and on Monte Carlo-simulated data. The Z-model was found to provide better estimates of the concentration of endogenous agonist, the EC₅₀ values, and the Hill value, in curves with Hill coefficient deviating from one. In conclusion, Z-analysis was suitable both to determine the concentration of endogenous agonists and to determine true EC₅₀ values. We found several advantages with the Z-model compared to traditional E₀-model for analysis of stimulation curves that contain basic intrinsic stimulation.

Keywords: Receptor; Curve analysis; Stimulation; Agonist concentration; EC; ₅₀; Hill coefficient

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Biochemical Pharmacology (v.70, #1)

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Biochemical Pharmacology (v.70, #1)