Regioselective 2-hydroxylation of 17beta-estradiol by rat cytochrome P4501B1

Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17beta-estradiol (E2) as a substrate. Km and Kcat values were estimated using the Michaelis-Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61+/-0.23 and 1.84+/-0.73 microM; the turnover numbers (Kcat) were 0.23+/-0.02 and 0.46+/-0.05 pmol/min/pmol P450; and the catalytic efficiencies (Kcat/Km) were 0.37 and 0.25, respectively. For human CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 1.22+/-0.25 and 1.10+/-0.26; the turnover numbers were 1.23+/-0.06 and 0.33+/-0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4- to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low Km E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3',5'-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 microM, respectively. The Ki values of 7,8-benzoflavone (alpha-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 microM, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.     

N6-methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates

In this study we present the identification and characterization of the enzyme involved in the N6-cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (N6-cyclopropyl-PMEDAP) conversion to biologically active 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) as well as abacavir 5'-phosphate to carbovir 5'-phosphate. This enzyme was purified from rat liver to homogeneity; it appears to be composed from six 42 kDa subunits and its native form has the molecular weight 260 kDa. This so far unknown enzyme catalyzes conversion of both N6-methyl-AMP and N6-methyl-dAMP to IMP and/or dIMP, respectively. The enzyme acts as 6-(N-substituted amino)purine 5'-nucleotide aminohydrolase with the reaction mechanism very similar to AMP deaminase. The enzyme does not deaminate AMP and dAMP, or the corresponding nucleosides. It is inhibited by deoxycoformycin 5'-phosphate but not by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).     

Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases

Amdoxovir [(-)-beta-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.     

Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs,inhibitors and implications for therapy

Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.     

Showdomycin and its reactive moiety,maleimide: A comparison in selective toxicity and mechanism of action in vitro

Showdomycin, a C-nucleoside antibiotic, was twice as toxic to L1210 murine leukemia cells as to murine bone narrow progenitor cells of the granulocyte-macrophage series. Its aglycone, maleimide, was equally toxic to both cell lines. Cysteine, adenosine and the potent nucleoside transport inhibitor 6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine (HNBMPR) reversed the early stages of toxicity of showdomycin to L1210 cells, but did not reduce the toxicity of maleimide. At cytotoxic concentrations, showdomycin progressively inactivated the nucleoside uptake system to completion. This inhibition of nucleoside uptake was reversed by cysteine under conditions where it reversed cytotoxicity. The binding of 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine (NBMPR) by L1210 cells was also inhibited by showdomycin, indicating that the antibiotic inactivated the nucleoside transport site. The data suggest that the C-nucleoside structure confers some selectivity to the cytotoxic action of maleimide, directing it toward the nucleoside transport system of the tumor cell.     

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl-N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2-oxoquinolyl oxy)] -butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 micrograms/ml collagen was 5 microM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 microM, respectively. We purified over 480-fold the soluble low Km form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sepharose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a Km value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 microM respectively, and a Vmax value of 85.3 and 19.8 nmole/min/mg protein, respectively. Ki value of the OPC-13135 for the enzyme was 0.015 microM and was of competitive fashion against cyclic AMP.     

Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.     

Functional calcium coupling with the human metabotropic glutamate receptor subtypes 2 and 4 by stable co-expression with a calcium pathway facilitating G-protein chimera in Chinese hamster ovary cells

The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Galphaq with the C-terminal five amino acids replaced with those from Galphai3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of L-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740>L-CCG-I=DCG-IV>L-glutamate>/=(2R,4R)-APDC>/=(1S,3R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was L-AP4>L-SOP>/=ACPT-I=L-CCG-I>/=L-glutamate=(R,S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [35S]GTPgammaS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.     

Hypoxanthine Transport in Human Glioblastoma Cells and Effect on Cell Susceptibility to Methotrexate

PURPOSE: Cancer cells may circumvent the cytotoxic effect of antimetabolite drugs that inhibit de novo nucleotide synthesis via the uptake of extracellular preformed nucleobases or nucleosides. The goal of this study was to investigate the nucleobase transport mechanism in human U-118 glioblastoma cells and to determine whether the purine nucleobase hypoxanthine affects cell susceptibility to methotrexate. METHODS: Uptake experiments were performed using 3H-labeled hypoxanthine. RT-PCR was used to determine the expression of nucleoside transporters. Methotrexate-induced apoptosis was analyzed using annexin V staining and FACScan analysis. RESULTS: Hypoxanthine transport in U-118 cells involved both carrier-mediated (Km = 10.5 +/- 6.3 microM, Vmax = 1.45 +/- 0.69 pmol/10(5) cells/60 s) and simple diffusion processes (Kd = 0.36 +/- 0.009 microm/10(5) cells/60 s). Uptake was sensitive to Na+ and inhibited by nucleobases but not nucleosides or nucleoside transport inhibitors. In contrast, uptake of a nucleoside, uridine, was inhibited by nucleosides but not nucleobases. RT-PCR analysis suggested the presence of hENT1, hENT2, and hCNTI nucleoside transporters in U-118 cells. In the absence of hypoxanthine, methotrexate inhibited U-118 cell proliferation and induced apoptosis. These toxic effects were diminished when hypoxanthine was present at physiologically relevant concentrations. CONCLUSIONS: Hypoxanthine transport in U-118 cells involves a Na+-dependent, high-affinity nucleobase transport system functionally distinct from nucleoside transporters. At physiologic concentrations, hypoxanthine protects glioblastoma cells from the cytotoxicity of methotrexate.     

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5)

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.     

Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: High-affinity inhibitors of human concentrative nucleoside transporter 1

Human concentrative nucleoside transporters (hCNTs) mediate electrogenic secondary active transport of physiological nucleosides and nucleoside drugs into cells. Six fused-pyrimidine ribonucleosides and one 2′-deoxynucleoside were assessed for their abilities to inhibit [³H]uridine transport in the yeast Saccharomyces cerevisiae producing recombinant hCNT1, hCNT2 or hCNT3. Six of the analogs inhibited hCNT1 with K_i values < 1 μM whereas only two analogs inhibited hCNT3 with K_i values < 1 μM and none inhibited hCNT2. To assess if the inhibitory analogs were also permeants, currents evoked were measured in oocytes of Xenopus laevis producing recombinant hCNT1, hCNT2 or hCNT3. Significant inward currents, indicating permeant activity, were generated with (i) three of the analogs in hCNT1-producing oocytes, (ii) none of the analogs in hCNT2-producing oocytes and (iii) all of the analogs in hCNT3-producing oocytes. Four were not, or were only very weakly, transported by hCNT1. The thienopyrimidine 2′-deoxynucleoside (dMeThPmR, 3) and ribonucleoside (MeThPmR, 4) were the most active inhibitors of uridine transport in hCNT1-producing oocytes and were an order of magnitude more effective than adenosine, a known low-capacity transport inhibitor of hCNT1. Neither was toxic to cultured human leukemic CEM cells, and both protected CEM cell lines with hCNT1 but not with hENT1 against gemcitabine cytotoxicity. In summary, dMeThPmR (3) and MeThPmR (4) were potent inhibitors of hCNT1 with negligible transportability and little apparent cytotoxicity, suggesting that pending further evaluation for toxicity against normal cells, they may have utility in protecting normal hCNT1-producing tissues from toxicities resulting from anti-cancer nucleoside drugs that enter via hCNT1.     

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.     

Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: a significant role of specific binding by serum albumin in the improved potency and stability

AS-3201 [(3R)-2'-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4'(1'H)-pyrrolo[1,2-a]pyrazine]-1',2,3',5(2'H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 (R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode (S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA-R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation. (13)C NMR observations of the succinimide ring (5-(13)C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and (19)F NMR of the pendent benzyl ring (2-(19)F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.     

Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.     

Bis(pivaloyloxymethyl) thymidine 5'-phosphate is a cell membrane-permeable precursor of thymidine 5'-phosphate in thymidine kinase deficient CCRF CEM cells

Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM(2)-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM(2)-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk-). After 72 h incubation at 37 degrees C, TdR showed significant antiproliferative activity (IC(50)=27 microM) against CEM cells but was weakly effective (IC(50)=730 microM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5'-monophosphate (POM(2)-dTMP) was equally inhibitory (IC(50)=5 microM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-(3)H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk- with 5 microM POM(2)-[methyl-(3)H]dTMP. The incorporation of [methyl-(3)H]TdR into HClO(4)-insoluble cell residue by CEM tk- was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk- incorporated radioactivity from POM(2)-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM(2)-dTMP can penetrate into cells and serve as a source of dTMP.     

Counteracting effects of NADPH oxidase and the Na+/Ca2+ exchanger on membrane repolarisation and store-operated uptake of Ca2+ by chemoattractant-activated human neutrophils

This study was designed to investigate the possible involvement of NADPH oxidase and the Na(+)/Ca(2+) exchanger in regulating membrane repolarisation and store-operated uptake of Ca(2+) by FMLP (1 microM)-activated human neutrophils. Diphenyleneiodonium chloride (DPI, 5-10 microM) and KB-R7943 (2.5-10 microM), inhibitors of NADPH oxidase and the reverse mode of the Na(+)/Ca(2+) exchanger respectively, were used as pharmacological probes. Transmembrane fluxes of Ca(2+), K(+) and Na(+) were determined radiometrically, while alterations in membrane potential and cytosolic Ca(2+) were evaluated using spectrofluorimetric procedures. DPI, added to the cells at the time of maximum FMLP-activated membrane depolarisation, accelerated the rates of both membrane repolarisation and influx of Ca(2+), while KB-R7943 effectively antagonised these processes. SKF 96365 (10 microM), an antagonist of store-operated Ca(2+) channels, abolished the influx of Ca(2+) into FMLP-activated neutrophils, but had no effects on membrane repolarisation, suggesting that the Na(+)/Ca(2+) exchanger is primarily involved in mediating membrane repolarisation, thereby facilitating uptake of Ca(2+) via store-operated channels. These observations are compatible with prominent negative and positive regulatory roles for NADPH oxidase and the Na(+)/Ca(2+) exchanger respectively in regulating the rates of membrane repolarisation and store-operated uptake of Ca(2+) by chemoattractant-activated neutrophils.     

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors

The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.