Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O⁶-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O⁶-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.     

Quantitative relationship between guanine O-alkyl lesions produced by Onrigin™ and tumor resistance by O-alkylguanine-DNA alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O⁶-chloroethyl (Onrigin™ and carmustine) and O⁶-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O⁶-methylguanines/cell and ∼300 O⁶-chloroethylguanines/cell, respectively. AGT repaired O⁶-methylguanines until the AGT pool was exhausted, while its repair of O⁶-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O⁶-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O⁶-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.     

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O⁶-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC₅₀ value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O⁶-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.     

Growth Inhibitory Effect of (E)-2,4-bis(p-hydroxyphenyl)-2-Butenal Diacetate through Induction of Apoptotic Cell Death by Increasing DR3 Expression in Human Lung Cancer Cells

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-κB (NF-κB) inactivation and G₂/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.     

Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.     

Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction

Epidemiological studies indicated an enhancement of cigarette smoke-induced carcinogenicity, including hepatocellular carcinoma, by arsenic. We believe that arsenic will enhance the expression of hepatic CYP2A enzyme and NNK metabolism (a cigarette smoke component), thus its metabolites, and carcinogenic DNA adducts. Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential.     

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant’s leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.     

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).     

N6-Substituted adenosine derivatives: selectivity,efficacy, and species differences at A3 adenosine receptors

The activation of the human A(3) adenosine receptor (AR) by a wide range of N(6)-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N(6)-alkyl substitutions, small N(6)-alkyl groups were associated with selectivity for human A(3)ARs vs. rat A(3)ARs, and multiple points of branching were associated with decreased hA(3)AR efficacy. N(6)-Cycloalkyl-substituted adenosines were full (/=6 carbons) hA(3)AR agonists. N(6)-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A(1)ARs. Numerous N(6)-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A(1) and A(3) vs. A(2A)ARs (with variable degrees of partial to full A(3)AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A(3)AR affinity and efficacy of N(6)-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N(6)-(R-1-phenylethyl)adenosine vs. N(6)-(S-1-phenylethyl)adenosine, as well as for the N(6)-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A(3)AR. Interestingly, DPMA, a potent agonist for the A(2A)AR (K(i)=4nM), was demonstrated to be a moderately potent antagonist for the human A(3)AR (K(i)=106nM). N(6)-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (K(i)=0.63nM) than rat A(3)ARs. Dual acting A(1)/A(3) agonists (N(6)-3-chlorobenzyl- 29, N(6)-(S-1-phenylethyl)- 39, and 2-chloro-N(6)-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.     

Anti-arthritis effects of (E)-2,4-bis(p-hydroxyphenyl)- 2-butenal are mediated by inhibition of the STAT3 pathway

Background and Purpose

Products of Maillard reactions between aminoacids and reducing sugars are known to have anti-inflammatory properties. Here we have assessed the anti-arthritis effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal and its possible mechanisms of action.

Experimental Approach

We used cultures of LPS-activated macrophages (RAW264.7 cells) and human synoviocytes from patients with rheumatoid arthritis for in vitro assays and the collagen-induced arthritis model in mice. NO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities were measured in vitro and in joint tissues of arthritic mice, along with clinical scores and histopathological assessments. Binding of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal to STAT3 was evaluated by a pull-down assay and its binding site was predicted using molecular docking studies with Autodock VINA.

Key Results

(E)-2,4-bis(p-hydroxyphenyl)-2-butenal (2.5–10 μg·mL⁻¹) inhibited LPS-inducedNO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities in macrophage and human synoviocytes. This compound also suppressedcollagen-induced arthritic responses in mice by inhibiting expression of iNOS and COX2, and NF-κB/IKK and STAT3 activities; it also reduced bone destruction and fibrosis in joint tissues. A pull-down assay showed that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal interfered with binding of ATP to STAT3. Docking studies suggested that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal bound to the DNA-binding interface of STAT3 possibly inhibiting ATP binding to STAT3 in an allosteric manner.

Conclusions and Implications

(E)-2,4-bis(p-hydroxyphenyl)-2-butenal exerted anti-inflammatory and anti-arthritic effects through inhibition of the NF-κB/STAT3 pathway by direct binding to STAT3. This compound could be a useful agent for the treatment of arthritic disease.     

Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e₆ monoethylenediamine (Mce₆) conjugates was explored to aid in the design of second-generation PDT delivery systems. Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce₆ was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce₆, P-NLS(FITC)-GG-Mce₆) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce₆, P-NLS(FITC)-GFLG-Mce₆). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC₅₀), cell associated drug concentration (CAD) and confocal microscopy. Results. P-GFLG-Mce₆ possessed enhanced chemotherapeutic activity compared to P-GG-Mce₆ indicating enzymatically released Mce₆ was more active than copolymer-bound Mce₆. Lysosomes appeared less sensitive to photodamage as observed by a higher IC₅₀. Nuclear-directed HPMA copolymer-Mce₆ conjugates (P-NLS(FITC)-GG-Mce₆ P-NLS(FITC)-GFLG-Mce₆) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce₆ conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce₆) or amino groups (P-NH₂-GG-Mce₆) also displayed increased chemotherapeutic activity. Conclusions. Nuclear delivery was observed for P-NLS(FITC)-GG-Mce₆ and P-NLS(FITC)-GFLG-Mce₆ indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.     

Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase

The human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) is an important source of resistance to some therapeutic alkylating agents and attempts to circumvent this resistance by the use of hAGT inhibitors have reached clinical trials. Several human polymorphisms in the MGMT gene that encodes hAGT have been described including L84F and the linked double alteration I143V/K178R. We have investigated the inactivation of these variants and the much rarer variant W65C by O(6)-benzylguanine, which is currently in clinical trials, and a number of other second generation hAGT inhibitors that contain folate derivatives (O(4)-benzylfolic acid, the 3' and 5' folate esters of O(6)-benzyl-2'-deoxyguanosine and the folic acid gamma ester of O(6)-(p-hydroxymethyl)benzylguanine). The I143V/K178R variant was resistant to all of these compounds. The resistance was due solely to the I143V change. These results suggest that the frequency of the I143V/K178R variant among patients in the clinical trials with hAGT inhibitors and the correlation with response should be considered.     

Preparation and characterization of dialkylphosphoryl-obidoxime conjugates, potent anticholinesterase derivatives that are quickly hydrolyzed by human paraoxonase (PON1192Q)

The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1192Q being about 50-fold more active than PON1192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 degrees C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.