• Design and synthesis of cyclodiene insecticide biosensors and fluorescent probes

      Brummel, Kathleen Elizabeth; Wright, Jeremy, Ph.D. (1997)
      A fiber optic evanescent fluorosensor has been developed for the detection of cyclodiene insecticides which possess hexachlorobicyclic ring systems. Four polyclonal (pAb) and one monoclonal (mAb) antibodies have been generated from synthesized haptens by conjugation with bovine serum albumin (BSA). These include, 6-(1,4,5,6,7,7-hexachloro-bicyclo(2.2.1) heptane-2, 3b-succinimido)-caproic acid (CCA), 3-(1,4,5,6,7,7-hexachloro-bicyclo(2.2.1) heptane-2, 3b-succinimido)-alanine acid (CAPA), 6,7-dihydro-6-carboxyaldrin (ALD-COOH) and N-(5-carboxypentanyl)-5-norbornene-2,3-dicarboxyimide(2,3b) bicyclo-1,4,5,6,7,7-hexachloro-cycloheptane (HC-NBCA). Their respective Abs were anti-CCA, anti-CAPA, anti-ALD, anti-HCNBCA and mAb IF11. Fluorescein conjugates of 1,4,5,6,7,7 hexachloro-5-norbornene 2,3 dicarboxylic anhydride, CCA and ALD-COOH were synthesized and designated as FL-CCA, FL-CCA long chain and FL-ALD. Antibodies immobilized covalently on quartz fibers were used to detect chlordane, heptachlor, dieldrin, endrin, aldrin and endosul fan by competitive inhibition of binding of the fluorescein conjugates (designated FL-probes). FL-ALD (10 nM) demonstrated affinity for anti-ALD, anti-HCNBCA and anti-CCA Ab-coated fibers at 50 mug/mL. In competition studies with anti-ALD, 0.1 muM aldrin and CCA inhibited fluorescence by 50% and 45%, respectively. FL-ALD was inhibited by 53% with heptachlor and 62% with aldrin at 0.1 muM on anti-HCNBCA fibers and CCA caused 73% and aldrin 40% inhibition on anti-CCA fibers. Anti-CAPA demonstrated affinity towards FL-CCA long chain with a slow dissociating component upon removal of the FL-probe from the flow buffer. FL-CCA binds to anti-CCA with high affinity (K subscript D=1.9 nM) and reversibility. Competitive inhibition of FL-CCA by the different cyclodiene insecticides showed CCA > chlordane > heptachlor > dieldrin > aldrin. Detection limits were 0.04 ng/mL, 0.4 ng/mL, 4 ng/mL, 4 ng/mL and 370 ng/mL, respectively. Since CCA was used to generate the antisera, cross-reactivity with other chlorinated hydrocarbons was expected. The level used to test cross-reactivity was 0.1 muM, representing the lowest concentration causing a significant change in fluorescence between compounds. The biosensor showed cross-reactivities for chlordane, heptachlor, dieldrin, endrin, endosulfan and aldrin to be 64, 60, 51, 20, 9 and 7, respectively, where CCA = 100. These experiments show that the biosensor can recognize various chlorinated insecticides based upon their structural similarities to the hapten and the fluorescein conjugates used. Extension of the research could ultimately result in an on-site detection system for cyclodiene insecticides.
    • Photosensitization by diaziquone: Correlation between diaziquone cytotoxicity and photoinduced free radicals in MCF-7 cells

      Al-Nabulsi, Isaf; Wright, Jeremy, Ph.D. (1991)
      The ability of visible light to enhance the activity of diaziquone (AZQ) was evaluated in MCF-7 human breast cancer cells. Exponentially growing monolayers of MCF-7 cells were incubated for 1 hr with AZQ (IC{dollar}\sb{lcub}90{rcub}{dollar}, 0.05 {dollar}\mu{dollar}M, IC{dollar}\sb{lcub}50{rcub}{dollar}, 0.3 {dollar}\mu{dollar}M, or various concentrations of AZQ) prior to variable time intervals of visible light irradiation. Irradiations were performed using a 100W quartz-halogen lamp or 100W mercury arc lamp with a dose rate of 30 or 170 mW/m{dollar}\sp2{dollar}, respectively. The effect of visible light and/or AZQ on cellular growth was determined by clonogenic assay. The results show that MCF-7 cells were sensitive to growth inhibition by AZQ. Without AZQ, visible light irradiation had no effect on cell survival, while with AZQ, visible light potentiated its cytotoxicity by a factor of 1.6 at 10% survival. This potentiation of AZQ activity is correlated with the formation of free radicals (hydroxyl radicals and AZQ semiquinone) and with the production of DNA strand breaks as measured by electron paramagnetic resonance and gel electrophoresis, respectively. These results support the hypothesis that free radical formation is part of the mechanism of action of AZQ. Moreover, they indicate that visible light irradiation can increase the activity of AZQ and may allow its use in the treatment of tumor in human patients.
    • Structure-activity relationship of cocaine analogs: The synthesis of 3-beta-substituted cocaine analogs and computer-aided prediction of the pharmacological activity of cocaine analogs

      Yang, Biao; Wright, Jeremy, Ph.D. (1994)
      Ten 3{dollar}\beta{dollar}-ecgonine analogs were synthesized and characterized by {dollar}\sp1{dollar}H and {dollar}\sp{lcub}13{rcub}{dollar}C NMR, MS and elemental analysis. The compounds were synthesized as ({dollar}-{dollar})-stereoisomers from ({dollar}-{dollar})-cocaine. These compounds were assessed for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to rat striatal tissue and to inhibit ({dollar}\sp3{dollar}H) dopamine uptake into rat striatal synaptosomes. In this series of compounds, the length of the spacer between the aryl group and the tropane skeleton ranged from 1 to 4 bond distances, and conformational flexibility of the linkage and orientation of the aryl ring system were controlled by various types of linkage. The results showed that the potencies of these 3{dollar}\beta{dollar} substituted ecgonine methyl esters were acutely sensitive to the distance between the aryl ring and the tropane skeleton and to the orientation of the aryl ring system. The most potent of the analogs was (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo (3.2.1) octyl 3-{dollar}\beta{dollar}-styrene. One of the less potent compounds was found to inhibit ({dollar}\sp3{dollar}H) cocaine binding and ({dollar}\sp3{dollar}H) dopamine uptake by a mechanism apparently different from that of the nine other analogs. The current study provided a clearer picture of the shape and size of the putative hydrophobic binding pocket at the cocaine receptor. Conformational properties of cocaine, WIN 32 065-2 and the WIN vinyl analog were studied via Austin Model 1 (AM1) semi-empirical calculations. The conformational space of the molecules, as defined by the dihedral angles representing the orientation of the 2{dollar}\beta{dollar} and 3{dollar}\beta{dollar} sidechains, was calculated on a 13 x 13 grid in the gas phase and included full geometry optimization at each grid point. Aqueous solvation free energy surfaces were obtained using the AM1-Solvation Model 2 (AM1-SM2). The lowest energy points from the surfaces were fully optimized in both the gas and aqueous phases. Results predict the minimum 2{dollar}\beta{dollar} and 3{dollar}\beta{dollar} sidechain conformations to be similar between the gas and aqueous phases, between the neutral and protonated forms of the molecules and between the three molecules. A detailed analysis of the relationship of structural contributions to changes in conformational properties is presented. Population analysis of the minimum energy regions indicates the neutral forms of WIN and the WIN vinyl analog to be more conformationally restricted than cocaine. This conformational restriction is related to steric interactions between the 2{dollar}\beta{dollar} and 3{dollar}\beta{dollar} moieties of the WIN compounds. Cocaine is calculated to be more favorably solvated than the WIN compounds due to the presence of the ester groups in the 2{dollar}\beta{dollar} and 3{dollar}\beta{dollar} moieties. The calculations predict the pK{dollar}\sb{lcub}\rm a{rcub}{dollar}s of the tropane nitrogen of the WIN compounds to be higher than in cocaine. A model is presented relating the decreased conformational flexibility, decreased aqueous solvation and increased pK{dollar}\sb{lcub}\rm a{rcub}{dollar}s of the WIN compounds to their increased binding affinity to the cocaine receptor. The present results suggest that both the protonated form and the neutral form of the compounds may bind with high affinity to the cocaine receptor. The binding of either form of the drugs could not be eliminated based on the current study. (Abstract shortened by UMI.)
    • The synthesis and pharmacological activity of 3-arylecgonine methyl ester and 3-carbamoyloxyecgonine methyl ester analogues: Probes for the characterization of the dopaminergic cocaine receptor

      Kline, Richard Harry, Jr.; Wright, Jeremy, Ph.D. (1991)
      Several analogues of 3-arylecgonine methyl ester were designed, synthesized and characterized by {dollar}\sp1{dollar}H and {dollar}\sp{lcub}13{rcub}{dollar}C NMR, IR and MS. The compounds were synthesized as racemates from 2,4,6-cycloheptatriene carboxylic acid or as enantiomerically pure compounds from 1 R-cocaine hydrochloride. These analogues were assessed for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to bovine striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into striatal synaptosomes. Methyl(1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo (3.2.1) octane-2 carboxylate was the most potent showing IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of ({dollar}\sp3{dollar}H) cocaine binding and ({dollar}\sp3{dollar}H) dopamine uptake of 22 and 133 nM, respectively. The racemates and the 1R-isomers proved to be equally potent inhibitors of binding and uptake. The 1RS-{dollar}\alpha{dollar}-dinitrophenylecgonine methyl ester analogue had the lowest potency. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of binding and uptake were 11 and 30 {dollar}\mu{dollar}M, respectively. One of these compounds, 1R-{dollar}\beta{dollar}-(p-aminophenyl)ecgonine methyl ester, was designed and synthesized as a potential ligand for affinity chromatography. Several (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-phenylcarbamoyloxy)ecgonine methyl ester analogues were also designed, synthesized, and spectroscopically characterized. Many of these compounds were synthesized as 1{dollar}R{dollar}-stereoisomers from 1{dollar}R{dollar}-ecgonine methyl ester in good yields. These compounds were tested for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to rat striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into synaptosomes prepared from the same tissue. The most potent of these analogues was (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-3{dollar}\sp\prime {dollar}-nitrophenylcarbamoyloxy)-8-methyl-8-azabicyclo (3.2.1) octane-2-carboxylic acid methyl ester. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition binding and uptake were 37 and 178 nM, respectively. In general, compounds which contained meta substituents on the phenyl ring of the C{dollar}\sb3{dollar} phenylcarbamate side chain were more potent than those with para substituents. Also, analogues which contained electron withdrawing groups on the same phenyl ring had considerably higher potencies than those with electron donating groups. Meta and para substituted amino 3-carbamoyloxyecgonine methyl ester analogues were found to be potential affinity chromatography ligands. Two isothiocyanato derivatives proved to bind irreversibly to the receptor, and two azido compounds were shown to function as photoaffinity ligands. These probes may be instrumental in the eventual isolation and purification of the cocaine receptor/dopamine transporter.