RESUMO
We have identified a series of hydrophilic indium-labeled DOTA and DO3A conjugates of naltrindole (NTI) that are suited to in vivo studies of peripheral delta opioid receptors. Indium(III) complexes, linked to the indole nitrogen of NTI by six- to nine-atom spacers, display high affinities (0.1-0.2 nM) and excellent selectivities for binding to delta sites in vitro. The [111In]-labeled complexes can be prepared in good isolated yields ( approximately 65%) with high specific radioactivities (>3300 mCi/mumol). The spacers serve as pharmacokinetic modifiers, and log D7.4 values range from -2.74 to -1.79. These radioligands exhibit a high level of specific binding (75-94%) to delta opioid receptors in mouse gut, heart, spleen, and pancreas in vivo. Uptakes of radioactivity are saturable by the non-radioactive complexes, inhibited by naltrexone, and blocked by NTI. Thus, these radiometal-labeled NTI analogues warrant further study by single-photon emission computed tomography.
Assuntos
Radioisótopos de Índio , Compostos Macrocíclicos/síntese química , Naltrexona/análogos & derivados , Compostos Radiofarmacêuticos/síntese química , Receptores Opioides delta/metabolismo , Aldeídos/química , Animais , Autorradiografia , Ligação Competitiva , Cobaias , Compostos Heterocíclicos com 1 Anel/química , Técnicas In Vitro , Ligantes , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Masculino , Camundongos , Naltrexona/síntese química , Naltrexona/química , Naltrexona/farmacocinética , Ensaio Radioligante , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.
Assuntos
Azidas/farmacologia , Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Radioisótopos do Iodo/farmacologia , Animais , Cocaína/farmacologia , Brometo de Cianogênio/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Epitopos/química , Humanos , Metionina/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Tripsina/químicaRESUMO
Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.
Assuntos
Annona/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Álcoois Graxos/metabolismo , Furanos/farmacologia , Lactonas/metabolismo , Acetogeninas , Annona/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Ésteres/química , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Furanos/química , Lactonas/química , Lactonas/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Rutênio/química , Sementes/metabolismo , Especificidade por SubstratoRESUMO
The introduction of a primary amine function on the terminal alpha,beta-unsaturated lactone of squamocin 1, a common structural hallmark of annonaceous acetogenins, shifted this specific inhibitor of mitochondrial complex I into a potent dual inhibitor of complexes I and III. The mechanism of action of beta-aminosquamocin 2, against these two respiratory targets, is studied and discussed in view of current structure-activity relationship knowledge in the acetogenin series.
Assuntos
Annonaceae/química , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Furanos/química , Furanos/metabolismo , Lactonas/química , Lactonas/metabolismo , Relação Estrutura-AtividadeRESUMO
Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.
Assuntos
Azidas/química , Cocaína/análogos & derivados , Cocaína/química , Isotiocianatos/química , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Catecolaminas , Cromatografia Líquida de Alta Pressão , Cocaína/síntese química , Cocaína/metabolismo , Radioisótopos do Iodo , Estrutura Molecular , RatosRESUMO
A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata, have been synthesized in two to three steps from protected (l)-aminoacids and squamocin 1 using standard coupling/deprotection procedures. These semisynthetic analogs were tested on submitochondrial particles (SMP) for their complex I inhibitory activities, and against KB 3-1 cells in vitro. All triesters derivatives exhibited a complete extinction of activity at the enzymatic level, correlated to a reduced though modulated cytotoxicity in comparison with squamocin 1. This activity can apparently be considered as a function of the amphipathy of the analogs, the more amphiphilic ones being the more cytotoxic.
Assuntos
Annona/química , Furanos/síntese química , Furanos/farmacologia , Lactonas/síntese química , Lactonas/farmacologia , Linhagem Celular , Ésteres , Furanos/química , Humanos , Lactonas/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Partículas Submitocôndricas/efeitos dos fármacosRESUMO
A new cytotoxic acetogenin, chamuvarinin (1), containing a tetrahydropyran ring with an adjacent bis-tetrahydrofuran ring, which corresponds to a novel carbon skeleton in this series, was isolated from the roots of Uvaria chamae, together with the previously reported acetogenins squamocin (2), desacetyluvaricin (3), and neoannonin (4). The structure determination of chamuvarinin (1) was based on extensive NMR studies and high-resolution mass spectral measurements. This new compound shows significant cytotoxicity toward the KB 3-1 cell line (IC50 = 8 x 10(-10) M). In addition, a biosynthetic relationship between 1 and 2 is briefly discussed.