RESUMO
Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique 'address' domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the 'message-address' concept, also facilitated our next generation ligand design and development.
Assuntos
Naltrexona/análogos & derivados , Receptores Opioides mu/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Naltrexona/síntese química , Naltrexona/química , Naltrexona/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Mu opioid receptor antagonists have clinical utility and are important research tools. To develop non-peptide and highly selective mu opioid receptor antagonist, a series of 14-O-heterocyclic-substituted naltrexone derivatives were designed, synthesized, and evaluated. These compounds showed subnanomolar-to-nanomolar binding affinity for the mu opioid receptor. Among them, compound 1 exhibited the highest selectivity for the mu opioid receptor over the delta and kappa receptors. These results implicated an alternative 'address' domain in the extracellular loops of the mu opioid receptor.
Assuntos
Química Farmacêutica/métodos , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/síntese química , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/química , Ligação Competitiva , Desenho de Fármacos , Humanos , Cinética , Ligantes , Conformação Molecular , Estrutura Molecular , Antagonistas de Entorpecentes/farmacologia , Nitrogênio/química , Peptídeos/química , Estrutura Terciária de ProteínaRESUMO
The CB1 cannabinoid receptor is a G-protein coupled receptor that has important physiological roles in synaptic plasticity, analgesia, appetite, and neuroprotection. We report the discovery of two structurally related CB1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB1. CRIP1a and CRIP1b are generated by alternative splicing of a gene located on chromosome 2 in humans, and orthologs of CRIP1a occur throughout the vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB1-mediated tonic inhibition of voltage-gated Ca2+ channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB1 activity has therapeutic potential (e.g., chronic pain, obesity, and epilepsy).
Assuntos
Proteínas de Transporte/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/fisiologia , Sequência de Aminoácidos , Animais , Canabinoides/metabolismo , Canabinoides/farmacologia , Proteínas de Transporte/genética , Linhagem Celular , Humanos , Proteínas com Domínio LIM , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Ratos , Receptor CB1 de Canabinoide/genética , Receptores de Canabinoides/metabolismoRESUMO
As important pharmacological probes, highly selective opioid receptor antagonists are essential in opioid receptor structural characterization and opioid agonist functional studies. At present, a nonpeptidyl, highly selective, and reversible mu opioid receptor antagonist is still not available. Among a series of novel naltrexamine derivatives that have been designed and synthesized following molecular modeling studies, two compounds, NAP and NAQ, were identified as leads based on the results of in vitro and in vivo pharmacological assays. Both of them displayed high binding affinity and selectivity to the mu opioid receptor. Further pharmacokinetic and functional characterization revealed that NAP seems to be a peripheral nervous system agent while NAQ seems to be a central one. Such characteristics provide two distinguished potential application routes for these two agents and their derivatives. These results also supported our hypothesis that they may serve as leads to develop more potent and selective antagonists for the mu opioid receptor.
Assuntos
Compostos Heterocíclicos/síntese química , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/síntese química , Receptores Opioides mu/antagonistas & inibidores , Animais , Células CHO , Células CACO-2 , Cricetinae , Cricetulus , Implantes de Medicamento , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Camundongos , Morfina/administração & dosagem , Dependência de Morfina/psicologia , Naltrexona/síntese química , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/administração & dosagem , Ratos , Síndrome de Abstinência a Substâncias/psicologia , Tálamo/efeitos dos fármacos , Tálamo/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) and cannabinoid receptors are G-protein-coupled receptors that mediate the effects of S1P and endocannabinoids, respectively. Cannabinoid receptors also mediate the effects of Delta9-tetrahydrocannabinol, the primary psychoactive ingredient in marijuana, whereas S1P receptors contribute to the immunosuppressant effects of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720). FTY720 is a sphingosine analog that can prevent renal graft rejections and suppress a variety of autoimmune disorders in animal models and clinical trials. We now report that both FTY720 and sphingosine interact with CB1 but not CB2 cannabinoid receptors. FTY720 and sphingosine inhibited the binding of the CB1-selective antagonist [3H]N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide ([3H]SR141716A) and the cannabinoid agonist [3H](-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP55,940) in a concentration-dependent manner in both CB1-expressing cell lines and mouse cerebellum. However, these compounds did not significantly alter [3H]CP55,940 binding to CB2 receptors. In G-protein activation assays, FTY720 and sphingosine inhibited the maximal stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding by the cannabinoid agonist R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) in a concentration-dependent manner, and this antagonist effect was not mimicked by S1P. FTY720 and sphingosine also inhibited activation of extracellular signal-regulated kinases 1 and 2 and Akt by WIN55,212-2 in intact Chinese hamster ovary (CHO) cells expressing CB1 receptors and attenuated WIN55,212-2-stimulated internalization of a fluorescence-tagged CB1 receptor in CHO cells. Moreover, both FTY720 and sphingosine produced rightward shifts in the concentration-effect curves of cannabinoid agonists for G-protein activation, indicating that they act as competitive CB1 antagonists. These results suggest that the CB1 receptor could be a novel target of FTY720 and that sphingosine could be an endogenous CB11 antagonist.
Assuntos
Propilenoglicóis/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Animais , Benzoxazinas , Ligação Competitiva/efeitos dos fármacos , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Cicloexanos/metabolismo , Cicloexanos/farmacologia , Cicloexanóis , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Cloridrato de Fingolimode , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/metabolismo , Morfolinas/farmacologia , Naftalenos/metabolismo , Naftalenos/farmacologia , Fenóis/metabolismo , Fenóis/farmacologia , Piperidinas/metabolismo , Piperidinas/farmacologia , Propilenoglicóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Ensaio Radioligante , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/metabolismo , Rimonabanto , Esfingosina/metabolismoRESUMO
A new class of cannabimimetic indoles, with 3-phenylacetyl or substituted 3-phenylacetyl substituents, has been prepared and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. In general those compounds with a 2-substituted phenylacetyl group have good affinity for both receptors. The 4-substituted analogs have little affinity for either receptor, while the 3-substituted compounds are intermediate in their affinities. Two of these compounds, 1-pentyl-3-(2-methylphenylacetyl)indole (JWH-251) and 1-pentyl-3-(3-methoxyphenylacetyl)indole (JWH-302), have 5-fold selectivity for the CB1 receptor with modest affinity for the CB2 receptor. GTPgammaS determinations indicate that both compounds are highly efficacious agonists at the CB1 receptor and partial agonists at the CB2 receptor.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Canabinoides/química , Indóis/química , Indóis/farmacologia , Pentanos/química , Acetilação , Materiais Biomiméticos/classificação , Materiais Biomiméticos/metabolismo , Indóis/síntese química , Indóis/classificação , Estrutura Molecular , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Relação Estrutura-AtividadeRESUMO
Protein kinase B (PKB)/Akt and its upstream signal transducer, phosphatidylinosito-3 kinase (PI3K) play an essential role in control of transcription and translation, which impact cell growth, survival, and metabolism. Transcription factor E2F is a component of the downstream proliferative machinery regulated by Akt. Hyperphosphorylation of retinoblastoma protein (pRb), a pocket protein, leads to release of E2F1, resulting in transition from G1 to S phase. The present study shows that in normal C141 cells, vanadate treatment increased the percentage of cells at S phase and elevated cyclin E and cyclin A expression. Vanadate treatment triggered phosphorylation of pRb and release of E2F1. Furthermore, vanadate increased Akt kinase activity and caused its phosphorylation at Ser473 and Thr308. Inhibition of Akt by either inhibitors or transfected cells with dominant negative kinase mutant or dominant negative phosphorylation mutant decreased the percentage of the cells at the S phase induced by vanadate, and reduced both cyclin E and E2F1 expression and phosphorylation of pRb. The present study indicates that Akt plays an essential role in vanadate-induced increase in cell number at S phase and transition from G1 to S phase through E2F-pRb pathway.
Assuntos
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S/efeitos dos fármacos , Vanadatos/farmacologia , Animais , Divisão Celular , Linhagem Celular , Ciclina E/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Phosphatidylinositil-3 kinase (PI3K) is a heterodimer of catalytic and regulatory subunits. It is involved in various signaling pathways and key functions of the cells. The present study investigated the role of PI3K in vanadate-induced alteration in cell cycle regulation in C141 mouse epidermal cells. Vanadate caused a time- and dose-dependent increase in PI3K activity and phosphorylation of p70 S6 kinase (p70S6K) at Thr421/Ser424 and Thr389 sites. The phosphorylation at these sites was inhibited by PI3K inhibitor, LY294002, and p70S6K mutation. Vanadate promoted S phase entry and this promotion was inhibited by LY294002 and rapmycin, a p70S6K inhibitor. Vanadate-induced enhancement in S phase entry was also inhibited in transfection with dominant negative p70S6K mutant cells. The results obtained show that vanadate is able to increase PI3K activity through phosphorylation. PI3K activated p70S6K, which phosphated protein S6, and promoted S phase entry.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fase S/efeitos dos fármacos , Vanadatos/farmacologia , Animais , Linhagem Celular , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Mutação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Arsenite is widely distributed environmental toxicant in water, food and air. It is a known human carcinogen, which is strongly associated with human cancers originated from liver, nasal cavity, lung, skin, bladder, kidney, and prostate. In this study, we investigated whether arsenite induces expression of hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric basic helix-loop-helix transcription factor, composed of HIF-1alpha and HIF-1beta/ARNT subunits; and is involved in tumor growth and angiogenesis. Here we demonstrate that arsenite induces the expression of HIF-1alpha but not HIF-1beta subunit in DU145 human prostate carcinoma cells. Arsenite also increases the expression of VEGF through the induction of HIF-1. We also found that arsenite activates PI3K and Akt that are required for arsenite-induced expression of HIF-1alpha and VEGF. The induction of HIF-1 and VEGF by arsenite can not be inhibited by MAP kinase inhibitors. Arsenite causes production of reactive oxygen species (ROS). The major species of ROS required for the induction of HIF-1 and VEGF is H2O2. These data indicate that the arsenite-induced activation of PI3K/Akt signaling and the expression of HIF-1 and VEGF through the generation of ROS could be an important mechanism in the arsenite-induced carcinogenesis.