RESUMO
Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit ß-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids.
Assuntos
Mitragyna/química , Antagonistas de Entorpecentes/síntese química , Receptores Opioides mu/agonistas , Alcaloides de Triptamina e Secologanina/química , Agonismo Parcial de Drogas , Humanos , Simulação de Acoplamento Molecular , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Ligação Proteica , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/antagonistas & inibidores , Alcaloides de Triptamina e Secologanina/isolamento & purificação , Alcaloides de Triptamina e Secologanina/farmacologia , Relação Estrutura-AtividadeRESUMO
Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , GMP Cíclico/análogos & derivados , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , GMP Cíclico/química , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Conformação de Ácido Nucleico , Riboswitch/genética , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
Mitragyna speciosa, more commonly known as kratom, is a plant native to Southeast Asia, the leaves of which have been used traditionally as a stimulant, analgesic, and treatment for opioid addiction. Recently, growing use of the plant in the United States and concerns that kratom represents an uncontrolled drug with potential abuse liability, have highlighted the need for more careful study of its pharmacological activity. The major active alkaloid found in kratom, mitragynine, has been reported to have opioid agonist and analgesic activity in vitro and in animal models, consistent with the purported effects of kratom leaf in humans. However, preliminary research has provided some evidence that mitragynine and related compounds may act as atypical opioid agonists, inducing therapeutic effects such as analgesia, while limiting the negative side effects typical of classical opioids. Here we report evidence that an active metabolite plays an important role in mediating the analgesic effects of mitragynine. We find that mitragynine is converted in vitro in both mouse and human liver preparations to the much more potent mu-opioid receptor agonist 7-hydroxymitragynine and that this conversion is mediated by cytochrome P450 3A isoforms. Further, we show that 7-hydroxymitragynine is formed from mitragynine in mice and that brain concentrations of this metabolite are sufficient to explain most or all of the opioid-receptor-mediated analgesic activity of mitragynine. At the same time, mitragynine is found in the brains of mice at very high concentrations relative to its opioid receptor binding affinity, suggesting that it does not directly activate opioid receptors. The results presented here provide a metabolism-dependent mechanism for the analgesic effects of mitragynine and clarify the importance of route of administration for determining the activity of this compound. Further, they raise important questions about the interpretation of existing data on mitragynine and highlight critical areas for further research in animals and humans.
RESUMO
Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), have emerged as key regulators of brain plasticity and represent disease-modifying targets for several brain disorders, including Alzheimer's disease and major depressive disorder. Because of poor pharmacokinetic properties of BDNF, the interest in small-molecule TrkB agonists and modulators is high. Several compounds have been reported to act as TrkB agonists, and their increasing use in various nervous system disorder models creates the perception that these are reliable probes. To examine key pharmacological parameters of these compounds in detail, we have developed and optimized a series of complementary quantitative assays that measure TrkB receptor activation, TrkB-dependent downstream signaling, and gene expression in different cellular contexts. Although BDNF and other neurotrophic factors elicited robust and dose-dependent receptor activation and downstream signaling, we were unable to reproduce these activities using the reported small-molecule TrkB agonists. Our findings indicate that experimental results obtained with these compounds must be carefully interpreted and highlight the challenge of developing reliable pharmacological activators of this key molecular target.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glicoproteínas de Membrana/agonistas , Neuroblastoma/tratamento farmacológico , Neurônios/efeitos dos fármacos , Receptor trkB/agonistas , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Ensaio de Imunoadsorção Enzimática , Flavanonas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Receptor trkB/metabolismoRESUMO
Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.
Assuntos
Antidepressivos Tricíclicos/farmacologia , Receptores Opioides mu/metabolismo , Tiazepinas/farmacologia , Analgésicos Opioides/farmacologia , Animais , Antidepressivos Tricíclicos/metabolismo , Antidepressivos Tricíclicos/farmacocinética , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/metabolismo , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Morfina/farmacologia , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Tiazepinas/metabolismo , Tiazepinas/farmacocinéticaRESUMO
Modulation of growth factor signaling pathways in the brain represents a new experimental approach to treating neuropsychiatric disorders such as depression, anxiety, and addiction. Neurotrophins and growth factors exert synaptic, neuronal, and circuit level effects on a wide temporal range, which suggests a possibility of rapid and lasting therapeutic effects. Consequently, identification of small molecules that can either enhance the release of growth factors or potentiate their respective pathways will provide a drug-like alternative to direct neurotrophin administration or viral gene delivery and thus represents an important frontier in chemical biology and drug design. Glial cell line-derived neurotrophic factor (GDNF), in particular, has been implicated in marked reduction of alcohol consumption in rodent addiction models, and the natural product ibogaine, a substance used traditionally in ritualistic ceremonies, has been suggested to increase the synthesis and release of GDNF in the dopaminergic system in rats. In this report, we describe a novel iboga analog, XL-008, created by unraveling the medium size ring of the ibogamine skeleton, and its ability to induce release of GDNF in C6 glioma cells. Additionally, XL-008 potentiates the release of GDNF induced by fibroblast growth factor 2 (FGF2), another neurotrophin implicated in major depressive disorder, increasing potency more than 2-fold (from 7.85 ± 2.59 ng/mL to 3.31 ± 0.98 ng/mL) and efficacy more than 3-fold. The GDNF release by both XL-008 and the FGF2/XL-008 mixture was found to be mediated through the MEK and PI3K signaling pathways but not through PLCγ in C6 glioma cells.