RESUMO
Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.
Assuntos
Interleucina-6 , Alcamidas Poli-Insaturadas , Alcaloides , Benzodioxóis/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperidinas , Alcamidas Poli-Insaturadas/metabolismoRESUMO
The bioactive piperine, a compound found in some pepper species, has been widely studied because of its therapeutic properties that include the inhibition of an important inflammation pathway triggered by interleukin-1 beta (IL-1ß). However, investigation into the molecular interactions between IL-1ß and piperine is not reported in the literature. Here, we present for the first time the characterisation of the complex formed by IL-1ß and piperine through experimental and computational molecular biophysical analyses. Fluorescence spectroscopy unveiled the presence of one binding site for piperine with an affinity constant of 14.3 × 104 M-1 at 298 K. The thermodynamic analysis indicated that the interaction with IL-1ß was spontaneous (∆G = -25 kJ/mol) and, when split into enthalpic and entropic contributions, the latter was more significant. Circular dichroism spectroscopy showed that piperine did not affect IL-1ß secondary structure (~2%) and therefore its stability. The set of experimental data parameterized the computational biophysical approach. Through molecular docking, the binding site micro-environment was revealed to be composed mostly by non-polar amino acids. Furthermore, molecular dynamics, along with umbrella sampling, are in agreement with the thermodynamic parameters obtained by fluorescence assays and showed that large protein movements are not present in IL-1ß, corroborating the circular dichroism data.
Assuntos
Alcaloides/química , Benzodioxóis/química , Interleucina-1beta/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperidinas/química , Alcamidas Poli-Insaturadas/química , Conformação Proteica , Algoritmos , Alcaloides/metabolismo , Benzodioxóis/metabolismo , Sítios de Ligação , Fenômenos Biofísicos , Dicroísmo Circular , Interleucina-1beta/metabolismo , Cinética , Modelos Químicos , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ligação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
BACKGROUND: Xylazine is an α2 adrenoceptor agonist that is extensively used in veterinary medicine and animal experimentation procedures to produce analgesia, sedation and muscle relaxation without causing general anesthesia. Considering the lack of knowledge of the mechanisms involved in peripheral antinociception induced by xylazine and the potential interactions between the adrenergic and endocannabinoid systems, the present study investigated the contribution of the latter system in the mechanism of xylazine. METHODS: The rat paw pressure test, in which hyperalgesia was induced by the intraplantar injection of prostaglandin E2, was performed. RESULTS: Xylazine administered via an intraplantar injection (25, 50 and 100 µg) induced a peripheral antinociceptive effect against prostaglandin E2 (2 µg)-induced hyperalgesia. This effect was blocked by treatment with the selective CB1 cannabinoid antagonist AM251 (20, 40 and 80 µg) but not by the selective CB2 cannabinoid antagonist AM630 (100 µg). The anandamide reuptake inhibitor VDM11 (2.5 µg) intensified the peripheral antinociceptive effect of a submaximal dose of xylazine (25 µg), and the inhibitor of endocannabinoid enzymatic hydrolysis, MAFP (0.5 µg), showed a tendency towards this same effect. In addition, liquid-chromatography mass spectrometric analysis indicated that xylazine (100 µg) treatment was associated with an increase in anandamide levels in the rat paws treated with PGE2. CONCLUSIONS: The present results provides evidence that the peripheral antinociceptive effect of the α2 adrenoceptor agonist xylazine probably results from anandamide release and subsequent CB1 cannabinoid receptor activation.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Hiperalgesia/tratamento farmacológico , Alcamidas Poli-Insaturadas/metabolismo , Xilazina/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Espectrometria de Massas , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Xilazina/administração & dosagemRESUMO
The activity of rifampin (RIF) and piperine was evaluated at the relative transcript levels of 12 efflux pumps (EPs), and an additional mechanism was proposed to be behind the synergic interactions of piperine plus RIF in Mycobacterium tuberculosis AutoDock v4.2.3 and Molegro v6 programs were used to evaluate PIP binding in M. tuberculosis RNA polymerase (RNAP). A hypothesis has been raised that piperine interferes in M. tuberculosis growth through RNAP inhibition, differently from what was previously endorsed for EP inhibition only.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Rifampina/farmacologia , Alcaloides/administração & dosagem , Alcaloides/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Benzodioxóis/administração & dosagem , Benzodioxóis/metabolismo , Sítios de Ligação , Sinergismo Farmacológico , Quimioterapia Combinada , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Piperidinas/administração & dosagem , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/administração & dosagem , Alcamidas Poli-Insaturadas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rifampina/administração & dosagem , Rifampina/metabolismoRESUMO
The bioactive piperine (1-piperoyl piperidine) compound found in some pepper species (Piper nigrum linn and Piper sarmentosum Roxb) has been shown to have therapeutic properties and to be useful for well-being. The tests used to validate these properties were performed in vitro or with small rats. However, in all these assays, the molecular approach was absent. Although the first therapeutic trials relied on the use of rats, no proposal was mentioned either experimentally or computationally at the molecular level regarding the interaction between piperine and rat serum albumin (RSA). In the present study, several spectroscopic techniques were employed to characterize rat serum albumin and, aided by computational techniques, the protein modeling was proposed. From the spectroscopic results, it was possible to estimate the binding constant (3.9 × 104 M-1 at 288 K) using the Stern-Volmer model and the number of ligands (three) associated with the protein applying interaction density function model. The Gibbs free energy, an important thermodynamic parameter, was determined (-25 kJ/mol), indicating that the interaction was spontaneous. This important set of experimental results served to parameterize the computational simulations. The results of molecular docking and molecular dynamics matched appropriately made it possible to have detailed microenvironments of RSA accessed by piperine.
Assuntos
Alcaloides/química , Benzodioxóis/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperidinas/química , Alcamidas Poli-Insaturadas/química , Albumina Sérica/química , Algoritmos , Alcaloides/metabolismo , Animais , Benzodioxóis/metabolismo , Sítios de Ligação , Dicroísmo Circular , Ligação de Hidrogênio , Conformação Molecular , Estrutura Molecular , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ligação Proteica , Ratos , Albumina Sérica/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Studies suggest that the endocannabinoid and endovanilloid systems are implicated in the pathophysiology of schizophrenia. The Spontaneously Hypertensive Rats (SHR) strain displays impaired contextual fear conditioning (CFC) attenuated by antipsychotic drugs and worsened by pro-psychotic manipulations. Therefore, SHR strain is used to study emotional processing/associative learning impairments associated with schizophrenia and effects of potential antipsychotic drugs. Here, we evaluated the expression of CB1 and TRPV1 receptors in some brain regions related to the pathophysiology of schizophrenia. We also assessed the effects of drugs that act on the endocannabinoid/endovanilloid systems on the CFC task in SHRs and control animals (Wistar rats - WRs). The following drugs were used: AM404 (anandamide uptake/metabolism inhibitor), WIN55-212,2 (non-selective CB1 agonist), capsaicin (TRPV1 agonist), and capsazepine (TRPV1 antagonist). SHRs displayed increased CB1 expression in prelimbic cortex and cingulate cortex area 1 and in CA3 region of the dorsal hippocampus. Conversely, SHRs exhibited decreases in TRPV1 expression in prelimbic and CA1 region of dorsal hippocampus and increases in the basolateral amygdala. AM404, WIN 55,212-2 and capsaicin attenuated SHRs CFC deficit, although WIN 55,212-2 worsened SHRs CFC deficit in higher doses. WRs and SHRs CFC were modulated by distinct doses, suggesting that these strains display different responsiveness to cannabinoid and vanilloid drugs. Treatment with capsazepine did not modify CFC in either strains. The effects of AM404 on SHRs CFC deficit was not blocked by pretreatment with rimonabant (CB1 antagonist) or capsazepine. These results reinforce the involvement of the endocannabinoid/endovanilloid systems in the SHRs CFC deficit and point to these systems as targets to treat the emotional processing/cognitive symptoms of schizophrenia.
Assuntos
Sintomas Afetivos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Disfunção Cognitiva/metabolismo , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Esquizofrenia/metabolismo , Sintomas Afetivos/induzido quimicamente , Animais , Ácidos Araquidônicos/agonistas , Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Ácidos Araquidônicos/uso terapêutico , Agonistas de Receptores de Canabinoides/farmacologia , Agonistas de Receptores de Canabinoides/uso terapêutico , Disfunção Cognitiva/induzido quimicamente , Endocanabinoides/agonistas , Endocanabinoides/antagonistas & inibidores , Masculino , Alcamidas Poli-Insaturadas/agonistas , Alcamidas Poli-Insaturadas/antagonistas & inibidores , Alcamidas Poli-Insaturadas/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Esquizofrenia/induzido quimicamente , Esquizofrenia/prevenção & controleRESUMO
BACKGROUND AND PURPOSE: The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) bind to CB1 and CB2 cannabinoid receptors in the brain and modulate the mesolimbic dopaminergic pathway. This neurocircuitry is engaged by psychostimulant drugs, including cocaine. Although CB1 receptor antagonism and CB2 receptor activation are known to inhibit certain effects of cocaine, they have been investigated separately. Here, we tested the hypothesis that there is a reciprocal interaction between CB1 receptor blockade and CB2 receptor activation in modulating behavioural responses to cocaine. EXPERIMENTAL APPROACH: Male Swiss mice received i.p. injections of cannabinoid-related drugs followed by cocaine, and were then tested for cocaine-induced hyperlocomotion, c-Fos expression in the nucleus accumbens and conditioned place preference. Levels of endocannabinoids after cocaine injections were also analysed. KEY RESULTS: The CB1 receptor antagonist, rimonabant, and the CB2 receptor agonist, JWH133, prevented cocaine-induced hyperlocomotion. The same results were obtained by combining sub-effective doses of both compounds. The CB2 receptor antagonist, AM630, reversed the inhibitory effects of rimonabant in cocaine-induced hyperlocomotion and c-Fos expression in the nucleus accumbens. Selective inhibitors of anandamide and 2-AG hydrolysis (URB597 and JZL184, respectively) failed to modify this response. However, JZL184 prevented cocaine-induced hyperlocomotion when given after a sub-effective dose of rimonabant. Cocaine did not change brain endocannabinoid levels. Finally, CB2 receptor blockade reversed the inhibitory effect of rimonabant in the acquisition of cocaine-induced conditioned place preference. CONCLUSION AND IMPLICATIONS: The present data support the hypothesis that CB1 and CB2 receptors work in concert with opposing functions to modulate certain addiction-related effects of cocaine. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.
Assuntos
Ácidos Araquidônicos/metabolismo , Cocaína/farmacologia , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/agonistas , Recompensa , Animais , Comportamento Animal/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Condicionamento Clássico , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Tuberculosis (TB) is an important public health problem worldwide and the emergence of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB worsened the global context. The resistance in Mycobacterium tuberculosis, the causative agent of TB, can partially derive from efflux pumps (EPs) activity in plasma membrane. Due to the recent discovery of piperine (PIP), an organic alkaloid compound, increasing the bioavailability of various drugs, the current assay evaluated the combined activity of PIP and anti-TB drugs in susceptible and resistant M. tuberculosis clinical isolates. The minimum inhibitory concentrations for isoniazid, rifampicin, ethambutol, streptomycin and PIP were determined by resazurin microtiter assay and the combined effects of anti-TB drugs with PIP determined by resazurin drug combination microtiter assay and time-kill curve. The efflux pump inhibitor activity of PIP was determined by bromide accumulation assay and cytotoxicity carried out in VERO cells and J774. A1 macrophages. PIP showed to have EPI activity and RIF + PIP and SM + PIP combinations showed synergistic effect, but low effect in enhancing the killing in M. tuberculosis H37Rv and in the clinical isolates studied, which had different resistance profiles. Future studies are needed to further clarify the importance of PIP as an adjunctive drug in the therapy against TB.
Assuntos
Alcaloides/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Benzodioxóis/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Alcaloides/metabolismo , Animais , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Benzodioxóis/metabolismo , Chlorocebus aethiops , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Células VeroRESUMO
Ketamine has been widely used as an analgesic and produces dissociative anesthetic effects. The antinociceptive effects of ketamine have been studied, but the involvement of endocannabinoids in these effects has not yet been investigated. In this study, we evaluated the involvement of the endocannabinoid system in the peripheral antinociceptive effects induced by ketamine. All drugs were administered via the intraplantar route. To induce hyperalgesia, rat paws were injected with prostaglandin E2 (2 µg per paw). The nociceptive threshold for mechanical stimulation was measured in the right hind paw of Wistar rats using the Randall-Selitto test. The tissue levels of anandamide (AEA), 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide were measured using liquid chromatography coupled to single quadrupole mass spectrometry. The administration of the cannabinoid receptor type 1 (CB1) antagonist, N(piperidine-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl 1 pyrazolcarboxamide (20, 40, and 80 µg per paw), but not the cannabinoid receptor type 2 antagonist, 6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-indol-3-yl) (4-methoxyphenyl) methanone (100 µg per paw), antagonized the ketamine-induced peripheral antinociception in a dose-dependent manner. Additionally, the administration of the endocannabinoid metabolizing enzyme inhibitor (.5 µg per paw) or an AEA reuptake inhibitor, (5Z,8Z,11Z,14Z)N(4Hydroxy2methylphenyl)5,8,11,14 eicosatetraenamide (2.5 µg per paw) significantly enhanced low-dose ketamine-induced peripheral antinociception. AEA paw levels were increased only after ketamine administration to prostaglandin E2-injected paws. These data suggest that ketamine, in the presence of a nociceptive stimulus, induces a selective release of AEA levels and subsequent CB1 cannabinoid activation at the peripheral level. PERSPECTIVE: This study suggests that ketamine antinociception depends at least in part on AEA release and CB1 cannabinoid receptor activation in inflammatory conditions. This study could potentially help clinicians in the use of ketamine as a peripheral analgesic for inflammatory pain.
Assuntos
Analgésicos/uso terapêutico , Endocanabinoides/metabolismo , Ketamina/uso terapêutico , Dor/tratamento farmacológico , Dor/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Analgésicos/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Agonistas de Receptores de Canabinoides/farmacologia , Agonistas de Receptores de Canabinoides/uso terapêutico , Antagonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/uso terapêutico , Ketamina/farmacologia , Masculino , Alcamidas Poli-Insaturadas/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/agonistasRESUMO
Activation of the hypothalamic-pituitary-adrenal axis (HPA) is critical for survival when the organism is exposed to a stressful stimulus. The endocannabinoid system (ECS) is currently considered an important neuromodulator involved in numerous pathophysiological processes and whose primary function is to maintain homeostasis. In the tissues constituting the HPA axis, all the components of the ECS are present and the activation of this system acts in parallel with changes in the activity of numerous neurotransmitters, including nitric oxide (NO). NO is widely distributed in the brain and adrenal glands and recent studies have shown that free radicals, and in particular NO, may play a crucial role in the regulation of stress response. Our objective was to determine the participation of the endocannabinoid and NOergic systems as probable mediators of the neuroendocrine HPA axis response to a psychophysical acute stress model in the adult male rat. Animals were pre-treated with cannabinoid receptors agonists and antagonists at central and systemic level prior to acute restraint exposure. We also performed in vitro studies incubating adrenal glands in the presence of ACTH and pharmacological compounds that modifies ECS components. Our results showed that the increase in corticosterone observed after acute restraint stress is blocked by anandamide administered at both central and peripheral level. At hypothalamic level both cannabinoid receptors (CB1 and CB2) are involved, while in the adrenal gland, anandamide has a very potent effect in suppressing ACTH-induced corticosterone release that is mainly mediated by vanilloid TRPV1 receptors. We also observed that stress significantly increased hypothalamic mRNA levels of CB1 as well as adrenal mRNA levels of TRPV1 receptor. In addition, anandamide reduced the activity of the nitric oxide synthase enzyme during stress, indicating that the anti-stress action of endocannabinoids may involve a reduction in NO production at hypothalamic and adrenal levels. In conclusion, an endogenous cannabinoid tone maintains the HPA axis in a stable basal state, which is lost with a noxious stimulus. In this case, the ECS dampens the response to stress allowing the recovery of homeostasis. Moreover, our work further contributes to in vitro evidence for a participation of the endocannabinoid system by inhibiting corticosterone release directly at the adrenal gland level.