RESUMO
Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC50 = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (â60% at 10 µM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC50 = 90 ± 7 nM) surpassing NNGH with MMP-9 over -2 and MMP-10 over -13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC50 by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Pulmonares , Metaloproteinase 9 da Matriz , Tiazóis , Triazóis , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Relação Estrutura-Atividade , Metaloproteinase 9 da Matriz/metabolismo , Tiazóis/química , Tiazóis/farmacologia , Tiazóis/síntese química , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/síntese química , Cisplatino/farmacologia , Cisplatino/química , Canais de Cálcio Tipo T/metabolismoRESUMO
The ventrolateral periaqueductal gray (vlPAG) serves as a central hub for descending pain modulation. It receives upstream projections from the medial prefrontal cortex (mPFC) and the ventrolateral orbitofrontal cortex (vlOFC), and projects downstream to the locus coeruleus (LC) and the rostroventral medulla (RVM). While much research has focused on upstream circuits and the LC-RVM connection, less is known about the PAG-LC circuit and its involvement in neuropathic pain. Here we examined the intrinsic electrophysiological properties of vlPAG-LC projecting neurons in Sham and spared nerve injury (SNI) operated mice. Injection of the retrotracer Cholera Toxin Subunit B (CTB-488) into the LC allowed the identification of LC-projecting neurons in the vlPAG. Electrophysiological recordings from CTB-488 positive cells revealed that both GABAergic and glutamatergic cells that project to the LC exhibited reduced intrinsic excitability after peripheral nerve injury. By contrast, CTB-488 negative cells did not exhibit alterations in firing properties after SNI surgery. An SNI-induced reduction of LC projecting cells was confirmed with c-fos labeling. Hence, SNI induces plasticity changes in the vlPAG that are consistent with a reduction in the descending modulation of pain signals.
Assuntos
Locus Cerúleo , Camundongos Endogâmicos C57BL , Neurônios , Substância Cinzenta Periaquedutal , Animais , Substância Cinzenta Periaquedutal/fisiopatologia , Substância Cinzenta Periaquedutal/fisiologia , Locus Cerúleo/fisiopatologia , Locus Cerúleo/patologia , Locus Cerúleo/fisiologia , Neurônios/fisiologia , Masculino , Camundongos , Potenciais de Ação/fisiologia , Vias Neurais/fisiopatologia , Neuralgia/fisiopatologia , Neuralgia/patologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Traumatismos dos Nervos Periféricos/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
BACKGROUND: More than 80% of patients may experience acute pain after a surgical procedure, and this is often refractory to pharmacological intervention. The identification of new targets to treat postoperative pain is necessary. There is an association of polymorphisms in the Cav2.3 gene with postoperative pain and opioid consumption. Our study aimed to identify Cav2.3 as a potential target to treat postoperative pain and to reduce opioid-related side effects. EXPERIMENTAL APPROACH: A plantar incision model was established in adult male and female C57BL/6 mice. Cav2.3 expression was detected by qPCR and suppressed by siRNA treatment. The antinociceptive efficacy and safety of a Cav2.3 blocker-alone or together with morphine-was also assessed after surgery. KEY RESULTS: Paw incision in female and male mice caused acute nociception and increased Cav2.3 mRNA expression in the spinal cord but not in the incised tissue. Intrathecal treatment with siRNA against Cav2.3, but not with a scrambled siRNA, prevented the development of surgery-induced nociception in both male and female mice, with female mice experiencing long-lasting effects. High doses of i.t. SNX-482, a Cav2.3 channel blocker, or morphine injected alone, reversed postoperative nociception but also induced side effects. A combination of lower doses of morphine and SNX-482 mediated a long-lasting reversal of postsurgical pain in female and male mice. CONCLUSION: Our results demonstrate that Cav2.3 has a pronociceptive role in the induction of postoperative pain, indicating that it is a potential target for the development of therapeutic approaches for the treatment of postoperative pain.
RESUMO
The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.
Assuntos
Caveolina 1 , Receptores Purinérgicos P2X7 , Animais , Camundongos , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMO
Artemisia is one of the largest genera in the plant family Asteraceae and has long been used in traditional medicine for its antitussive, analgesic, antihypertensive, antitoxic, antiviral, antimalarial, and anti-inflammatory properties. However, the anti-diabetic activity of Artemisia montana has not been broadly studied. The goal of this study was to determine whether extracts of the aerial parts of A. montana and its main constituents inhibit protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase activities. We isolated nine compounds from A. montana including ursonic acid (UNA) and ursolic acid (ULA), which significantly inhibited PTP1B with IC50 values of 11.68 and 8.73 µM, respectively. In addition, UNA showed potent inhibitory activity against α-glucosidase (IC50 = 61.85 µM). Kinetic analysis of PTP1B and α-glucosidase inhibition revealed that UNA was a non-competitive inhibitor of both enzymes. Docking simulations of UNA demonstrated negative binding energies and close proximity to residues in the binding pockets of PTP1B and α-glucosidase. Molecular docking simulations between UNA and human serum albumin (HSA) revealed that UNA binds tightly to all three domains of HSA. Furthermore, UNA significantly inhibited fluorescent AGE formation (IC50 = 4.16 µM) in a glucose-fructose-induced HSA glycation model over the course of four weeks. Additionally, we investigated the molecular mechanisms underlying the anti-diabetic effects of UNA in insulin-resistant C2C12 skeletal muscle cells and discovered that UNA significantly increased glucose uptake and decreased PTP1B expression. Further, UNA increased GLUT-4 expression level by activating the IRS-1/PI3K/Akt/GSK-3 signaling pathway. These findings clearly demonstrate that UNA from A. montana shows great potential for treatment of diabetes and its complications.
Assuntos
Artemisia , Diabetes Mellitus , Insulinas , Humanos , Lactente , Hipoglicemiantes/farmacologia , alfa-Glucosidases/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Cinética , Artemisia/química , Artemisia/metabolismo , Simulação de Acoplamento Molecular , Quinase 3 da Glicogênio Sintase/metabolismo , Montana , Diabetes Mellitus/tratamento farmacológico , Transdução de Sinais , Proteína Tirosina Fosfatase não Receptora Tipo 1RESUMO
The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Citocinas/metabolismo , Neoplasias Pulmonares , Animais , Humanos , Hiperalgesia/metabolismo , Inflamação/patologia , Ligantes , Camundongos , Dor/tratamento farmacológico , Receptores Proteína Tirosina Quinases , Células Receptoras Sensoriais/metabolismo , Corno Dorsal da Medula Espinal/patologiaRESUMO
Trigeminal neuropathic pain has been modeled in rodents through the constriction of the infraorbital nerve (CCI-ION). Sensory alterations, including spontaneous pain, and thermal and mechanical hyperalgesia are well characterized, but there is a notable lack of evidence about the affective pain component in this model. Evaluation of the emotional component of pain in rats has been proposed as a way to optimize potential translational value of non-clinical studies. In rats, 22 and 50 kHz ultrasonic vocalizations (USVs) are considered well-established measures of negative and positive emotional states, respectively. Thus, this study tested the hypothesis that trigeminal neuropathic pain would result, in addition to the sensory alterations, in a decrease of 50 kHz USV, which may be related to altered function of brain areas involved in emotional pain processing. CCI-ION surgery was performed on 60-day-old male Wistar rats. 15 days after surgery, von Frey filaments were applied to detect mechanical hyperalgesia, and USV was recorded. At the same timepoint, systemic treatment with d,l-amphetamine (1 mg/kg) allowed investigation of the involvement of the dopaminergic system in USV emission. Finally, brain tissue was collected to assess the change in tyrosine hydroxylase (TH) expression in the nucleus accumbens (NAc) and c-Fos expression in brain areas involved in emotional pain processing, including the prefrontal cortex (PFC), amygdala, and NAc. The results showed that CCI-ION rats presented mechanical hyperalgesia and a significant reduction of environmental-induced 50 kHz USV. Amphetamine caused a marked increase in 50 kHz USV emission in CCI-ION rats. In addition, TH expression was lower in constricted animals and c-Fos analysis revealed an increase in neuronal activation. Taken together, these data indicate that CCI-ION causes a reduction in the emission of environmental-induced appetitive calls concomitantly with facial mechanical hyperalgesia and that both changes may be related to a reduction in the mesolimbic dopaminergic activity.
Assuntos
Neuralgia , Neuralgia do Trigêmeo , Animais , Hiperalgesia/tratamento farmacológico , Masculino , Neuralgia/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Neuralgia do Trigêmeo/complicaçõesRESUMO
BACKGROUND & AIMS: Chronic abdominal pain is a common symptom of inflammatory bowel diseases (IBDs). Peripheral and central mechanisms contribute to the transition from acute to chronic pain during active disease and clinical remission. Lower mechanical threshold and hyperexcitability of visceral afferents induce gliosis in central pain circuits, leading to persistent visceral hypersensitivity (VHS). In the spinal cord, microglia, the immune sentinels of the central nervous system, undergo activation in multiple models of VHS. Here, we investigated the mechanisms of microglia activation to identify centrally acting analgesics for chronic IBD pain. METHODS: Using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) expressed in transient receptor potential vanilloid member 1-expressing visceral neurons that sense colonic inflammation, we tested whether neuronal activity was indispensable to control microglia activation and VHS. We then investigated the neuron-microglia signaling system involved in visceral pain chronification. RESULTS: We found that chemogenetic inhibition of transient receptor potential vanilloid member 1+ visceral afferents prevents microglial activation in the spinal cord and subsequent VHS in colitis mice. In contrast, chemogenetic activation, in the absence of colitis, enhanced microglial activation associated with VHS. We identified a purinergic signaling mechanism mediated by neuronal adenosine triphosphate (ATP) and microglial P2Y12 receptor, triggering VHS in colitis. Inhibition of P2RY12 prevented microglial reactivity and chronic VHS post-colitis. CONCLUSIONS: Overall, these data provide novel insights into the central mechanisms of chronic visceral pain and suggest that targeting microglial P2RY12 signaling could be harnessed to relieve pain in patients with IBD who are in remission.
Assuntos
Dor Crônica , Colite , Doenças Inflamatórias Intestinais , Dor Visceral , Animais , Humanos , Camundongos , Microglia , Neurônios , Antagonistas do Receptor Purinérgico P2Y , Canais de Cátion TRPVRESUMO
T-type calcium channels are considered potential drug targets to combat cancer. Combining T-type calcium channel blockers with conventional chemotherapy drugs represents a promising strategy towards successful cancer treatment. From this perspective, we report in this study the design and synthesis of a novel series of N3-sustituted dihydropyrimidines (DHPMs) as anticancer adjuvants to cisplatin (Cis) and etoposide (Eto). Full spectral characterization of the new compounds was done using FT-IR, 1H NMR, 13C NMR, and HRMS. Structure elucidation was confirmed by 2D NMR 1H-H COSY, HSQC and NOESY experiments. Novel derivatives were tested for their Ca2+ channel blocking activity by employing the whole cell patch-clamp technique. Results demonstrated that most compounds were potential T-type calcium channel blockers with the triazole-based C12 and C13 being the most selective agents against CaV3.2 channel. Further electrophysiological studies demonstrated that C12 and C13 inhibited CaV3.2 currents with respective affinity of 2.26 and 1.27 µM, and induced 5 mV hyperpolarizing shifts in the half-inactivation potential. Subsequently, C12 and C13 were evaluated for their anticancer activities alone and in combination with Cis and Eto against A549 and MDA-MB 231 cancer cells. Interestingly, both compounds exhibited potential anticancer effects with IC50 values < 5 µM. Combination studies revealed that both compounds had synergistic effects (combination index CI < 1) on Cis and Eto through induction of apoptosis (p53 activation and up-regulation of BAX and p21 gene expression). Importantly, in silico physicochemical and ADMET assessment of both compounds revealed their potential drug-like properties with decreased risk of cardiac toxicity. Hence, C12 and C13 are promising anticancer adjuvants through inhibition of CaV3.2 T-type calcium channels, thereby serving as eminent leads for further modification.
Assuntos
Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Cisplatino/farmacologia , Etoposídeo/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Etoposídeo/química , Humanos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Acute pain that persists for a few days is associated with a reduction in patients' quality of life. Orofacial persistent pain promotes psychological disorders such as anxiety, impairs daily essential activities such as eating, and results in decreased social interaction. Here, we investigated whether rats subjected to orofacial formalin injection or intraoral incision surgery display persistent facial heat hyperalgesia, ongoing pain, anxiety-like behavior, and changes in ultrasonic vocalization. Orofacial formalin injection or intraoral incision caused facial heat hyperalgesia for 3 days compared with saline-injected and sham animals. In addition, both experimental groups showed a reduction in the number of entries and in the time spent in the open arms in the elevated plus maze test on day 3, suggesting that anxiety-like behavior developed as a consequence of persistent pain. At this time point, both groups also displayed a reduction in the number of 50-kHz calls, specifically in the flat subtype, which suggests a decrease in social communication. Moreover, on day 3 after surgery, systemic morphine produced robust conditioned place preference in rats subjected to intraoral incision compared with sham, and the former group also presented increased spontaneous facial grooming, revealing the presence of ongoing pain. Finally, Western blot and immunohistochemistry analysis showed a reduction in tyrosine hydroxylase expression in the nucleus accumbens, which may reflect a decrease in mesolimbic dopaminergic activity. Altogether, the results demonstrate that acute orofacial pain causes prolonged changes in behavioral and affective pain components, which may be related to dopaminergic changes in the nucleus accumbens.
Assuntos
Dor Aguda , Animais , Modelos Animais de Doenças , Dor Facial , Humanos , Hiperalgesia/etiologia , Qualidade de Vida , Ratos , Ratos WistarRESUMO
In our previous work, a series of 2-amino-3,4-dihydroquinazoline derivativesusing an electron acceptor group was reported to be potent T-type calcium channel blockers and exhibit strong cytotoxic effects against various cancerous cell lines. To investigate the role of the guanidine moiety in the 2-amino-3,4-dihydroquinazoline scaffold as a pharmacophore for dual biological activity, a new series of 2-thio-3,4-dihydroquniazoline derivatives using an electron donor group at the C2-position was synthesized and evaluated for T-type calcium channel blocking activity and cytotoxic effects against two human cancerous cell lines (lung cancer A549 and colon cancer HCT-116). Among them, compound 6g showed potent inhibition of Cav3.2 currents (83% inhibition) at 10 µM concentrations. The compound also exhibited IC50 values of 5.0 and 6.4 µM against A549 and HCT-116 cell lines, respectively, which are comparable to the parental lead compound KYS05090. These results indicate that the isothiourea moiety similar to the guanidine moiety of 2-amino-3,4-dihydroquinazoline derivatives may be an essential pharmacophore for the desired biological activities. Therefore, our preliminary work can provide the opportunity to expand a chemical repertoire to improve affinity and selectivity for T-type calcium channels.
Assuntos
Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Quinazolinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
1,4-Dihydropyridines (DHPs) are an important class of blockers targeting different calcium channel subtypes and have great therapeutic value against cardiovascular and neurophysiologic conditions. Here, we present the design of DHP-based hexahydroquinoline derivatives as either selective or covalent inhibitors of calcium channels. These compounds were synthesized via a modified Hantzsch reaction under microwave irradiation and characterized by IR, 1H NMR, 13C NMR and mass spectra. Additionally, the proposed structure of HM12 was resolved by single crystal X-ray analysis. The abilities of the target compounds to block both L- and T-type calcium channels were evaluated by utilizing the whole-cell patch clamp technique. Our results identified covalent inhibitors of calcium channels for the first time, which could be achieved by introducing a Michael acceptor group into the ester side chain of the compounds. The proposed covalent binding between the compounds and the cysteine amino acid (Cys1492) within the DHP binding pocket of L-type calcium channel was supported by docking and pharmacophore analysis as well as a glutathione reactivity assay.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo T/química , Di-Hidropiridinas/farmacologia , Descoberta de Drogas , Glutationa/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Drugs targeting different calcium channel subtypes have strong therapeutic potential for future drug development for cardiovascular disorders, neuropsychiatric diseases and cancer. This study aims to design and synthesize a new series of C2 substituted dihydropyrimidines to mimic the structure features of third generation long acting dihydropyridine calcium channel blockers and dihydropyrimidines analogues. The target compounds have been evaluated as blockers for CaV1.2 and CaV3.2 utilizing the whole-cell patch clamp technique. Among the tested compounds, compound 7a showed moderate calcium channel blockade activity against CaV3.2. Moreover, the predicted physicochemical properties and pharmacokinetic profiles of the target compounds recommend that they can be considered as drug-like candidates. The results highlight some significant information for the future design of lead compounds as calcium channel blockers.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Pirimidinas/farmacologia , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/farmacocinética , Linhagem Celular , Simulação por Computador , Desenho de Fármacos , Eletrofisiologia/métodos , Humanos , Técnicas de Patch-Clamp , Pirimidinas/síntese química , Pirimidinas/farmacocinéticaRESUMO
The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo T/metabolismo , Infecções por HIV/complicações , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Paclitaxel/toxicidade , Triterpenos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Células CHO , Cricetulus , Diprenorfina/farmacocinética , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Triterpenos Pentacíclicos , Traumatismos dos Nervos Periféricos/induzido quimicamente , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/etiologia , Traumatismos dos Nervos Periféricos/virologia , Ratos , Ratos Sprague-Dawley , Trítio/farmacocinética , Ácido BetulínicoRESUMO
Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.
Assuntos
Gânglios Espinais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sumoilação , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Animais , Comportamento Animal , Membrana Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Hiperalgesia/sangue , Hiperalgesia/complicações , Ativação do Canal Iônico , Masculino , Proteínas Mutantes/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Fator de Necrose Tumoral alfa/sangueRESUMO
Cav3.2 T-type calcium channels are important for the signaling of nociceptive information in the primary afferent pain pathway. During neuropathy and peripheral inflammation, Cav3.2 channels are upregulated due to an increased association with the deubiquitinase USP5. Disrupting these interactions in male mice by the use of cell permeant peptides reverses mechanical and thermal hypersensitivity. Here we explore the effects of interfering with USP5 binding to the channel in female mice with synchronized estrous cycle. We show that intrathecal delivery of a cell-penetrating TAT peptide corresponding to the UBPc domain of USP5 fully reverses mechanical hypersensitivity in mice intraplantarly injected with Complete Freund's Adjuvant. Hence, the USP5 mediated dysregulation of Cav3.2 channel activity does not exhibit sex differences, and potential therapeutics targeting this interaction should be effective in both male and female subjects.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Hiperalgesia/metabolismo , Inflamação/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Dor Crônica/metabolismo , Dor Crônica/patologia , Feminino , Hiperalgesia/patologia , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Peptídeos/metabolismoRESUMO
Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1ß (IL-1ß) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.
Assuntos
Artralgia/prevenção & controle , Artrite Experimental/prevenção & controle , Conexinas/metabolismo , Conexinas/fisiologia , Hiperalgesia/prevenção & controle , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Doenças da Medula Espinal/prevenção & controle , Animais , Artralgia/etiologia , Artrite Experimental/etiologia , Conexinas/genética , Hiperalgesia/etiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Doenças da Medula Espinal/etiologiaRESUMO
Pain-sensing sensory neurons of the dorsal root ganglion (DRG) can become sensitized or hyperexcitable in response to surgically induced peripheral tissue injury. We investigated the potential role and molecular mechanisms of nociceptive ion channel dysregulation in acute pain conditions such as those resulting from skin and soft tissue incision. We used selective pharmacology, electrophysiology, and mouse genetics to link increased current densities arising from the CaV3.2 isoform of T-type calcium channels (T-channels) to nociceptive sensitization using a clinically relevant rodent model of skin and deep tissue incision. Furthermore, knockdown of the CaV3.2-targeting deubiquitinating enzyme USP5 or disruption of USP5 binding to CaV3.2 channels in peripheral nociceptors resulted in a robust antihyperalgesic effect in vivo and substantial T-current reduction in vitro. Our study provides mechanistic insight into the role of plasticity in CaV3.2 channel activity after surgical incision and identifies potential targets for perioperative pain that may greatly decrease the need for narcotics and potential for drug abuse.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Nociceptores/metabolismo , Dor/metabolismo , Complicações Pós-Operatórias/metabolismo , Animais , Canais de Cálcio Tipo T/genética , Procedimentos Cirúrgicos Dermatológicos/efeitos adversos , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Temperatura Alta , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/fisiopatologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/fisiopatologia , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
INTRODUCTION: Voltage-gated calcium channels (VGCC) have been found to be differentially expressed in several different tumor types, but their role in tumor growth, malignant invasion, metastases and impact on clinical outcomes has not been clarified. MATERIALS AND METHODS: From a cohort database of 193 patients with early-stage NSCLC, 163 formalin-fixed paraffin-embedded specimens were available for analysis to construct tissue microarrays. Cav3.1 protein expression was detected using fluorescence immunohistochemistry, and quantified using automated image acquisition and analysis. RESULTS: Among the cohort of 193 NSCLC patients, adenocarcinoma (53.9%) and squamous cell carcinoma (SCC) (30.1%) were the most common histologies. There was no difference between SCC and non-SCC subtypes in overall survival (OS) or relapse-free survival (RFS); 74.2 vs 90.1 months (p = 0.543) and 48.8 vs 52.6 months (p = 0.766), respectively. T-type VGCC 3.1 (Cav3.1) overexpression was assessed by tissue microarray immunohistochemistry analysis from 163 available patient samples. Eighteen (11.0%) NSCLC primaries were found to have Cav3.1 overexpression levels, and were significantly associated with SCC histology (p < 0.001), larger tumor size (p < 0.001) and later stage disease at diagnosis (p = 0.019). Median OS was 48.6 vs 106.7 months for Cav3.1 overexpressing and non-overexpressing patients, respectively (p = 0.032). Regression analysis revealed a significantly negative effect for Cav3.1 overexpression on RFS (Hazard ratio [HR] = 2.02, p = 0.048). CONCLUSIONS: Cav3.1 overexpression is a potential biomarker for poorer patient outcomes. These results bring supportive evidence for calcium channels inducing an aggressive phenotype in NSCLC and potentially may serve as a therapeutic target in overexpressing tumors.