RESUMO
The Ca2+-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca2+, and was initially known for its regulation of parathyroid hormone (PTH) release. Ubiquitous expression of CaSR in different tissues and organs was later noted and CaSR participation in various physiological functions was demonstrated. Accumulating evidence has suggested that CaSR functionally interacts with transient receptor potential (TRP) channels, which are mostly non-selective cation channels involved in sensing temperature, pain and stress. This review describes the interactions of CaSR with TRP channels in diverse cell types to trigger a variety of biological responses. CaSR has been known to interact with different types of G proteins. Possible involvements of G proteins, other signaling and scaffolding protein intermediates in CaSR-TRP interaction are discussed. In addition, an attempt will be made to extend the current understanding of biased agonism of CaSR.
Assuntos
Sinalização do Cálcio , Receptores de Detecção de Cálcio , Canais de Potencial de Receptor Transitório , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/agonistas , Humanos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Host ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that treatment with ML2-SA1, an agonist for the human endolysosomal cation channel TRPML2, impairs ZIKV replication. Upon ML2-SA1 treatment, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced and cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound's negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV and many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed that ML2-SA1 also negatively impacted HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides indicating that TRPML2 may be a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.
Assuntos
Antivirais , Canais de Potencial de Receptor Transitório , Replicação Viral , Infecção por Zika virus , Zika virus , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Replicação Viral/efeitos dos fármacos , Humanos , Antivirais/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo , Infecção por Zika virus/virologia , Infecção por Zika virus/tratamento farmacológico , Chlorocebus aethiops , Animais , Células Vero , Colesterol/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Linhagem Celular , Células HEK293 , Ftalimidas , QuinolinasRESUMO
The TRPA1 ion channel is a sensitive detector of reactive chemicals, found primarily on sensory neurons. The phenotype exhibited by mice lacking TRPA1 suggests its potential as a target for pharmacological intervention. Antibody-based detection for distribution analysis is a standard technique. In the case of TRPA1, however, there is no antibody with a plausible validation in knockout animals or functional studies, but many that have failed in this regard. To this end we employed the single molecule in situ hybridization technique RNAscope on sensory neurons immediately after detection of calcium responses to the TRPA1 agonist allyl isothiocyanate. There is a clearly positive correlation between TRPA1 calcium imaging and RNAscope detection (R = 0.43), although less than what might have been expected. Thus, the technique of choice should be carefully considered to suit the research question. The marginal correlation between TRPV1 RNAscope and the specific agonist capsaicin indicates that such validation is advisable for every RNAscope target. Given the recent description of a long-awaited TRPA1 reporter mouse, TRPA1 RNAscope detection might still have its use cases, for detection of RNA at particular sites, for example, defined structurally or by other molecular markers.
Assuntos
Cálcio , Isotiocianatos , Canal de Cátion TRPA1 , Animais , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genética , Isotiocianatos/farmacologia , Camundongos , Cálcio/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/agonistas , Capsaicina/farmacologia , Hibridização In Situ , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/agonistas , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , MasculinoRESUMO
Glioblastoma multiforme represents a substantial clinical challenge. Transient receptor potential channel (TRPC) antagonists might provide new therapeutic options for this aggressive cancer. In this study, a series of N-alkyl-N-benzoyl and N-alkyl-N-benzyl thiazoles were designed and prepared using a scaffold-hopping strategy and evaluated as TRPC6 antagonists. This resulted in the discovery of 15g, a potent TRPC antagonist that exhibited suitable inhibitory micromolar activities against TRPC3, TRPC4, TRPC5, TPRC6, and TRPC7 and displayed noteworthy anti-glioblastoma efficacy in vitro against U87 cell lines. In addition, 15g featured an acceptable pharmacokinetic profile and exhibited better in vivo potency (25 mg/kg/d) than the frontline therapeutic agent temozolomide (50 mg/kg/d) in xenograft models. Taken together, the TRPC antagonist 15g represents a promising lead compound for developing new anti-glioblastoma agents.
Assuntos
Glioblastoma , Canais de Potencial de Receptor Transitório , Humanos , Linhagem Celular , Glioblastoma/tratamento farmacológico , Temozolomida , Canais de Potencial de Receptor Transitório/agonistas , Canais de Cátion TRPC/metabolismoRESUMO
The transient receptor potential (TRP) channel family, including TRPA1, is known to be involved in temperature sensing and response. Previous studies have shown that intragastric administration of cinnamaldehyde (a typical TRPA1 agonist) can change body temperature, but the role of TRPA1 in this response is not clear. In this study, we found that intragastric administration of cinnamaldehyde increased in the intrascapular brown adipose tissue (IBAT) and rectal temperatures. However, this effect was not observed in TRPA1 knockout mice, suggesting that TRPA1 is involved in these temperature changes. Intravenous cinnamaldehyde also increased IBAT and rectal temperatures, only in the presence of TRPA1. We also explored the contribution of the vagus nerve to these temperature changes and found that it played a limited role. These results suggest that cinnamaldehyde can affect body temperature through TRPA1 activation, with the vagus nerve having a minor influence.
Assuntos
Temperatura Corporal , Canais de Potencial de Receptor Transitório , Camundongos , Animais , Canal de Cátion TRPA1/genética , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/agonistas , Acroleína/farmacologiaRESUMO
Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.
Assuntos
Tetrazóis , Canais de Potencial de Receptor Transitório , Canais de Potencial de Receptor Transitório/agonistas , Relação Estrutura-Atividade , Tetrazóis/química , Tetrazóis/farmacologiaRESUMO
Artemisinin and its derivatives are the main therapeutic drugs against Plasmodium protists, the causative agents of malaria. While several putative mechanisms of action have been proposed, the precise molecular targets of these compounds have not been fully elucidated. In addition to their antimalarial properties, artemisinins have been reported to act as anti-tumour agents and certain antinociceptive effects have also been proposed. We investigated the effect of the parent compound, artemisinin, on a number of temperature-gated Transient Receptor Potential ion channels (so called thermoTRPs), given their demonstrated roles in pain-sensing and cancer. We report that artemisinin acts as an agonist of the Transient Receptor Potential Ankyrin type 1 (TRPA1) receptor channel. Artemisinin was able to evoke calcium transients in HEK293T cells expressing recombinant human TRPA1, as well as in a subpopulation of mouse dorsal root ganglion (DRG) neurons which also responded to the selective TRPA1 agonist allyl isothiocyanate (AITC) and these responses were reversibly abolished by the selective TRPA1 antagonist A967079. Artemisinin also triggered whole-cell currents in HEK293T cells transiently transfected with human TRPA1, as well as in TRPA1-expressing DRG neurons, and these currents were inhibited by A967079. Interestingly, using human TRPA1 mutants, we demonstrate that artemisinin acts as a non-electrophilic agonist of TRPA1, activating the channel in a similar manner to carvacrol and menthol. These results may provide a better understanding of the biological actions of the very important antimalarial and anti-tumour agent artemisinin.
Assuntos
Antimaláricos , Artemisininas , Canais de Potencial de Receptor Transitório , Animais , Humanos , Camundongos , Anquirinas/química , Anquirinas/farmacologia , Antimaláricos/química , Antimaláricos/farmacologia , Artemisininas/química , Artemisininas/farmacologia , Gânglios Espinais , Células HEK293 , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/química , Canal de Cátion TRPA1RESUMO
BACKGROUND: Bioactive dietary constituents activating Transient receptor potential (TRP) channels have emerged as promising candidates for the prevention of metabolic disorders. OBJECTIVE: The present study is an attempt to evaluate anti-obesity potential of a dietary TRP-based tri-agonist, combination of sub-effective doses of capsaicin (TRPV1 agonist), menthol (TRPM8 agonist), and cinnamaldehyde (TRPA1 agonist) in high-fat diet (HFD)-fed mice. DESIGN: Male C57BL/6 J mice divided into three groups (n = 8), were fed on normal pellet diet (NPD), or high-fat diet (HFD) (60% energy by fat) and HFD + CB (combination of capsaicin 0.4 mg/Kg, menthol 20 mg/Kg, and cinnamaldehyde 2 mg/Kg; p.o) for 12 weeks. Effects on HFD-induced weight gain, biochemical, histological and genomic changes in the WAT, BAT, liver and hypothalamus tissues were studied. RESULTS: Administration of tri-agonist prevented HFD-induced increase in weight gain, improved altered morphometric parameters, glucose homeostasis, and adipose tissue hypertrophy. Tri-agonist supplementation was found to induce browning of white adipose tissue and promote brown adipose tissue activation. Enhanced glucose utilization and prevention of lipid accumulation and insulin resistance in the liver was observed in mice supplemented with a tri-agonist. CONCLUSION: The present work provides evidence that the new approach based on combination of sub-effective doses of TRP channel agonists (TRI-AGONIST) can be employed to develop concept-based functional food for therapeutic and preventive strategies against HFD-associated pathological complications.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/agonistas , Acroleína/administração & dosagem , Acroleína/análogos & derivados , Acroleína/uso terapêutico , Animais , Capsaicina/administração & dosagem , Capsaicina/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Dieta Hiperlipídica/métodos , Modelos Animais de Doenças , Mentol/administração & dosagem , Mentol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL/metabolismo , Fenótipo , Canais de Potencial de Receptor Transitório/farmacologiaRESUMO
Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.
Assuntos
Antivirais/farmacologia , Ftalimidas/farmacologia , Quinolinas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Zika virus/efeitos dos fármacos , Células A549 , Animais , Autofagia , Chlorocebus aethiops , Endossomos/enzimologia , Endossomos/metabolismo , Humanos , Lisossomos/enzimologia , Lisossomos/metabolismo , Células Vero , Infecção por Zika virus/virologiaRESUMO
During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.
Assuntos
Antineoplásicos/farmacologia , Lisossomos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Canais de Potencial de Receptor Transitório/agonistas , Zinco/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/secundário , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic pain is a common symptom experienced during cancer progression. Additionally, some patients experience bone pain caused by cancer metastasis, which further complicates the prognosis. Cancer pain is often treated using opioid-based pharmacotherapy, but these drugs possess several adverse effects. Accordingly, new mechanisms for cancer pain management are being explored, including transient receptor potential channels (TRPs). TRP ion channels are expressed in several tissues and play a key role in pain detection, especially TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1). In the present review, we describe the role of TRPV1 and TRPA1 involved in cancer pain mechanisms. Several studies have revealed that the administration of TRPV1 or TRPA1 agonists/antagonists and TRPV1 or TRPA1 knockdown reduced sensitivity to nociception in cancer pain models. TRPV1 was also found to be involved in various models of cancer-induced bone pain (CIBP), with TRPV1 expression reportedly enhanced in some models. These studies have demonstrated the TRPV1 or TRPA1 association with cancer pain in models induced by tumour cell inoculation into the bone cavity, hind paw, mammary fat pad, and sciatic nerve in mice or rats. To date, only resiniferatoxin, a TRPV1 agonist, has been evaluated in clinical trials for cancer pain and showed preliminary positive results. Thus, TRP channels are potential targets for managing cancer-related pain syndromes.
Assuntos
Dor do Câncer/tratamento farmacológico , Dor do Câncer/fisiopatologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Ensaios Clínicos como Assunto , Humanos , Manejo da Dor , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Recent findings have revealed that exposure to environmental contaminants may result in obesity and pose a health threat to the general public. As the activity of transient receptor potential channels (TRPs) plays a permissive role in adipogenesis, the interactions between TRPs and some food pollutants, i.e. bisphenol A, di (2-ethylhexyl) phthalate, zearalenone, and zeranol at 10 µM were investigated in the present study. TRP-V1,-V3, -C4 and -C6 are reported to be differentially expressed in the adipocyte differentiation, and immunoblotting was performed to quantify changes in these TRPs affected by the pollutants. Our result indicated that the mycoestrogen zeranol or α-zearalanol suppressed the expression of the V1 and C6 isoforms. Subsequently, confocal microscopy was used to measure the calcium inflow repressed by zeranol from 0.1 µM to 10 µM. Oil Red O staining was used to determine the differentiation of 3T3 L1 preadipocytes. Zeranol could suppress the expression of TRP-V1 and -C6 protein and inhibit the associated flow of calcium into the cytosol of 3T3 L1 cells. Its IC50 value for inhibiting calcium inflow stimulated by 40 µM capsaicin or 10 µM GSK1702934A was estimated to be around 6 µM. Reduced TRP-V1 or -C6 activity might result in promoting adipogenesis. In conclusion, this study demonstrated that zeranol could potentiate fat cell differentiation through antagonizing TRP-V1 and -C6 activities.
Assuntos
Estrogênios não Esteroides/toxicidade , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Zeranol/toxicidade , Células 3T3-L1 , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Capsaicina/farmacologia , Redução da Medicação , Estradiol/farmacologia , Estrogênios não Esteroides/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo , Zeranol/administração & dosagemRESUMO
Despite the introduction of antiretroviral (ARV) therapy (ART), approximately 30-50% of people living with human immunodeficiency virus-1 (HIV-1) will develop a spectrum of measurable neurocognitive dysfunction, collectively called HIV-associated neurocognitive disorder (HAND). While the clinical manifestations of HAND have changed with the advent of ART, certain pathological features have endured, including white matter alterations and dysfunction. The persistence of white matter alterations in the post-ART era suggests that ARV drugs themselves may contribute to HAND pathology. Our group has previously demonstrated that two ARV compounds from the protease inhibitor (PI) class, ritonavir and lopinavir, inhibit oligodendrocyte maturation and myelin protein production. We hypothesized that other members of the PI class, saquinavir and darunavir, could also negatively impact oligodendrocyte differentiation. Here we demonstrate that treating primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either ARV drug results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Furthermore, we show that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- and darunavir-induced inhibition of oligodendrocyte maturation. Moreover, our findings suggest, for the first time, an imperative role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against ARV-induced oligodendrocyte dysregulation. Graphical Abstract Treatment of primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either antiretroviral compound of the protease inhibitor class, darunavir or saquinavir, results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Additionally, in darunavir or saquinavir-treated cultures we observed a concentration-dependent decrease in the number of acidic lysosomes, via immunostaining with LysoTracker Red, compared with vehicle-treated cultures. Finally, we showed that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- or darunavir-induced inhibition of oligodendrocyte maturation. Our findings suggest, for the first time, a critical role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against antiretroviral-induced oligodendrocyte dysregulation.
Assuntos
Darunavir/farmacologia , Endossomos/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Lisossomos/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Saquinavir/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Darunavir/toxicidade , Depressão Química , Relação Dose-Resposta a Droga , Endossomos/química , Inibidores da Protease de HIV/toxicidade , Concentração de Íons de Hidrogênio , Lisossomos/química , Proteínas da Mielina/biossíntese , Estresse Oxidativo , Ftalimidas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Saquinavir/toxicidade , Canais de Potencial de Receptor Transitório/agonistasRESUMO
Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Ftalimidas/farmacologia , Quinolinas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Zika virus/efeitos dos fármacos , Células A549 , Autofagia , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Hepáticas , Lisossomos/enzimologia , Lisossomos/metabolismo , Proteólise , Infecção por Zika virus/virologiaRESUMO
Pungent substances, such as capsaicin and gingerol, activate the transient receptor potential (TRP)-V1 channel and affect the feeding behaviors of animals. To gain insight into how living organisms have acquired a sense for pungent substances, we explored the response to TRP agonists in a protist, Chlamydomonas reinhardtii When capsaicin or gingerol was applied to wild-type cells, they became immotile, with flagella detaching from the cell body. The degree of deflagellation was nearly halved in a mutant defective in the TRP channel ADF1. Deflagellation in the adf1 mutant was inhibited further by Ruthenium Red, indicating ADF1 and another TRP channel are involved in the deflagellation response. The response to capsaicin and gingerol was not inhibited by TRPV1-specific blockers such as 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) and capsazepine. When capsaicin or gingerol was applied to wild-type cells in the presence of Ruthenium Red, a large proportion lost motility while flagella remained attached, suggesting that flagella stop contributing to motility, at least in part, through a TRP-channel-independent pathway. These results indicate that pungent compounds such as capsaicin and gingerol induce loss of flagellar motility and flagellar detachment in C.reinhardtii cells.
Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Capsaicina/farmacologia , Catecóis/farmacologia , Álcoois Graxos/farmacologia , Flagelos/efeitos dos fármacos , Flagelos/metabolismo , Concentração de Íons de Hidrogênio , Transdução de Sinais , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Two-pore channels (TPCs) have been a hot topic in recent literature. Their involvement in various diseases such as viral infections and cancer is of great interest for drug research. Due to their localization in the endolysosomal system and the lack of cell-permeable activators, complex techniques were required for studying channel functions. Here, we review the first published lipophilic small-molecule activators of TPCs. In independent high-throughput screens, several new agonists were discovered, which now allow simple and fast investigation of TPCs in more detail in intact cells and in vivo. Zhang et al. identified tricyclic and phenothiazine antidepressants as TPC1 and TPC2 activators by screening a library of approved drugs. In contrast, Gerndt et al. screened an extensive compound library with mostly new chemotypes and drug structures. The latter resulted in two structurally distinct high-affinity agonists, which are able to selectively activate TPC2 in either an NAADP- or PI(3,5)P2 -like manner. Here, we discuss the advantages and drawbacks of the identified molecules and their structural features. The versatility by which TPCs can be activated indicates many opportunities for future studies.
Assuntos
Canais de Cálcio/química , Sinalização do Cálcio , Descoberta de Drogas , Ativação do Canal Iônico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Animais , HumanosRESUMO
Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca2+ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca2+ channels may represent a promising approach to treat DMD and related muscle diseases.
Assuntos
Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Animais , Biomarcadores , Biópsia , Modelos Animais de Doenças , Distrofina/genética , Imunofluorescência , Expressão Gênica , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.
Assuntos
Ceramidase Ácida/metabolismo , Calcificação Vascular/metabolismo , Ceramidase Ácida/genética , Animais , Aorta/metabolismo , Aorta/patologia , Sinalização do Cálcio , Células Cultivadas , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Lipogranulomatose de Farber/genética , Lipogranulomatose de Farber/metabolismo , Lisossomos/metabolismo , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Cardiovasculares , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Esfingolipídeos/metabolismo , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologiaRESUMO
In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both planarian species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. RNAi of DjTRPVa/DjTRPVb attenuated scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior's signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation.
Assuntos
Reação de Fuga/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/genética , Animais , Peróxido de Hidrogênio/farmacologia , Isotiocianatos/farmacologia , Nociceptividade/efeitos dos fármacos , PlanáriasRESUMO
The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKß and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.