RESUMEN
Our sensory adaptation to cold and chemically induced coolness is mediated by the intrinsic property of TRPM8 channels to desensitize. TRPM8 is also implicated in cold-evoked pain disorders and migraine, highlighting its inhibitors as an avenue for pain relief. Despite the importance, the mechanisms of TRPM8 desensitization and inhibition remained unclear. We found, using cryo-electron microscopy, electrophysiology, and molecular dynamics simulations, that TRPM8 inhibitors bind selectively to the desensitized state of the channel. These inhibitors were used to reveal the overlapping mechanisms of desensitization and inhibition and that cold and cooling agonists share a common desensitization pathway. Furthermore, we identified the structural determinants crucial for the conformational change in TRPM8 desensitization. Our study illustrates how receptor-level conformational changes alter cold sensation, providing insights into therapeutic development.
Asunto(s)
Frío , Mentol , Canales Catiónicos TRPM , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Humanos , Mentol/farmacología , Simulación de Dinámica Molecular , Adaptación Fisiológica , Microscopía por Crioelectrón , Células HEK293 , Conformación Proteica , AnimalesRESUMEN
Background: To investigate the effects of arsenic trioxide (ATO) on human colorectal cancer cells (HCT116) growth and the role of transient receptor potential melastatin 4 (TRPM4) channel in this process. Methods: The viability of HCT116 cells was assessed using the CCK-8 assay. Western blot analysis was employed to examine the protein expression of TRPM4. The apoptosis of HCT116 cells was determined using TUNEL and Flow cytometry. Cell migration was assessed through the cell scratch recovery assay and Transwell cell migration assay. Additionally, Transwell cell invasion assay was performed to determine the invasion ability of HCT116 cells. Results: ATO suppressed the viability of HCT116 cells in a dose-dependent manner, accompanied by a decline in cell migration and invasion, and an increase in apoptosis. 9-phenanthroline (9-Ph), a specific inhibitor of TRPM4, abrogated the ATO-induced upregulation of TRPM4 expression. Additionally, blocking TRPM4 reversed the effects of ATO on HCT116 cells proliferation, including restoration of cell viability, migration and invasion, as well as the inhibition of apoptosis. Conclusion: ATO inhibits CRC cell growth by inducing TRPM4 expression, our findings indicate that ATO is a promising therapeutic strategy and TRPM4 may be a novel target for the treatment of CRC.
Asunto(s)
Apoptosis , Trióxido de Arsénico , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Neoplasias Colorrectales , Canales Catiónicos TRPM , Humanos , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genética , Trióxido de Arsénico/farmacología , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Células HCT116 , Movimiento Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Óxidos/farmacología , Antineoplásicos/farmacología , Invasividad Neoplásica , Arsenicales/farmacologíaRESUMEN
Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.
Asunto(s)
Activación del Canal Iónico , Canales Catiónicos TRPM , Temperatura , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Calcio/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Especificidad por Sustrato , Canales Catiónicos TRPM/agonistas , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/metabolismo , Vanadatos/química , Vanadatos/farmacología , Vanadatos/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. EXPERIMENTAL APPROACH: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. KEY RESULTS: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. CONCLUSION AND IMPLICATIONS: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.
Asunto(s)
Inmunosupresores , Ratones Noqueados , Células Receptoras Sensoriales , Sirolimus , Canales Catiónicos TRPM , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Animales , Humanos , Células HEK293 , Sirolimus/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Inmunosupresores/farmacología , Ratas , Ratones , Masculino , Ratones Endogámicos C57BL , Células Cultivadas , Calcio/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , FríoRESUMEN
BACKGROUND AND PURPOSE: We extend the characterization of the TRPM8 antagonist VBJ103 with tests of selectivity, specificity and distribution, therapeutic efficacy of systemic administration against oxaliplatin-induced cold hyperalgesia and the impact of systemic administration on core body temperature (CBT). EXPERIMENTAL APPROACH: Selectivity at human TRPA1 and TRPV1 as well as in vitro safety profiling was determined. Effects of systemic administration of VBJ103 were evaluated in a model of oxaliplatin-induced cold hyperalgesia. Both peripheral and centrally mediated effects of VBJ103 on CBT were assessed with radiotelemetry. KEY RESULTS: VBJ103 had no antagonist activity at TRPV1 and TRPA1, but low potency TRPA1 activation. The only safety liability detected was partial inhibition of the dopamine transporter (DAT). VBJ103 delivered subcutaneously dose-dependently attenuated cold hypersensitivity in oxaliplatin-treated mice at 3, 10 and 30 mg·kg-1 (n = 7, P < 0.05). VBJ103 (30 mg·kg-1) antinociception was influenced by neither the TRPA1 antagonist HC-030031 nor the DAT antagonist GBR12909. Subcutaneous administration of VBJ103 (3, 10 and 30 mg·kg-1, but not 100 or 300 mg·kg-1, n = 7) decreased CBT (2°C). Intraperitoneal (i.p.) administration of VBJ103 (3, 10 and 30 mg·kg-1) dose-dependently decreased CBT to an extent larger than that detected with subcutaneous administration. Intracerebroventricular (i.c.v.) administration (306 nmol/1 µL; n = 5) did not alter CBT. CONCLUSIONS AND IMPLICATIONS: We achieve therapeutic efficacy with subcutaneous administration of a novel TRPM8 antagonist that attenuates deleterious influences on CBT, a side effect that has largely prevented the translation of TRPM8 as a target.
Asunto(s)
Hiperalgesia , Oxaliplatino , Canales Catiónicos TRPM , Animales , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Masculino , Ratones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/inducido químicamente , Humanos , Oxaliplatino/administración & dosificación , Inyecciones Subcutáneas , Temperatura Corporal/efectos de los fármacos , Ratones Endogámicos C57BL , Relación Dosis-Respuesta a Droga , Células HEK293 , Síndromes Periódicos Asociados a CriopirinaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron death and neuroinflammation. Emerging evidence points to the involvement of the transient receptor potential melastatin 2 (TRPM2) channel in neuron death and glial activation in several neurodegenerative diseases. However, the involvement of TRPM2 in PD and specifically its relation to the neuroinflammation aspect of the disease remains poorly understood. Here, we hypothesized that AG490, a TRPM2 inhibitor, can be used as a treatment in a mouse model of PD. Mice underwent stereotaxic surgery for 6-hydroxydopamine (6-OHDA) administration in the right striatum. Motor behavioral tests (apomorphine, cylinder, and rotarod) were performed on day 3 post-injection to confirm the PD model induction. AG490 was then daily injected i.p. between days 3 to 6 after surgery. On day 6, motor behavior was assessed again. Substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry, immunoblotting, and RT-qPCR analysis on day 7. Our results revealed that AG490 post-treatment reduced motor behavior impairment and nigrostriatal neurodegeneration. In addition, the compound prevented TRPM2 upregulation and changes of the Akt/GSK-3ß/caspase-3 signaling pathway. The TRPM2 inhibition also avoids the glial morphology changes observed in the PD group. Remarkably, the morphometrical analysis revealed that the ameboid-shaped microglia, found in 6-OHDA-injected animals, were no longer present in the AG490-treated group. These results indicate that AG490 treatment can reduce dopaminergic neuronal death and suppress neuroinflammation in a PD mouse model. Inhibition of TRPM2 by AG490 could then represent a potential therapeutical strategy to be evaluated for PD treatment.
Asunto(s)
Ratones Endogámicos C57BL , Neuroglía , Canales Catiónicos TRPM , Tirfostinos , Animales , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Ratones , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Tirfostinos/farmacología , Tirfostinos/uso terapéutico , Progresión de la Enfermedad , Oxidopamina/toxicidad , Modelos Animales de Enfermedad , Degeneración Nerviosa/patología , Degeneración Nerviosa/tratamiento farmacológico , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/prevención & control , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología , Sustancia Negra/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
TRP channels are implicated in various diseases, but high structural similarity between them makes selective pharmacological modulation challenging. Here, we study the molecular mechanism underlying specific inhibition of the TRPM7 channel, which is essential for cancer cell proliferation, by the anticancer agent CCT128930 (CCT). Using cryo-EM, functional analysis, and MD simulations, we show that CCT binds to a vanilloid-like (VL) site, stabilizing TRPM7 in the closed non-conducting state. Similar to other allosteric inhibitors of TRPM7, NS8593 and VER155008, binding of CCT is accompanied by displacement of a lipid that resides in the VL site in the apo condition. Moreover, we demonstrate the principal role of several residues in the VL site enabling CCT to inhibit TRPM7 without impacting the homologous TRPM6 channel. Hence, our results uncover the central role of the VL site for the selective interaction of TRPM7 with small molecules that can be explored in future drug design.
Asunto(s)
1-Naftilamina/análogos & derivados , Antineoplásicos , Canales Catiónicos TRPM , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células HEK293 , Simulación de Dinámica Molecular , Sitios de Unión , Unión Proteica , Microscopía por CrioelectrónRESUMEN
OBJECTIVES: To elucidate the association between the anticancer activities of piperlongumine (PL) and its potential target, transient receptor potential melastatin 7 channel (TRPM7), in oral squamous cell carcinoma (OSCC). METHODS: The expression levels and electrical characteristics of TRPM7 as well as cell viability in response to various PL treatments were investigated in the OSCC cell line Cal27. RESULTS: PL treatment resulted in a concentration- and time-dependent reduction in TRPM7 mRNA and protein expression in Cal27 cells. Furthermore, PL treatment inhibited TRPM7-like rectifying currents in Cal27 cells; however, this inhibition was less effective than that of the TRPM7 antagonist waixenicin A. Rapid perfusion and washout experiments revealed an immediate inhibitory effect of PL on TRPM7-like currents. The antagonistic effect of PL occurred within 1 min and was not completely reversed following washout. Notably, the extracellular Ca2+ concentration still influenced PL-induced changes in the TRPM7-like current, indicating that PL can directly but gently antagonize the TRPM7 channel. Functional changes in TRPM7 correlated with the observed antiproliferative and cytotoxic effects of PL in Cal27 cells. CONCLUSIONS: These findings suggest that PL exhibits potent inhibitory effects on TRPM7 and exerts its anti-cancer effects by downregulating TRPM7 expression and antagonizing channel currents.
Asunto(s)
Carcinoma de Células Escamosas , Supervivencia Celular , Dioxolanos , Neoplasias de la Boca , Canales Catiónicos TRPM , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/genética , Humanos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Línea Celular Tumoral , Dioxolanos/farmacología , Supervivencia Celular/efectos de los fármacos , Antineoplásicos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Acetatos , Diterpenos , PiperidonasRESUMEN
Waixenicin A, a xenicane diterpene from the octocoral Sarcothelia edmondsoni, is a selective, potent inhibitor of the TRPM7 ion channel. To study the structure-activity relationship (SAR) of waixenicin A, we isolated and assayed related diterpenes from S. edmondsoni. In addition to known waixenicins A (1) and B (2), we purified six xenicane diterpenes, 7S,8S-epoxywaixenicins A (3) and B (4), 12-deacetylwaixenicin A (5), waixenicin E (6), waixenicin F (7), and 20-acetoxyxeniafaraunol B (8). We elucidated the structures of 3-8 by NMR and MS analyses. Compounds 1, 2, 3, 4, and 6 inhibited TRPM7 activity in a cell-based assay, while 5, 7, and 8 were inactive. A preliminary SAR emerged showing that alterations to the nine-membered ring of 1 did not reduce activity, while the 12-acetoxy group, in combination with the dihydropyran, appears to be necessary for TRPM7 inhibition. The bioactive compounds are proposed to be latent electrophiles by formation of a conjugated oxocarbenium ion intermediate. Whole-cell patch-clamp experiments demonstrated that waixenicin A inhibition is irreversible, consistent with a covalent inhibitor, and showed nanomolar potency for waixenicin B (2). Conformational analysis (DFT) of 1, 3, 7, and 8 revealed insights into the conformation of waixenicin A and congeners and provided information regarding the stabilization of the proposed pharmacophore.
Asunto(s)
Acetatos , Antozoos , Diterpenos , Proteínas Serina-Treonina Quinasas , Canales Catiónicos TRPM , Animales , Humanos , Antozoos/química , Diterpenos/farmacología , Diterpenos/química , Diterpenos/aislamiento & purificación , Conformación Molecular , Estructura Molecular , Relación Estructura-Actividad , Canales Catiónicos TRPM/antagonistas & inhibidoresRESUMEN
BACKGROUND: Allergies often present challenges in managing itch and the effects of histamine. Cooling agents that act via transient receptor potential melastatin 8 (TRPM8) agonism have shown potential in itch management. However, animal studies on itch have limitations, as animals cannot communicate subjective events and their fur-coated skin differs from that of humans. Human studies offer more direct and reliable information. OBJECTIVES: To investigate the effects of a specific TRPM8 agonist gel (cryosim-1) on itch induced by various pruritogens in human skin. METHODS: Calcium imaging experiments determined the binding of cryosim-1 and histamine to their respective receptors. Thirty healthy volunteers underwent skin prick tests with pruritogens and a control vehicle. Itch and pain intensity were measured using a numerical rating scale (NRS) across 10â min. Participants were randomly assigned to pretreatments with vehicle or TRPM8 agonist gel. Tests were repeated at a later date, and skin moisture, transepidermal water loss and mechanical sensitivity were measured. RESULTS: The in vitro study confirmed that histamine is not a TRPM8 agonist and cryosim-1 does not act as an agonist or antagonist on the human histamine 1 receptor. The TRPM8 agonist gel significantly reduced the itch intensity for all pruritogens compared with the vehicle-only gel. It also reduced itch NRS and the integrated itch score. Mechanical sensitivity was also reduced. CONCLUSIONS: The specific TRPM8 agonist gel effectively suppressed human skin itch induced by various pruritogens. These versatile actions suggest that cooling agents may be promising treatments for multiple forms of itch stimuli.
Managing itching and the effects of histamine can be difficult for people with allergies. Cooling the skin or applying menthol provides some relief from itch, but the way they work is not fully understood. Cooling agents interact with a protein called TRPM8 (also known as the 'cold and menthol receptor') and have shown potential for the management of itch. However, much of the research has been done on animals and has limitations when compared with human studies. Antihistamine medications can help with histamine-induced itching, but they may not work for other causes of itch. This study investigated the effects of a specific TRPM8 agonist (a chemical that activates a receptor to produce a biologic response) gel called cryosim-1 on itch in human skin. To do this, we conducted tests on 30 healthy people using five different substances that cause itching. Participants rated the itch intensity and pain using a scale and we measured various aspects of their skin. The results showed that all substances caused significant itching compared to a control substance, but itchiness gradually decreased over time. Histamine and compound 48/80 also caused pain. However, when participants applied the TRPM8 activator gel before exposure, they experienced less itching and lower itch intensity versus the gel without the activator. There were no significant differences in pain between the TRPM8 activator and the gel without it. In summary, our findings showed that activating TRPM8 receptors with a specific substance effectively relieved itching caused by various irritants on human skin. This suggests its potential as a treatment for itch-related conditions. Further research is needed to understand its mechanisms better and evaluate its effectiveness in real-life situations.
Asunto(s)
Histamina , Prurito , Canales Catiónicos TRPM , Humanos , Prurito/tratamiento farmacológico , Prurito/inducido químicamente , Canales Catiónicos TRPM/agonistas , Canales Catiónicos TRPM/antagonistas & inhibidores , Adulto , Masculino , Histamina/administración & dosificación , Histamina/efectos adversos , Femenino , Adulto Joven , Geles , Persona de Mediana Edad , Antipruriginosos/administración & dosificación , Antipruriginosos/farmacología , Antipruriginosos/efectos adversos , Método Doble Ciego , Administración CutáneaRESUMEN
The first detailed phytochemical analysis of the cannabigerol (CBG)-rich chemotype IV of Cannabis sativa L. resulted in the isolation of the expected cannabigerolic acid/cannabigerol (CBGA/CBG) and cannabidiolic acid/cannabidiol (CBDA/CBD) and of nine new phytocannabinoids (5-13), which were fully characterized by HR-ESIMS and 1D and 2D NMR. These included mono- or dihydroxylated CBGA/CBG analogues, a congener with a truncated side chain (10), cyclocannabigerol B (11), and the CBD derivatives named cannabifuranols (12 and 13). Cyclocannabigerol B and cannabifuranols are characterized by a novel phytocannabinoid structural architecture. The isolated phytocannabinoids were assayed on the receptor channels TRPA1 and TRPM8, unveiling a potent dual TRPA1 agonist/TRPM8 antagonist profile for compounds 6, 7, and 14. Chiral separation of the two enantiomers of 5 resulted in the discovery of a synergistic effect of the two enantiomers on TRPA1.
Asunto(s)
Cannabinoides , Cannabis , Canal Catiónico TRPA1 , Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Cannabis/química , Canal Catiónico TRPA1/antagonistas & inhibidores , Cannabinoides/farmacología , Cannabinoides/química , Cannabinoides/aislamiento & purificación , Canales Catiónicos TRPM/antagonistas & inhibidores , Estructura Molecular , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificación , Fitoquímicos/química , Humanos , Cannabidiol/farmacología , Cannabidiol/química , Canales de Calcio/metabolismoRESUMEN
Cannabinoids are a major class of compounds produced by the plant Cannabis sativa. Previous work has demonstrated that the main cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC) can have some beneficial effects on pain, inflammation, epilepsy, and chemotherapy-induced nausea and vomiting. While CBD and THC represent the two major plant cannabinoids, some hemp varieties with enzymatic deficiencies produce mainly cannabigerolic acid (CBGA). We recently reported that CBGA has a potent inhibitory effect on both Store-Operated Calcium Entry (SOCE) via inhibition of Calcium Release-Activated Calcium (CRAC) channels as well as currents carried by the channel-kinase TRPM7. Importantly, CBGA prevented kidney damage and suppressed mRNA expression of inflammatory cytokines through inhibition of these mechanisms in an acute nephropathic mouse model. In the present study, we investigate the most common major and minor cannabinoids to determine their potential efficacy on TRPM7 channel function. We find that approximately half of the tested cannabinoids suppress TRPM7 currents to some degree, with CBGA having the strongest inhibitory effect on TRPM7. We determined that the CBGA-mediated inhibition of TRPM7 requires a functional kinase domain, is sensitized by both intracellular Mgâ ATP and free Mg2+ and reduced by increases in intracellular Ca2+. Finally, we demonstrate that CBGA inhibits native TRPM7 channels in a B lymphocyte cell line. In conclusion, we demonstrate that CBGA is the most potent cannabinoid in suppressing TRPM7 activity and possesses therapeutic potential for diseases in which TRPM7 is known to play an important role such as cancer, stroke, and kidney disease.
Asunto(s)
Cannabinoides , Canales Catiónicos TRPM , Animales , Ratones , Cannabinoides/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidoresRESUMEN
BACKGROUND AND PURPOSE: Interstitial cystitis (=painful bladder syndrome) is a chronic bladder syndrome characterised by pelvic and bladder pain, urinary frequency and urgency, and nocturia. Transient receptor potential (TRP) channels are an attractive target in reducing the pain associated with interstitial cystitis. The current study aims to determine the efficacy of combination of TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8) channel inhibition in reducing the pain associated with experimental cystitis in guinea pigs. EXPERIMENTAL APPROACH: A novel animal model of non-ulcerative interstitial cystitis has been developed using protamine sulfate/zymosan in female guinea pigs. Continuous voiding cystometry was performed in conscious guinea pigs. Ex vivo "close-to-target" single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder. Visceromotor responses in vivo were used to determine the effects of TRP channel antagonists on cystitis-induced bladder hypersensitivity. KEY RESULTS: Protamine sulfate/zymosan treatment evoked mild inflammation in the bladder and increased micturition frequency in conscious animals. In cystitis, high threshold muscular afferents were sensitised via up-regulation of TRPV1 channels, high threshold muscular-mucosal afferents were sensitised via TRPM8 channels, and mucosal afferents by both. Visceromotor responses evoked by noxious bladder distension were significantly enhanced in cystitis and were returned to control levels upon administration of combination of low doses of TRPV1 and TRPM8 antagonists. CONCLUSIONS AND IMPLICATIONS: The data demonstrate the therapeutic promises of combination of TRPV1 and TRPM8 antagonists for the treatment of bladder hypersensitivity in cystitis.
Asunto(s)
Cistitis Intersticial , Cistitis , Hipersensibilidad , Canales de Potencial de Receptor Transitorio , Animales , Femenino , Cobayas , Cistitis/tratamiento farmacológico , Dolor , Canales Catiónicos TRPV , Vejiga Urinaria , Zimosan/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidoresRESUMEN
Melanoma continues to be the most aggressive and devastating form of skin cancer for which the development of novel therapies is required. The present study aimed to determine the effects of antagonism of the transient receptor potential melastatin2 (TRPM2) ion channel in primary human malignant melanoma cells. TRPM2 antagonism via use of the antifungal agent, clotrimazole, led to decreases in cell proliferation, as well as dosedependent increases in cell death in all melanoma cell lines investigated. The targeting of TRPM2 channels was verified using TRPM2 knockdown, where treatment with TRPM2 smallinterfering RNA led to similar levels of cell death in all melanoma cell lines when compared with clotrimazole treatment. Minimal effects on proliferation and cell death were observed following antagonism or knockdown of TRPM2 in noncancerous human keratinocytes. Moreover, characteristics of TRPM2 were explored in these melanoma cells and the results demonstrated that TRPM2, localized to the plasma membrane as a nonspecific ion channel in noncancerous cells, displayed a nuclear localization in all human melanoma cell lines analyzed. Additional characterization of these melanoma cell lines confirmed that each expressed one or more established multidrug resistance genes. Results of the present study therefore indicated that antagonism of the TRPM2 channel led to antitumor effects in human melanoma cells, including those that are potentially unresponsive to current treatments due to the expression of drug resistance genes. The unique cellular localization of TRPM2 and the specificity of the antitumor effects elicited by TRPM2 antagonism suggested that TRPM2 possesses a unique role in melanoma cells. Collectively, the targeting of TRPM2 represents a potentially novel, efficacious and readily accessible treatment option for patients with melanoma.
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Línea Celular Tumoral/metabolismo , Melanoma/genética , Melanoma/prevención & control , Canales Catiónicos TRPM/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral/fisiología , Proliferación Celular/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológicoRESUMEN
Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug-protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC50 1.25 ± 0.26 µM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 µM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.
Asunto(s)
Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Descubrimiento de Drogas/métodos , Femenino , Ratones , Ratones Endogámicos C57BL , Relación Estructura-ActividadRESUMEN
The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+. The possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV.
Asunto(s)
Antioxidantes/farmacología , Cimenos/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPV/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de OxígenoRESUMEN
The roles of ion channels, miRNAs and, neurotransmitters in the pathophysiology of major depressive disorder (MDD) are not yet fully elucidated. The current study aims to investigate ion channel gene expressions in the brain, the therapeutic efficacies of TRPC1, TRPM4, and CHRNA6 inhibitors, miRNAs specific to these ion channels and, neurotransmitter interactions in a chronic unpredictable mild stress (CUMS) induced MDD rat model. 48 two-month-old male albino Wistar rats were divided into Control, CUMS, Sham, CUMS+Pico145 (TRPC1 inhibitor), CUMS+ 9-Phe (TRPM4 inhibitor), and CUMS+BPiDl (CHRNA6 inhibitor) groups. Seven-week CUMS was used to induce MDD. Inhibitors were administered subacutely on the final of CUMS. Rats were subjected to behavioral tests. Gene expression levels were analyzed using qRT-PCR and neurotransmitter levels using ELISA. CUMS lead to a significant upregulation in the expression of channels in the hippocampus, and channels in the prefrontal cortex. Behavioral experiments determined the antidepressant effects as follows: Pico145 > BPiDl > 9-Phe. Compared to the Control, serotonin and noradrenaline levels remained unchanged, whereas dopamine levels increased. Acetylcholine levels decreased in CUMS and CUMS+Pico145 groups. CUMS significantly altered the expression of 6 miRNAs in the brain. BPiDl upregulated the expression of miR-6334 and Pico145 upregulated the expression of miR-135b-5p and miR-875 in the prefrontal cortex. The interactions of ion channels, miRNAs, and disruptions of neurotransmitter networks can play an important role in the pathophysiology of MDD. Moreover, as shown in this study, ion channel inhibitors have significant potential in the treatment of this disease.
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Encéfalo/efectos de los fármacos , Depresión/tratamiento farmacológico , Canales Iónicos/efectos de los fármacos , MicroARNs/efectos de los fármacos , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Estrés Psicológico/tratamiento farmacológico , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Depresión/metabolismo , Modelos Animales de Enfermedad , Masculino , MicroARNs/metabolismo , Ratas , Ratas Wistar , Estrés Psicológico/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
Fingolimod (FTY720) inhibits Ca2+-permeable, Mg2+-sensitive channels called transient receptor potential melastatin 7 (TRPM7), but its effects on Ca2+ paradox (CP) - induced myocardial damage has not been evaluated. We studied the effect of FTY720 on CP-induced myocardial damage and used other TRPM7 channel inhibitors nordihydroguaiaretic acid (NDGA) and Mg2+ to test if any effect of FTY720 was via TRPM7 inhibition. Langendorff-perfused Wistar rat hearts were treated with FTY720 or NDGA and subjected to a CP protocol consisting of Ca2+ depletion followed by Ca2+ repletion. Hearts of rats pre-treated with MgSO4 were also subjected to CP. Hemodynamic parameters were measured using an intraventricular balloon, and myocardial infarct size was quantified using triphenyltetrazolium chloride stain. TRPM7 proteins in ventricular tissue were detected using immunoblot analysis. FTY720, but not NDGA, decreased CP-induced infarct size. Both FTY720 and NDGA minimized the CP-induced elevation of left ventricular end-diastolic pressure, but only FTY720 ultimately improved ventricular developed pressure. Mg2+ pre-treatment had no effect on CP-induced infarct size, nor hemodynamic parameters during CP, nor the level of TRPM7 protein expression in ventricular tissue. Overall, FTY720 attenuated CP-induced myocardial damage, with potential therapeutic implications on Ca2+-mediated cardiotoxicity; however, the cardioprotective mechanism of FTY720 seems to be unrelated to TRPM7 channel modulation.
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Calcio/efectos adversos , Calcio/metabolismo , Cardiotónicos , Clorhidrato de Fingolimod/farmacología , Infarto del Miocardio/tratamiento farmacológico , Animales , Clorhidrato de Fingolimod/uso terapéutico , Técnicas In Vitro , Magnesio/metabolismo , Masculino , Masoprocol/farmacología , Masoprocol/uso terapéutico , Infarto del Miocardio/etiología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Ratas Wistar , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismoRESUMEN
TRPM7 is a non-selective divalent cation channel with an alpha-kinase domain. Corresponding with its broad expression, TRPM7 has a role in a wide range of cell functions, including proliferation, migration, and survival. Growing evidence shows that TRPM7 is also aberrantly expressed in various cancers, including brain cancers. Because ion channels have widespread tissue distribution and result in extensive physiological consequences when dysfunctional, these proteins can be compelling drug targets. In fact, ion channels comprise the third-largest drug target type, following enzymes and receptors. Literature has shown that suppression of TRPM7 results in inhibition of migration, invasion, and proliferation in several human brain tumours. Therefore, TRPM7 presents a potential target for therapeutic brain tumour interventions. This article reviews current literature on TRPM7 as a potential drug target in the context of brain tumours and provides an overview of various selective and non-selective modulators of the channel relevant to pharmacology, oncology, and ion channel function.
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Antineoplásicos/farmacología , Neoplasias Encefálicas , Canales Catiónicos TRPM , Neoplasias Encefálicas/tratamiento farmacológico , Humanos , Proteínas Serina-Treonina Quinasas , Canales Catiónicos TRPM/antagonistas & inhibidoresRESUMEN
Transient receptor potential melastatin-8 (TRPM8) represents an emerging target in prostate cancer, although its mechanism of action remains unclear. Here, we have characterized and investigated the effects of TRPM8 modulators in prostate cancer aggressiveness disclosing the molecular mechanism underlying their biological activity. Patch-clamp and calcium fluorometric assays were used to characterize the synthesized compounds. Androgen-stimulated prostate cancer-derived cells were challenged with the compounds and the DNA synthesis was investigated in a preliminary screening. The most effective compounds were then employed to inhibit the pro-metastatic behavior of in various PC-derived cells, at different degree of malignancy. The effect of the compounds was then assayed in prostate cancer cell-derived 3D model and the molecular targets of selected compounds were lastly identified using transcriptional and non-transcriptional reporter assays. TRPM8 antagonists inhibit the androgen-dependent prostate cancer cell proliferation, migration and invasiveness. They are highly effective in reverting the androgen-induced increase in prostate cancer cell spheroid size. The compounds also revert the proliferation of castrate-resistant prostate cancer cells, provided they express the androgen receptor. In contrast, no effects were recorded in prostate cancer cells devoid of the receptor. Selected antagonists interfere in non-genomic androgen action and abolish the androgen-induced androgen receptor/TRPM8 complex assembly as well as the increase in intracellular calcium levels in prostate cancer cells. Our results shed light in the processes controlling prostate cancer progression and make the transient receptor potential melastatin-8 as a 'druggable' target in the androgen receptor-expressing prostate cancers.