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1.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281152

RESUMO

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.


Assuntos
Anoctamina-1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Dietilestilbestrol/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Anoctamina-1/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dietilestilbestrol/metabolismo , Humanos , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Transdução de Sinais
2.
Toxicol Appl Pharmacol ; 421: 115543, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33872679

RESUMO

Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca2+ influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca2+-activated Cl- channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC50 value of ~5 µM. Short-circuit current analysis also showed that nimodipine prevented Ca2+-activated Cl- current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca2+ concentrations. In the absence of extracellular Ca2+, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl- currents. Nimodipine inhibited basolateral CCh-activated K+ channel activity with no effect on Na+/K+-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and Eact. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by Eact, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca2+ influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.


Assuntos
Anoctamina-1/antagonistas & inibidores , Bloqueadores dos Canais de Cálcio/toxicidade , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso/efeitos dos fármacos , Nimodipina/toxicidade , Vasoconstrição/efeitos dos fármacos , Animais , Anoctamina-1/metabolismo , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/metabolismo , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células HT29 , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
3.
Biomed Pharmacother ; 139: 111583, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33901875

RESUMO

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.


Assuntos
Anoctamina-1/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mucinas/metabolismo , Animais , Anoctamina-1/metabolismo , Anoctamina-1/fisiologia , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Ratos Endogâmicos F344 , Sistema Respiratório/citologia , Talaromyces
4.
Kidney Int ; 100(2): 311-320, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33836171

RESUMO

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.


Assuntos
Anoctamina-1/antagonistas & inibidores , Hipertensão , Músculo Liso Vascular , Vasoconstrição , Animais , Pressão Sanguínea/efeitos dos fármacos , Canais de Cloreto , Hipertensão/tratamento farmacológico , Contração Muscular/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR
5.
J Adv Res ; 29: 23-32, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33842002

RESUMO

Introduction: Ca2+-activated Cl- channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells. Objective: This study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis. Methods: Whole-cell patch clamp techniques were used to record Ca2+-activated Cl- currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively. Results: TMEM16A mediates the Ca2+-activated Cl- channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC50 of 0.1209 µM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased in vitro tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect. Conclusion: This study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.


Assuntos
Anoctamina-1/antagonistas & inibidores , Canais de Cloreto/metabolismo , Colesterol/farmacologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Patológica/metabolismo , Anoctamina-1/metabolismo , Aorta/metabolismo , Barreira Hematoencefálica/metabolismo , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Hipertensão/metabolismo , Técnicas de Patch-Clamp
6.
Eur J Pharmacol ; 895: 173881, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33476655

RESUMO

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.


Assuntos
Anoctamina-1/metabolismo , Proliferação de Células , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Neoplasias da Língua/metabolismo , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/genética , Antineoplásicos/farmacologia , Apoptose , Bestrofinas/genética , Bestrofinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Ciclopentanos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Indanos/farmacologia , Ativação do Canal Iônico , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Ligação Proteica , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/secundário , Neoplasias da Língua/tratamento farmacológico , Neoplasias da Língua/genética , Neoplasias da Língua/patologia
7.
Arch Biochem Biophys ; 695: 108650, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33132191

RESUMO

TMEM16A is a calcium-activated chloride channel that is associate with several diseases, including pulmonary diseases, hypertension, diarrhea and cancer. The CaCCinh-A01 (A01) is widely recognized as an efficient blocker of TMEM16A and has been used as a tool drug to inhibit TMEM16A currents in the laboratory. A01 also has excellent pharmacokinetic properties and can be developed as a drug to target TMEM16A. However, the molecular mechanism how A01 inhibits TMEM16A is still elusive, which slows down its drug development process. Here, calculations identified that the binding pocket of A01 was located above the pore, and it was also discovered that the binding of A01 to TMEM16A not only blocked the pore but also led to its collapse. The interaction model analysis predicted that R515/K603/E623 were crucial residues for the binding between TMEM16A and A01, and the site-directed mutagenesis studies confirmed the above results. The binding mode and quantum chemical calculations showed that the carboxyl and the amide oxygen atom of A01 were the key interaction sites between TMEM16A and A01. Therefore, our study proposed the inhibitory mechanism of TMEM16A current by A01 and revealed how A01 inhibits TMEM16A at the molecular level. These findings will shed light on both the development of A01 as a potential drug for TMEM16A dysfunction-related disorders and drug screening targeting the pocket.


Assuntos
Anoctamina-1 , Simulação de Acoplamento Molecular , Proteínas de Neoplasias , Tiofenos/química , Substituição de Aminoácidos , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/química , Anoctamina-1/genética , Anoctamina-1/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
8.
Molecules ; 25(21)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172169

RESUMO

Pyrimidine is a privileged scaffold in many synthetic compounds exhibiting diverse pharmacological activities, and is used for therapeutic applications in a broad spectrum of human diseases. In this study, we prepared a small set of pyrimidine libraries based on the structure of two hit compounds that were identified through the screening of an in-house library in order to identify an inhibitor of anoctamin 1 (ANO1). ANO1 is amplified in various types of human malignant tumors, such as head and neck, parathyroid, and gastrointestinal stromal tumors, as well as in breast, lung, and prostate cancers. After initial screening and further structure optimization, we identified Aa3 as a dose-dependent ANO1 blocker. This compound exhibited more potent anti-cancer activity in the NCI-H460 cell line, expressing high levels of ANO1 compared with that in A549 cells that express low levels of ANO1. Our results open a new direction for the development of small-molecule ANO1 blockers composed of a pyrimidine scaffold and a nitrogen-containing heterocyclic moiety, with drug-like properties.


Assuntos
Anoctamina-1/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Pirimidinas/química , Animais , Anoctamina-1/metabolismo , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Proteínas de Neoplasias/metabolismo , Pirimidinas/farmacologia , Ratos
9.
Fitoterapia ; 147: 104736, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33010370

RESUMO

Calcium-activated chloride channels (CaCCs) as a kind of widely expressed ion channels play crucial roles in a variety of physiological regulation. TMEM16A has been identified as the molecular basis of CaCCs in numerous cell types and is considered a new drug target for many diseases. Regulating the function of TMEM16A through small molecule modulators has become a new strategy to improve respiratory and digestive dysfunction and even tumor therapy. Herein, we obtained 5 sesquiterpenoids, named curzerenone, curdione, furanodienone, curcumol and germacrone with TMEM16A inhibition and revealed their mechanism of action by fluorescent and electrophysiological assays. Cell-based YFP fluorescence data demonstrated that 5 compounds inhibited TMEM16A-mediated I- influx in a dose-dependent manner. To explore the mechanism of 5 compounds on CaCCs, FRT cells with high expression of TMEM16A, HBE, HT-29 and T84 cells and mouse colons were used in short-circuit current assay. Our results showed that 5 compounds inhibited the Ca2+-activated Cl- currents generated by the Eact, ATP and UTP stimulation, and this inhibitory effect was related not only to the direct inhibition of channel opening, but also the inhibition of intracellular Ca2+ concentration and K+ channel activity. In addition to CaCCs, these 5 compounds also had definite inhibitory activities against cystic fibrosis transmembrane regulator (CFTR) at the cellular level. In summary, these compounds have the potential to regulate the activites of TMEM16A/CaCCs and CFTR channels in vitro, providing a new class of lead compounds for the development of drugs for diseases related to chloride channel dysfunction.


Assuntos
Agonistas dos Canais de Cloreto/farmacologia , Canais de Cloreto/metabolismo , Sesquiterpenos/farmacologia , Animais , Anoctamina-1/antagonistas & inibidores , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Furanos , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/antagonistas & inibidores , Ratos , Sesquiterpenos de Germacrano
10.
Eur J Med Chem ; 208: 112688, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32906067

RESUMO

Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with various physiological functions including cancer progression and metastasis/invasion. ANO1 has been considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly migration/invasion of GBM cells than reference compounds. We, for the first time, found that the combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and potent ANO1 inhibitors aiming at GBM treatment.


Assuntos
Anoctamina-1/antagonistas & inibidores , Antineoplásicos/farmacologia , Glioblastoma/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Temozolomida/farmacologia , Tiofenos/síntese química
11.
Int J Mol Sci ; 21(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899792

RESUMO

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3-6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 µM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.


Assuntos
Anoctamina-1/antagonistas & inibidores , Mallotus (Planta)/metabolismo , Extratos Vegetais/farmacologia , Animais , Anoctamina-1/metabolismo , Anoctamina-1/fisiologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Canais de Cloreto/metabolismo , Humanos , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Células PC-3 , Ratos
12.
Bioorg Chem ; 102: 104058, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32683177

RESUMO

Truxinic acid sucrose diesters analogs possess interesting chemical structure by the presence of cyclobutane-ring and macrocyclic sucrose diesters moieties which are rarely found from natural sources. This paper describes the isolation and structural elucidation of four new sucrose diesters of substituted truxinic acids, trigohonbanosides A-D (1-4), from the leaves of Trigonostemon honbaensis. Their chemical structures were elucidated by HR-ESI-MS, NMR, and CD spectroscopic methods. At a concentration of 30 µM, compounds 1-4 moderately inhibited ANO-1 activity with inhibitory percentages of 27.7 ± 1.10%, 35.6 ± 0.92%, 43.7 ± 1.61%, and 40.8 ± 1.25%, respectively.


Assuntos
Anoctamina-1/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Fenantrenos/química , Folhas de Planta/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
13.
Biochem Pharmacol ; 178: 114062, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32492446

RESUMO

To overcome the adverse effects of conventional chemotherapy for cancers, various nanoparticles based drug delivery systems have been developed. However, nanoparticles delivering drugs directly to kill tumor cells still faced with challenges, because tumors possessed adopt complex mechanism to resist damages, which compromised the therapeutic efficacy. TMEM16A/CaCCs (Calcium activates chloride channels) has been identified to be overexpressed in lung adenocarcinoma which can serve as a novel tumor specific drug target in our previous work. Here, we developed a novel dual-targeted antitumor strategy via designing a novel nano-assembled, pH-sensitive drug-delivery system loading with specific inhibitors of TMEM16A against lung adenocarcinoma. For validation, we assayed the novel dual-targeting therapy on xenograft mouse model which exhibited significant antitumor activity and not affect mouse body weight. The dual targeting therapy accomplished in this study will shed light on the development of advanced antitumor strategy.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Pulmonares/metabolismo , Nanopartículas/administração & dosagem , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus
14.
Bioorg Chem ; 101: 104000, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32592976

RESUMO

Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly expressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 µM. 3n showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.


Assuntos
Anoctamina-1/antagonistas & inibidores , Benzopiranos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Anoctamina-1/metabolismo , Apoptose/efeitos dos fármacos , Benzopiranos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Proteínas de Neoplasias/metabolismo
15.
Mol Pharmacol ; 98(1): 61-71, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358165

RESUMO

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.


Assuntos
Anoctamina-1/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ácido Niflúmico/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
16.
Int J Mol Sci ; 21(7)2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32235608

RESUMO

The concept that increasing airway hydration leads to improvements in mucus clearance and lung function in cystic fibrosis has been clinically validated with osmotic agents such as hypertonic saline and more convincingly with cystic fibrosis transmembrane conductance regulator (CFTR) repair therapies. Although rapidly becoming the standard of care in cystic fibrosis (CF), current CFTR modulators do not treat all patients nor do they restore the rate of decline in lung function to normal levels. As such, novel approaches are still required to ensure all with CF have effective therapies. Although CFTR plays a fundamental role in the regulation of fluid secretion across the airway mucosa, there are other ion channels and transporters that represent viable targets for future therapeutics. In this review article we will summarise the current progress with CFTR-independent approaches to restoring mucosal hydration, including epithelial sodium channel (ENaC) blockade and modulators of SLC26A9. A particular emphasis is given to modulation of the airway epithelial calcium-activated chloride channel (CaCC), TMEM16A, as there is controversy regarding whether it should be positively or negatively modulated. This is discussed in light of a recent report describing for the first time bona fide TMEM16A potentiators and their positive effects upon epithelial fluid secretion and mucus clearance.


Assuntos
Anoctamina-1/metabolismo , Fibrose Cística/metabolismo , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Animais , Ânions/metabolismo , Anoctamina-1/antagonistas & inibidores , Antiporters/metabolismo , Fibrose Cística/patologia , Descoberta de Drogas , Canais Epiteliais de Sódio/metabolismo , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Mucosa Respiratória/patologia , Transportadores de Sulfato/metabolismo
17.
Sci Rep ; 10(1): 6644, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313203

RESUMO

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.


Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Cloretos/metabolismo , Proteínas Recombinantes de Fusão/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/genética , Antracenos/farmacologia , Cátions Bivalentes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Camundongos , Mutação , Técnicas de Patch-Clamp , Plasmídeos/química , Plasmídeos/metabolismo , Prótons , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Taninos/farmacologia , Transfecção
18.
Assay Drug Dev Technol ; 18(3): 134-147, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32319819

RESUMO

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC50s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents, but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC50s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.


Assuntos
Automação , Benzobromarona/análise , Desenvolvimento de Medicamentos , Ensaios de Triagem em Larga Escala , Ácido Niflúmico/análise , Bibliotecas de Moléculas Pequenas/análise , Anoctamina-1/antagonistas & inibidores , Benzobromarona/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fluorescência , Células HEK293 , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Ácido Niflúmico/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Software
19.
Pharmacol Res ; 156: 104763, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32201246

RESUMO

Transmembrane member 16A (TMEM16A) encoded Ca2+-activated Cl- channels were found to be involved in tumorigenesis. Previous studies suggest the effect of TMEM16A gene amplification on tumorigenic proliferation is exerted through its channel function. TMEM16A-specific and potent small molecule inhibitors have been proposed to potentially be useful for the treatment of cancer. Thus, we screened six analogues of avermectin for their inhibitory activities on TMEM16A mediated currents. A whole-cell patch technique was used to record the currents. The IC50 and Emax values for TMEM16A inhibition of five tested avermectins (avermectin B1, ivermectin, doramectin, selamectin, and moxidectin) were 0.15-1.32 µM and 65-87 %, respectively. In addition, these avermectins significantly inhibited endogenous TMEM16A mediated currents and thus, the proliferation, migration, inducing apoptosis of LA795 cancer cells. Eprinomectin (4"-(acetylamino)-4"-deoxy-avermectin B1) and two other important macrolides (erythromycin and azithromycin), which have minimal or no TMEM16A inhibitory effects, were used as negative control drugs. These drugs were found to have limited effects on the proliferation, migration, and apoptosis of LA795 cells. Finally, avermectin B1 and ivermectin dramatically inhibited the growth of xenograft tumors in mice. These data demonstrate that avermectins are novel TMEM16A inhibitors and are potentially useful in specific cancer therapies. These findings also provide a new opportunity to develop TMEM16A modulators.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Anoctamina-1/antagonistas & inibidores , Antineoplásicos/farmacologia , Ivermectina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Apoptose/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ivermectina/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos
20.
Pharmacol Res ; 155: 104721, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32097750

RESUMO

TMEM16A plays critical roles in physiological process and may serve as drug targets for diverse diseases. Recently, TMEM16A has started to be regarded as potential primary lung adenocarcinoma targets. Here, we identified that arctigenin, a natural compound, is a novel TMEM16A inhibitor, and it can suppress lung adenocarcinoma growth through inhibiting TMEM16A both in vitro and in vivo. Our data also showed that the IC50 of actigenin to TMEM16A whole-cell current was 19.29 ± 4.69 µM, and the putative binding sites of arctigenin in TMEM16A were R515 and R535. Arctigenin concentration-dependently inhibited the proliferation and migration of LA795, however, the inhibition effect can be abolished by knockdown of the endogenous TMEM16A with shRNA. Further, we injected arctigenin on xenograft mouse model which exhibited significant antitumor activity with no adverse effect. At last, western blotting results showed the mechanism of arctigenin inhibiting lung adenocarcinoma was through inhibiting MAPK pathway. In summary, TMEM16A is a novel drug target for lung adenocarcinoma treatment. Arctigenin can be used as a lead compound for the development of lung adenocarcinoma therapy drugs.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Anoctamina-1/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Furanos/uso terapêutico , Lignanas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Anoctamina-1/fisiologia , Antineoplásicos/farmacologia , Linhagem Celular , Furanos/farmacologia , Humanos , Lignanas/farmacologia , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C
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