Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2

Seo, Yohan; Lee, Ho K.; Park, Jin‐Hong; Jeon, Dong Kyu; Jo, Sungwoo; Jo, Min-Jae; Namkung, W.

doi:10.1371/journal.pone.0155771

Cited by 149 publications

(139 citation statements)

References 43 publications

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“…Recently, Seo et al (2016) identified a new inhibitor, Ani9 that seems to mainly inhibit TMEM16A current with negligible effects on CFTR, VRAC and TMEM16B.…”

Section: Discussionmentioning

confidence: 99%

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance

et al. 2017

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Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

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“…Recently, Seo et al (2016) identified a new inhibitor, Ani9 that seems to mainly inhibit TMEM16A current with negligible effects on CFTR, VRAC and TMEM16B.…”

Section: Discussionmentioning

confidence: 99%

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance

et al. 2017

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“…Immunoblotting was performed as described previously [18]. FRT and PC-3 cells were lysed with cell lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na 3 VO 4 , and protease inhibitor mixture).…”

Section: Methodsmentioning

confidence: 99%

“…To date, few ANO1 inhibitors have been identified, such as CaCC inh -A01 (IC 50 ~1 μM), tannic acid (IC 50 ~6 μM), T16A inh -A01 (IC 50 ~1 μM), MONNA (IC 50 ~1 μM) and idebenone (IC 50 ~9 μM) [13–17], and more recently we identified a novel small-molecule inhibitor of ANO1, Ani9 (IC 50 ~77 nM), showing high potency and selectivity for ANO1 [18]. However, the mechanisms of action and pharmacological properties of these inhibitors remain unclear, and the ANO1 inhibitors are still in the early phases of the drug discovery.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of ANO1 by luteolin and its cytotoxicity in human prostate cancer PC-3 cells

et al. 2017

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Anoctamin 1 (ANO1), a calcium-activated chloride channel, is highly amplified in prostate cancer, the most common form of cancer and leading causes of cancer death in men, and downregulation of ANO1 expression or its functional activity is known to inhibit cell proliferation, migration and invasion in prostate cancer cells. Here, we performed a cell-based screening for the identification of ANO1 inhibitors as potential anticancer therapeutic agents for prostate cancer. Screening of ~300 selected bioactive natural products revealed that luteolin is a novel potent inhibitor of ANO1. Electrophysiological studies indicated that luteolin potently inhibited ANO1 chloride channel activity in a dose-dependent manner with an IC50 value of 9.8 μM and luteolin did not alter intracellular calcium signaling in PC-3 prostate cancer cells. Luteolin inhibited cell proliferation and migration of PC-3 cells expressing high levels of ANO1 more potently than that of ANO1-deficient PC-3 cells. Notably, luteolin not only inhibited ANO1 channel activity, but also strongly decreased protein expression levels of ANO1. Our results suggest that downregulation of ANO1 by luteolin is a potential mechanism for the anticancer effect of luteolin.

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“…TMEM16A and TMEM16B are also blocked by other commonly used Cl À channel blockers such as 4,4 0 -diisothiocyano-2,2 0 -stilbenedisulfonic acid (DIDS) and niflumic acid with comparable potencies (Bradley et al, 2014;Liu et al, 2015;Pifferi et al, 2009). In contrast, a recently identified drug (Ani9) selectively inhibited TMEM16A, with no significant block of TMEM16B (Seo et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Contrasting effects of phosphatidylinositol 4,5‐bisphosphate on cloned TMEM16A and TMEM16B channels

Acheson

Rorsman

et al. 2017

British J Pharmacology

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Ca2+ -activated Cl À channels (CaCCs) are gated open by a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ), typically provoked by activation of G q -protein coupled receptors (G q PCR). G q PCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Here, we determined whether PIP 2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. EXPERIMENTAL APPROACHPatch-clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP 2 content, was used to define the mechanism of action of PIP 2 on TMEM16A and TMEM16B channels. KEY RESULTSA water-soluble PIP 2 analogue (diC8-PIP 2 ) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by~0.2-fold. The effects of diC8-PIP 2 on TMEM16A currents were especially pronounced at low [Ca 2+ ] i . In contrast, diC8-PIP 2 modulation of TMEM16B channels did not vary over a broad [Ca 2+ ] i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co-expression of TMEM16A or TMEM16B with a Danio rerio voltage-sensitive phosphatase (DrVSP), which degrades PIP 2 , led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co-expression of a phosphatidylinositol-4-phosphate 5-kinase that catalyses PIP 2 formation. CONCLUSIONS AND IMPLICATIONSPIP 2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP 2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy. AbbreviationsCaCC, calcium-activated chloride channel; DrVSP, Danio rerio voltage-sensitive phosphatase; E rev , reversal potential; G q PCR, G q -protein coupled receptors; IP 3 , inositol triphosphate; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PIPK, PIP 5-kinase type Iγ; V m , membrane potential

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Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2

Cited by 149 publications

References 43 publications

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance

Inhibition of ANO1 by luteolin and its cytotoxicity in human prostate cancer PC-3 cells

Contrasting effects of phosphatidylinositol 4,5‐bisphosphate on cloned TMEM16A and TMEM16B channels

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