MONNA, a Potent and Selective Blocker for Transmembrane Protein with Unknown Function 16/Anoctamin-1 s

Transmembrane protein with unknown function 16/anoctamin-1 (ANO1) is a protein widely expressed in mammalian tissues, and it has the properties of the classic calcium-activated chloride channel (CaCC). This protein has been implicated in numerous major physiological functions. However, the lack of effective and selective blockers has hindered a detailed study of the physiological functions of this channel. In this study, we have developed a potent and selective blocker for endogenous ANO1 in Xenopus laevis oocytes (xANO1) using a drug screening method we previously established (Oh et al., 2008). We have synthesized a number of anthranilic acid derivatives and have determined the correlation between biological activity and the nature and position of substituents in these derived compounds. A structure-activity relationship revealed novel chemical classes of xANO1 blockers. The derivatives contain a 2NO2 group on position 5 of a naphthyl group-substituted anthranilic acid, and they fully blocked xANO1 chloride currents with an IC50 , 10 mM. The most potent blocker, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), had an IC50 of 0.08 mM for xANO1. Selectivity tests revealed that other chloride channels such as bestrophin-1, chloride channel protein 2, and cystic fibrosis transmembrane conductance regulator were not appreciably blocked by 10∼30 mM MONNA. The potent and selective blockers for ANO1 identified here should permit pharmacological dissection of ANO1/CaCC function and serve as potential candidates for drug therapy of related diseases such as hypertension, cystic fibrosis, bronchitis, asthma, and hyperalgesia. Introduction Calcium-activated chloride channels (CaCCs) have been found in a wide range of organisms and tissues. They have fundamental and wide-ranging physiological roles in functions such as epithelial secretion, sensory transduction and adaptation, nociception, regulation of smooth muscle cell contraction and vascular tone, and control of neuronal and cardiac excitability (Hartzell et al., 2005). Therefore, CaCCs are potential drug targets for diarrhea, asthma, cystic fibrosis, and hypertension (Verkman and Galietta, 2009). CaCCs were first described in the early 1980s, in Xenopus laevis oocytes, where they generate the fertilization potential that generates a fast electrical inhibition to prevent polyspermy (Miledi, 1982; Barish, 1983). However, the molecular identity of these channels remained elusive until the transmembrane protein with unknown function 16/anoctamin-1 (ANO1) was identified as a CaCC in 2008 (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008). Since then, ANO1 has rapidly garnered attention, and a number of reports have described the properties and physiological roles of this protein (Huang et al., 2009). Notably, a recent study revealed that ANO1 acts as a heat sensor to detect nociceptive thermal stimuli in sensory neurons and possibly mediate nociception (Cho et al., 2012). The anoctamin family consists of 10 different protein subtypes. Among them, ANO1 has been the most extensively studied (Huang et al., 2009). ANO1 has very similar properties This work was supported by the Korea Institute of Science and Technology Institutional Program [Grant 2E24182]; the World Class Institute Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology of Korea [Grant WCI 2009-003]; the National Institutes of Health National Institute of General Medical Sciences [Grant R01 GM60448]; the National Institutes of Health National Eye Institute [Grant R01 EY11482]; and a pilot grant from the Emory Center for Cystic Fibrosis Research of Children’s Healthcare of Atlanta. S.-J.O. and S.J.H. contributed equally to this work. C.J.L. and E.J.R. contributed equally to this work. dx.doi.org/10.1124/mol.113.087502. s This article has supplemental material available at molpharm.aspetjournals. org. ABBREVIATIONS: ANO1, anoctamin-1; CaCC, calcium-activated chloride channel; CFTR, cystic fibrosis transmembrane conductance regulator; CLC2, chloride channel protein 2; cRNA, complementary RNA; DCM, dichloromethane; DIDS, 4,49-diisothiocyanatostilbene-2,29-disulfonic acid; DMSO, dimethylsulfoxide; DPC, diphenylamine-2-carboxylic acid; hANO1, human ANO1; HEK293, human embryonic kidney 293; HR-ESI-MS, high-resolution electrospray ionization mass spectrometry; mBest1, mouse bestrophin-1; mCLC2, mouse chloride channel protein 2; MONNA, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid; NFA, niflumic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; 4TFMPA, N-(4-trifluoromethylphenyl)anthranilic acid; 4TFP4NA, N-(4-trifluorophenyl)-4-nitroanthranilic acid; xANO1, Xenopus laevis ANO1. 726 http://molpharm.aspetjournals.org/content/suppl/2013/08/30/mol.113.087502.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from to endogenous CaCCs that have been observed in many different cells, tissues, and organisms. These properties include low-field-strength anion selectivity, Ca sensitivity, voltage dependence, and pharmacological profile. Despite the physiological importance of ANO1, the lack of a potent and selective blocker for this protein has impeded a better understanding of the channel at themolecular, biophysical, and pharmacological level. Currently available blockers for CaCCs, including ANO1, include niflumic acid (NFA), 4,49-diisothiocyanatostilbene-2,29disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and mefloquine, all of which must be applied at high concentrations to completely block ANO1. The halfmaximal concentrations for inhibition (IC50) of NFA, DIDS, and NPPB are reported to be 37.3, 10.7, and 32.3 mM, respectively (Oh et al., 2008). Furthermore, these blockers are known to cause undesirable side effects and block other channels. For example, NFA and DIDS also block the volumeregulated anion channel in some cell types (Xu et al., 1997; Greenwood and Large, 1998), whereas all three, NFA, DIDS, and NPPB, have a blocking effect on the K channel current (Wang et al., 1997; Greenwood and Leblanc, 2007). In addition, NFA, DIDS, and NPPB cause an elevation of intracellular Ca concentration in several cell types, which can elicit other cellular responses (Reinsprecht et al., 1995; Shaw et al., 1995; Schultheiss et al., 2000). More recently, several ANO1 inhibitors, such as dichlorophen, benzbromarone, and hexachlorophene, have been identified with high-throughput screening methods, having IC50 values of 5.49, 9.97, and 10.0 mM, respectively (Huang et al., 2012). These compounds showed somewhat improved potency over conventional blockers but still fell short of submicromolar potency. In another high-throughput screening study, an aminophenylthiazole (T16Ainh-A01; Namkung et al., 2011) was found to have an IC50 of around 1 mM, but no selectivity information was available. Thus, because of these issues related to low potency and selectivity, there is a very pressing need for improved ANO1 blockers. Many attempts have been made to uncover chemical compounds that block the endogenous CaCC in X. laevis oocytes. In a previous study, we established an optimized protocol for largescale drug screening using a two-electrode voltage-clamp recording system to search for better blockers for endogenous CaCCs inX. laevis oocytes (Oh et al., 2008), which were revealed to be dominantly mediated by endogenous ANO1 in X. laevis oocytes (xANO1) (Yang et al., 2008). In our previous study, we found a structural similarity between commercially available CaCC blockers and N-(4-trifluoromethylphenyl)anthranilic acid (4TFMPA), a novel potent blocker for xANO1, with an IC50 of 6.0 mM, which was synthesized based on structure-activity relationship analysis. In the present study, using the same screening method, we further examined the blocking effect of synthesized compounds using an in-depth structure-activity relationship analysis to discover new ANO1 blockers that have an IC50 less than 1 mM. Materials and Methods Preparation of Oocytes As described previously (Oh et al., 2008), mature stage V and VI oocytes were harvested from adult X. laevis females (Xenopus-I, Inc., Dexter, MI) that weremaintained in an automatedmaintenance system (Xenopus System; Aquatic Habitats, Apopoka, FL). All experimental procedures described later were performed in accordancewith theKorea Institute of Science and Technology (Seoul, Korea) institutional guidelines for humaneanimal handling.Animalswere anesthetized by cooling with ice. Surgically removed ovarian follicles were treated with 2 mg/ml collagenase type IA at room temperature for 90 minutes in Ca-free Barth’s solution containing 89mMNaCl, 1.0mMKCl, 2.4mMNaHCO3, 0.82mMMgSO4, and 10mMHEPES (pH 7.4). Oocytes were extensively rinsedwith normal Barth’s solution containing 88mMNaCl, 1.0mMKCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 1.41 mM CaCl2, and 5 mM HEPES (pH 7.4); placed in a culture of Barth’s solution containing 88mMNaCl, 1.0mMKCl, 2.4mMNaHCO3, 0.82mMMgSO4, 0.33 mM Ca(NO3)2, 0.91 mM CaCl2, 10 mM HEPES, 10 mg/ml streptomycin, and 10 mg/ml penicillin (pH 7.4); andmaintained at 18°C. Oocytes were used ∼1–4 days after isolation. Synthesis Some anthranilic acid derivatives (compound 4f in Figs. 1 and 2F, and six compounds that are not designated in Fig. 2F) had been previously synthesized and reported (Oh et al., 2008). The other anthranilic acid derivatives were synthesized as described in Fig. 1. Step A. II, IV, and VIII: Thionyl chloride was added dropwise to a solution of benzoic acid derivative (I, III, and VII) in anhydrous methyl alcohol at 0°C. After this addition, the mixture was stirred at reflux for 8∼12 hours. The reaction mixture was basified with 10% sodium bicarbonate, and ethyl acetate was added. The organic layer was dried over anhydrous MgSO4, filtered, and the solvent was evaporated to give the products II, IV, and VIII. Step B. IX: Triflic anhydride was added dropwise to a solution of 4-trifluoromethyl-2-hydroxybenzoicacid methyl ester (VIII) in pyridine and dichloromethane (DCM). This was stirred at room temperature for 6 hours and acidified by the addition of 1 M HCl(aq). The mixture was extracted with DCM and washed with brine. The organic layer was dried over anhydrous MgSO4, filtered, and the solvent was evaporated. IX was purified by column chromatography. Amine derivatives (compound 2a–q, Supplemental Methods, compound synthesis 2) were prepared by this method, except for the commercially available amines. Step C. V and X: Buchwald-Hartwig cross-coupling of amine (compound 2a–q, IV) and bromo [1b, II; or triflate (1a, IX)] gave anthanilic acid methyl ester (V, X). Tris (dibenzylideneacetone) dipalladium (0)-chloroform adduct (0.05 eq) and (6)-2,29-bis(diphenylphosphino)-1,19-binaphthalene (0.10 eq) in anhydrous toluene (3 ml) were stirred at room temperature for 30 minutes. Then, 2-bromo-N-nitrobenzoic acid methyl ester, Cs2CO3 (1.4 eq), and aniline derivative (1.2 eq) were added and stirred at 110–130°C for 5–10 hours. The reaction mixture was filtered through Celite (Yakuri Pure Chemicals Co., LTD, Kyoto, Japan) and concentrated in vacuo. Anthanilic acid methyl ester (V, X) was purified by column chromatography. Step D. VI: Iodination of bromo (V) gave VI. V, NaI (2 eq), CuI (0.05 eq), and trans-N,N9-dimethylcyclohexane-1,2-diamine (0.1 eq) in 1,4-dioxane were stirred at 110°C for 5 days. The reaction mixture was filtered through Celite and concentrated in vacuo. VI was purified by column chromatography. Step E. 3, 4, 5, 6: Hydrolysis of benzoic acid methyl ester derivative (V, VI, and X) was accomplished by refluxing tetrahydrofuran/ methanol (MeOH)/H2O (5:3:2) solution in the presence of lithium hydroxide. The reaction mixture was acidified by the addition of 1 M HCl(aq) then extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous MgSO4, filtered, and the solvent was evaporated to give anthranilic acid derivatives (3, 4, 5, and 6; Supplemental Methods, compound synthesis 1). Representative Examples of Active Compounds N-((3-Methoxy)naphthyl)-5-Nitroanthranilic Acid (5p). The final step yields 94.6% (orange powder). Proton nuclear magnetic resonance spectroscopy [H-NMR, 400MHz, dimethylsulfoxide (DMSO)– d6] d 10.64 (bs, 1H), 8.97 (d, J5 2.7 Hz, 1H), 8.06 (dd, J5 9.4, 2.6 Hz, Potent and Selective Blockers for ANO1 727 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from