Cystic Fibrosis: A New Target for 4-Imidazo[2,1-<i>b</i>]thiazole-1,4-dihydropyridines

The pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel has attracted significant interest in recent years with the aim to search for rational new therapies for diseases caused by CFTR malfunction. Mutations that abolish the function of CFTR cause the life-threatening genetic disease cystic fibrosis (CF). The most common cause of CF is the deletion of phenylalanine 508 (ΔF508) in the CFTR chloride channel. Felodipine, nifedipine, and other antihypertensive 1,4-dihydropyridines (1,4-DHPs) that block L-type Ca²⁺ channels are also effective potentiators of CFTR gating, able to correct the defective activity of ΔF508 and other CFTR mutants (Mol. Pharmacol. 2005, 68, 1736). For this purpose, we evaluated the ability of the previously and newly synthesized 4-imidazo[2,1-<i>b</i>]thiazoles-1,4-dihydropyridines without vascular activity and inotropic and/or chronotropic cardiac effects (J. Med. Chem. 2008, 51, 1592) to enhance the activity of ΔF508-CFTR. Our studies indicate compounds <b>17</b>, <b>18</b>, <b>20</b>, <b>21</b>, <b>38</b>, and <b>39</b> as 1,4-DHPs with an interesting profile of activity

Expression of TMEM16A in various nasal glands of TMEM16A^-/- and TMEM16A^+/+ littermate mice.

<p>Expression of TMEM16A and aquaporin 5 in the Bowman’s gland (BG), nasal septal gland (NSG) and lateral nasal gland (LNG) of TMEM16A^-/- and TMEM16A^+/+ littermate mice. <b>A, D:</b> aquaporin 5 immunopositive signals were seen in Bowman’s glands in P4 mice. Aquaporin 5 expression in glands and ducts is clearly visible. TMEM16A immunoreactivity (goat anti-TMEM16A) was not present in Bowman’s glands of TMEM16A^-/- nor of TMEM16A^+/+ littermate mice (<b>A, D)</b>. However, aquaporin 5 and TMEM16A were co-expressed in nasal septal glands (<b>B</b>) and lateral nasal glands (<b>C</b>) of TMEM16A^+/+ mice. No immunoreactivity to TMEM16A was detectable in TMEM16A^-/- mice (<b>E, F</b>). Glands marked by aquaporin 5 were similar in both types of mice. Images are averages of z-stacks of thickness of ~2.0 μm for <b>A, D</b>, or ~1 μm for <b>B, C, E, F</b>. Cell nuclei were stained by DAPI. Scale bars = 10 μm.</p

Expression of TMEM16A, cytokeratin 8 and ezrin in the olfactory epithelium of TMEM16A^-/- and TMEM16A^+/+ littermate mice.

<p>Confocal images of coronal sections of the olfactory epithelium from a region near the transition zone with the respiratory epithelium at E16.5 and P4 from TMEM16A^-/- (<b>A, C, E, G</b>) or TMEM16A^+/+ (<b>B, D, F, H)</b>. No immunoreactivity to TMEM16A was detectable in TMEM16A^-/- mice. <b>A-D:</b> Supporting cells marked by cytokeratin 8 were similar in both types of mice. In TMEM16A^+/+ mice, TMEM16A (goat anti-TMEM16A) and cytokeratin 8 immunoreactivity did not overlap (<b>B, D</b>), whereas TMEM16A expression (rabbit anti-TMEM16A) partially overlapped with ezrin immunopositive signals (<b>F, H</b>). Supporting cells marked by cytokeratin 8 and microvilli marked by ezrin were similar in both types of mice. Images are averages of z-stacks of ~1.5 μm thickness. Cell nuclei were stained by DAPI. Scale bars = 5 μm.</p

Markers for supporting cells.

<p><b>A-C:</b> Cytokeratin 8 marked cells with the typical morphology of supporting cells. Double staining of cytokeratin 8 with OMP showed no co-localization. <b>D:</b> Microvilli of supporting cells stained by ezrin were not stained by cytokeratin 8. <b>E:</b> Double staining of cytokeratin 8 with sox2 shows that sox2 is a nuclear marker for supporting cells whose nuclei are located in the apical region of the epithelium. Sox2 also stains nuclei of basal cells. Coronal sections of the olfactory epithelium of wild type mice at P60 (<b>A-C</b>), or P4 (<b>D, E</b>). Images are averages of z-stacks of ~1.5 μm thickness. Cell nuclei were stained by DAPI. Scale bars: A-C, D = 5 μm; E = 10 μm.</p

Olfactory sensory neurons in the developing olfactory epithelium of TMEM16A^-/- and TMEM16A^+/+ mice.

<p>Mature olfactory sensory neurons express the olfactory marker protein (OMP). Confocal images of coronal sections of the olfactory epithelium at E14.5, E16.5, E18.5 and P4 from TMEM16A^-/- (<b>A, C, E, G</b>) or TMEM16A^+/+ (<b>B, D, F, H)</b> mice. In both mice, at E14.5 a limited number of mature neurons was present (<b>A, B</b>), but the number progressively increased from E16.5 to P4 (<b>C-H</b>). ACIII signals were seen in the cilia protruding from the dendritic knob of mature olfactory sensory neurons (<b>A-H</b>). Mature neurons expressing OMP and cilia marked by ACIII were similar in TMEM16A^-/- and TMEM16A^+/+ littermates. Images are averages of z-stacks of ~1.5 μm thickness. Cell nuclei were stained by DAPI. Scale bars = 5 μm.</p

Cell densities in the olfactory epithelium of TMEM16A^-/- and TMEM16A^+/+ littermate mice.

<p>Comparison among the average number of supporting cells and olfactory sensory neurons in the olfactory epithelium of TMEM16A^-/- and TMEM16A^+/+ littermate mice. Average number of supporting cells (<b>A, B</b>) or neuronal cells (<b>C, D</b>) was calculated by counting nuclei in 150 x 150 μm² areas from several regions of the olfactory epithelium. <b>B, D:</b> Average number of cells calculated near the transition zone with the respiratory epithelium (TR), corresponding to TMEM16A expression in TMEM16A^+/+ mice, or far from the transition zone (OE). Counting was done in three different animals for each group and presented as average ± SEM.</p

Expression of cytokeratin 5 and Ki67 in the olfactory epithelium of TMEM16A^-/- and TMEM16A^+/+ littermate mice.

<p>Confocal images of coronal sections of the olfactory epithelium from a region near the transition zone with the respiratory epithelium at P4 from TMEM16A^+/+ (<b>A-C</b>) and TMEM16A^-/- (<b>D-F)</b> mice. Cell nuclei were stained by DAPI. Scale bars = 10 μm. <b>G:</b> Comparison among the average number of horizontal (HBC) and globose (GBC) basal cells in the olfactory epithelium of TMEM16A^+/+ and TMEM16A^-/- littermate mice. Average numbers of HBCs or GBCs were calculated by counting nuclei in 150 x 150 μm² areas from several regions of the olfactory epithelium. <b>H:</b> Average number of cells calculated near the transition zone with the respiratory epithelium (TR), corresponding to TMEM16A expression in TMEM16A^+/+ mice, or far from the transition zone (OE). Counting was done in three different animals for each group and presented as average ± SEM.</p

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

Substituted 2‑Acylamino­cycloalkyl­thiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A)

Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylamino­cycloalkyl­thiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (<b>10ab</b>–<b>10bw</b>) of the original AACT compound (<b>10aa</b>). Structure–activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (<b>10bm</b>), which contains an unusual bromodifluoro­acetamide at the thiophene 2-position, had IC₅₀ of ∼30 nM, ∼3.6-fold more potent than the most potent previously reported TMEM16A inhibitor <b>4</b> (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by <b>10bm</b> was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates

Analysis of pyocyanin mechanism of action.

<p><b>(A,B)</b> Cytosolic Ca²⁺ revealed with the fluorescent Fluo-4 probe in non-CF and CF bronchial epithelial cells, with and without treatment with pyocyanin (60 μM, 24 hours). Top graphs show the time-course of Fluo-4 fluorescence before and after acute stimulation with apical UTP (100 μM). Each dot is the mean of three experiments. Bottom graphs show the values of fluorescence measured at the indicated time points. Although the response to UTP in pyocyanin-treated CF cells was larger than in untreated cells, the difference was not statistically significant. <b>(C)</b> Western blot analysis of TMEM16A protein expression. Non-CF cells were treated with pyocyanin (60 μM) or IL-4 (10 ng/ml) for 24 hours. Pyocyanin and, to a larger extent, IL-4 increased TMEM16A expression. Each panel is representative of three similar experiments.</p