Synthetic Strategies for Improving Solubility: Optimization of Novel Pyrazolo[1,5-a]pyrimidine CFTR Activator That Ameliorates Dry Eye Disease.

Dry eye disease (DED) is one of the most prevalent ocular diseases but has limited treatment options. Cystic fibrosis transmembrane conductance regulator (CFTR), a major chloride channel that stimulates fluid secretion in the ocular surface, may pave the way for new therapeutic strategies for DED. Herein, we report the optimization of Cact-3, a potent CFTR activator with poor solubility, to 16d, a potent CFTR activator with suitable solubility for eye drop formulation. Notably, 16d was well distributed in target tissues including cornea and conjunctiva with minimal systemic exposure in rabbit. Topical ocular instillation of 16d significantly enhanced tear secretion and improved corneal erosion in a mouse model of DED. In addition, 16d significantly reduced mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-17, and TNF-α and MMP2 in cornea and conjunctiva of DED mice.

Novel CFTR Activator Cact-3 Ameliorates Ocular Surface Dysfunctions in Scopolamine-Induced Dry Eye Mice.

Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed on the ocular epithelium and plays a pivotal role in the fluid secretion driven by chloride transport. Dry eye disease is one of the most common diseases with limited therapeutic options. In this study, a high-throughput screening was performed to identify novel CFTR activators capable of inducing chloride secretion on the ocular surface. The screening of 50,000 small molecules revealed three novel CFTR activators. Among them, the most potent CFTR activator, Cact-3 (7-(3,4-dimethoxyphenyl)-N-(4-ethoxyphenyl)pyrazolo [1,5-α]pyrimidine-2-carboxamide), produced large and sustained Cl- currents in WT-CFTR-expressing FRT cells with no alterations of ANO1 and hERG channel activity. The application of Cact-3 strongly activated CFTR in the ocular epithelia of mice and it also significantly increased CFTR-mediated Cl- transport in a primary cultured human conjunctival epithelium. Cact-3 strongly stimulated tear secretion in normal mice. In addition, Cact-3 significantly reduced ocular surface damage and the expression of proinflammatory factors, including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in an experimental mouse model of dry eye disease. These results suggest that Cact-3, a novel CFTR activator, may be a potential development candidate for the treatment of dry eye disease.

Celastrol suppresses the growth of vestibular schwannoma in mice by promoting the degradation of ß-catenin.

Vestibular schwannoma (VS), one of characteristic tumors of neurofibromatosis type 2 (NF2), is an intracranial tumor that arises from Schwann cells of the vestibular nerve. VS results in hearing loss, tinnitus, dizziness, and even death, but there are currently no FDA-approved drugs for treatment. In this study, we established a high-throughput screening to discover effective compounds that could inhibit the viability of VS cells. Among 1019 natural products from the Korea Chemical Bank screened, we found that celastrol, a pentacyclic triterpene derived from a Tripterygium Wilfordi plant, exerted potent inhibitory effect on the viability of VS cells with an IC50 value of 0.5 µM. Celastrol (0.5, 1 µM) dose-dependently inhibited the proliferation of primary VS cells derived from VS patients. Celastrol also inhibited the growth, and induced apoptosis of two other VS cell lines (HEI-193 and SC4). Aberrant activation of Wnt/ß-catenin signaling has been found in VS isolated from clinically defined NF2 patients. In HEI-193 and SC4 cells, we demonstrated that celastrol (0.1, 0.5 µM) dose-dependently inhibited TOPFlash reporter activity and protein expression of ß-catenin, but not mRNA level of ß-catenin. Furthermore, celastrol accelerated the degradation of ß-catenin by promoting the formation of the ß-catenin destruction complex. In nude mice bearing VS cell line SC4 allografts, administration of celastrol (1.25 mg · kg-1 · d-1, i.p. once every 3 days for 2 weeks) significantly suppressed the tumor growth without showing toxicity. Collectively, this study demonstrates that celastrol can inhibit Wnt/ß-catenin signaling by promoting the degradation of ß-catenin, consequently inhibiting the growth of VS.

Isorhamnetin Ameliorates Dry Eye Disease via CFTR Activation in Mice.

Dry eye disease is one of the most common diseases, with increasing prevalence in many countries, but treatment options are limited. Cystic fibrosis transmembrane conductance regulator (CFTR) is a major ion channel that facilitates fluid secretion in ocular surface epithelium and is a potential target of therapeutic agent for the treatment of dry eye disease. In this study, we performed a cell-based, high-throughput screening for the identification of novel natural products that activate CFTR and restore the aqueous deficiency in dry eye. Screening of 1000 natural products revealed isorhamnetin, a flavonol aglycone, as a novel CFTR activator. Electrophysiological studies showed that isorhamnetin significantly increased CFTR chloride current, both wild type and ∆F508-CFTR. Isorhamnetin did not alter intracellular cAMP levels and the activity of other ion channels, including ANO1, ENaC, and hERG. Notably, application of isorhamnetin on mouse ocular surface induced CFTR activation and increased tear volume. In addition, isorhamnetin significantly reduced ocular surface damage and expression of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in an experimental mouse model of dry eye. These data suggest that isorhamnetin may be used to treat dry eye disease.

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

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.

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

Anoctamin1 (ANO1)/transmembrane protein 16A (TMEM16A), a calcium-activated chloride channel (CaCC), is involved in many physiological functions such as fluid secretion, smooth muscle contraction, nociception and cancer progression. To date, only a few ANO1 inhibitors have been described, and these have low potency and selectivity for ANO1. Here, we performed a high-throughput screening to identify highly potent and selective small molecule inhibitors of ANO1. Three novel ANO1 inhibitors were discovered from screening of 54,400 synthetic small molecules, and they were found to fully block ANO1 channel activity with an IC50 < 3 µM. Electrophysiological analysis revealed that the most potent inhibitor, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9), completely inhibited ANO1 chloride current with submicromolar potency. Notably, unlike previous small-molecule ANO1 inhibitors identified to date, Ani9 displayed high selectivity for ANO1 as compared to ANO2, which shares a high amino acid homology to ANO1. In addition, Ani9 did not affect the intracellular calcium signaling and CFTR chloride channel activity. Our results suggest that Ani9 may be a useful pharmacological tool for studying ANO1 and a potential development candidate for drug therapy of cancer, hypertension, pain, diarrhea and asthma.

Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines.

The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC50 value <10 µM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC50 values of 0.88 ± 0.11 and 4.45 ± 0.31 µM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.

Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines.

The expression levels of anoctamin 1 (ANO1, TMEM16A), a calcium-activated chloride channel (CaCC), are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy.

Imaging features of idiopathic intracranial hypertension, including a new finding: widening of the foramen ovale.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a clinical disorder of unknown etiology manifesting with increased intracranial pressure in the absence of hydrocephalus, an underlying mass lesion, and demonstrating normal cerebrospinal fluid composition. IIH may exhibit several non-specific imaging findings including: an empty sella, posterior globe flattening, tortuosity of the optic nerve, and optic nerve sheath distention. PURPOSE: To introduce widening of the foramen ovale as a new imaging marker for IIH. MATERIAL AND METHODS: IIH is a syndrome which may exhibit several previously described non-specific imaging findings including: an empty sella, posterior globe flattening, tortuosity of the optic nerve, and optic nerve sheath distention. We hypothesize that chronically elevated cerebrospinal fluid pressure can lead to osseous erosions and we propose widening of the foramen ovale as a new imaging marker for IIH. RESULTS: Average foramen ovale sizes were increased in patients with IIH compared to controls (30.03 ± 7.00 mm(2) vs. 24.21 ± 5.97 mm(2), P < 0.001). For a cut-off value of 30 mm(2), the sensitivity of FO area to detect IIH was 50%, with 81% specificity. Classic findings were significantly more common in patients with IIH compared to controls including: empty sella (65.9% vs. 0%), posterior globe flattening (65.9% vs. 4.5%), vertical tortuosity of the optic nerve (54.5% vs. 9.1%), and optic nerve sheath distention (52.3% vs. 11.4%, all P values < 0.001). CONCLUSION: Our study confirms the association of several classic imaging findings with IIH and supports widening of the foramen ovale as an additional imaging marker which may be incorporated into the evaluation of patients suspected to have this condition.

Magnetic resonance imaging of third cranial nerve palsy and trigeminal sensory loss caused by herpes zoster.

A 44-year-old man with right-sided herpes zoster ophthalmicus (HZO) developed ipsilateral third and sixth cranial nerve palsies and first-division trigeminal (fifth cranial nerve) sensory loss. MRI revealed contrast enhancement of the cisternal and cavernous portions of the third cranial nerve and high signal on a FLAIR sequence within the ipsilateral medulla at the presumed location of the trigeminal nucleus and tract. To our knowledge, this is the first report of the combination of these imaging findings in HZO.

Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model.

P110delta phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of certain inflammatory cells. Recent findings revealed that the activity of p110delta also contributes to allergen-IgE-induced mast cell activation and vascular permeability. We investigated the role of p110delta in allergic airway inflammation and hyperresponsiveness using IC87114, a selective p110delta inhibitor, in a mouse asthma model. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of ICAM-1 and VCAM-1 expression, and airway hyperresponsiveness. Intratracheal administration of IC87114 significantly (P<0.05) attenuated OVA-induced influx into lungs of total leukocytes, eosinophils, neutrophils, and lymphocytes, as well as levels of IL-4, IL-5, IL-13, and RANTES in a dose-dependent manner. IC87114 also significantly (P<0.05) reduced the serum levels of total IgE and OVA-specific IgE and LTC(4) release into the airspace. Histological studies show that IC87114 inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and inflammation score. In addition, IC87114 significantly (P<0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. Western blot analyses of whole lung tissue lysates shows that IC87114 markedly attenuated the OVA-induced increase in expression of IL-4, IL-5, IL-13, ICAM-1, VCAM-1, RANTES, and eotaxin. Furthermore, IC87114 treatment markedly attenuated OVA-induced serine phosphorylation of Akt, a downstream effector of PI3K signaling. Taken together, our findings implicate that inhibition of p110delta signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.