Discovery of Fungus-Derived Nornidulin as a Novel TMEM16A Inhibitor: A Potential Therapy to Inhibit Mucus Secretion in Asthma

Abstract

Introduction: Inhibition of Ca²⁺-activated transmembrane protein 16A (TMEM16A) Cl^- channels has been proposed to alleviate mucus secretion in asthma. In this study, we identified a novel class of TMEM16A inhibitors from natural sources in airway epithelial Calu-3 cells and determine anti-asthmatic efficacy of the most potent candidate in a mouse model of asthma.

Methods: For electrophysiological analyses, IL-4-primed Calu-3 cell monolayers were mounted in Ussing chamber and treated with various fungus-derived depsidones prior to the addition of UTP, ionomycin, thapsigargin, or E_act to stimulate TMEM16A Cl^- current. Ca²⁺-induced mucus secretion in Calu-3 cell monolayers was assessed by determining MUC5AC protein remaining in the cells using immunofluorescence staining. OVA-induced female BALB/c mice was used as an animal model of asthma. After the course of induction, cellular and mucus components in bronchoalveolar lavage were analyzed. Lungs were fixed and undergone with H&E and PAS staining for the evaluation of airway inflammation and mucus production, respectively.

Results: The screening of fungus-derived depsidones revealed that nornidulin completely abolished the UTP-activated TMEM16A current in Calu-3 cell monolayers with the IC₅₀ and a maximal effect being at ~0.8 µM and 10 µM, respectively. Neither cell viability nor barrier function was affected by nornidulin. Mechanistically, nornidulin (10 µM) suppressed Cl^- currents induced by ionomycin (a Ca²⁺-specific ionophore), thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ ATPase), and E_act (a putative TMEM16A activator) without interfering with intracellular Ca²⁺ ([Ca²⁺]_i) levels. These results suggest that nornidulin exerts its effect without changing [Ca²⁺]_i, possibly through direct effect on TMEM16A. Interestingly, nornidulin (at 10 µM) reduced Ca²⁺-dependent mucus release in the Calu-3 cell monolayers. In addition, nornidulin (20 mg/kg) inhibited bronchoalveolar mucus secretion without impeding airway inflammation in ovalbumin-induced asthmatic mice.

Discussion and conclusion: Our study revealed that nornidulin is a novel TMEM16A inhibitor that suppresses mucus secretion without compromising immunologic activity. Further development of nornidulin may provide a new remedy for asthma or other diseases associated with allergic mucus hypersecretion without causing opportunistic infections.

Keywords: TMEM16a; airway epithelium; asthma; mucus secretion; nornidulin.