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
• nornidulin
• asthma
• mucus secretion
• airway epithelium

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University (Protocol No. MUSC62-028-492).

Data Sharing Statement

Data supporting the findings of this study are available from the corresponding authors.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by Mahidol University (Basic Research Fund: fiscal year 2022), the NSTDA Chair Professor grant (Fourth Grant) of the Crown Property Bureau and the National Science and Technology Development Agency, the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B05F650041), and Tonkla Ramathibodi, Faculty of Medicine, Ramathibodi Hospital, Mahidol University.