A Repurposed Drug Screen for Compounds Regulating Aquaporin 5 Stability in Lung Epithelial Cells

Abstract

Aquaporin 5 (AQP5) is expressed in several cell types in the lung and regulates water transport, which contributes to barrier function during injury and the composition of glandular secretions. Reduced AQP5 expression is associated with barrier dysfunction during acute lung injury, and strategies to enhance its expression are associated with favorable phenotypes. Thus, pharmacologically enhancing AQP5 expression could be beneficial. Here, we optimized a high-throughput assay designed to detect AQP5 abundance using a cell line stably expressing bioluminescent-tagged AQP5. We then screened a library of 1153 compounds composed of FDA-approved drugs for their effects on AQP5 abundance. We show compounds Niclosamide, Panobinostat, and Candesartan Celexitil increased AQP5 abundance, and show that Niclosamide has favorable cellular toxicity profiles. We determine that AQP5 levels are regulated in part by ubiquitination and proteasomal degradation in lung epithelial cells, and mechanistically Niclosamide increases AQP5 levels by reducing AQP5 ubiquitination and proteasomal degradation. Functionally, Niclosamide stabilized AQP5 levels in response to hypotonic stress, a stimulus known to reduce AQP5 levels. In complementary assays, Niclosamide increased endogenous AQP5 in both A549 cells and in primary, polarized human bronchial epithelial cells compared to control-treated cells. Further, we measured rapid cell volume changes in A549 cells in response to osmotic stress, an effect controlled by aquaporin channels. Niclosamide-treated A549 cell volume changes occurred more rapidly compared to control-treated cells, suggesting that increased Niclosamide-mediated increases in AQP5 expression affects functional water transport. Taken together, we describe a strategy to identify repurposed compounds for their effect on AQP5 protein abundance. We validated the effects of Niclosamide on endogenous AQP5 levels and in regulating cell-volume changes in response to tonicity changes. Our findings highlight a unique approach to screen for drug effects on protein abundance, and our workflow can be applied broadly to study compound effects on protein abundance in lung epithelial cells.

Aquaporin-5 Protein Is Selectively Reduced in Rat Parotid Glands under Conditions of Fasting or a Liquid Diet

“…Another possible mechanism for the selective decrease of AQP5 is the ubiquitin-proteasome system [6]. Villandre et al [20] have demonstrated that AQP5 is ubiquitinated and degraded by the proteasome in a cultured bronchial epithelial cell line stably expressing human AQP5. The selective degradation of AQP5 by the ubiquitin-proteasome system under fasting or liquid diet conditions could be plausible if neither TMEM16A nor NKCC1 are ubiquitinated under the same conditions.…”

Aquaporins and Neuropathic Pain

“…Given the close relationship between AQP5 and tumors, AQP5 has been used as one of the biomarkers of some tumors and has been involved in some experiments on drug resistance and drug screening against cancer cells [129][130][131]. In addition, since its wide distribution in various systems of the body, AQP5 can be found to be involved in many glandular secretion-related diseases, such as poor salivary gland secretion in patients with Sjogren's syndrome, salivary gland dysfunction induced by ovariectomy in rats, and pulmonary edema in mice with allergic asthma induced by ovalbumin [128,132,133].…”

Lens Aquaporins in Health and Disease: Location is Everything!

“…AQP1 inhibitors have also been considered as a therapeutic target for the treatment of intraocular hypertension in glaucoma ( Patil et al, 2018 ). Thus, therapeutic targeting of aquaporins is an active area of research ( Villandre et al, 2022 ), including in ocular tissues, and we can expect continued discoveries of new AQP modulators. In the context of lens water transport and cataractogenesis, the lens microcirculation system needs to be tightly regulated and it is worth considering lens AQPs as a potential anti-cataract targets ( Pierscionek, 2021 ).…”