Small-Molecule CFTR Activators for Dry Eye Therapy

Abstract

Purpose: Therapies that augment the tear aqueous component have the potential to reduce the symptoms and pathology of dry eye disorders. We previously demonstrated substantial capacity for active ocular surface chloride (Cl^-) secretion through the cAMP-activated channel, CFTR (cystic fibrosis transmembrane conductance regulator). We are now evaluating novel CFTR activators to develop a first-in-class, pro-secretory dry eye therapy.

Methods: A cell-based, kinetic high-throughput screen of >75,000 compounds was performed to identify small-molecule activators of human CFTR. Highly active compounds were verified and prioritized by short-circuit current (I_SC) analysis on CFTR-transfected epithelial cells and primary cultures of human conjunctival epithelia. To confirm direct compound action on CFTR, intracellular cAMP levels and effects on other major ocular surface transporters were measured by immunoassay and cellular electrophysiology, respectively. CFTR activators emerging from in vitro assays and structure-activity analysis were tested in mice and rats following topical administration. Activity at the ocular surface was assessed by open-circuit potential difference (PD) and basal tear production measurements.

Results: Potent CFTR activators (EC₅₀ 70-500 nM) with diverse chemical scaffolds were identified. Ten compounds from 6 classes produced strong and sustained direct channel activation, achieving >90% activity of the non-specific cAMP agonist forskolin (I_SC). These compounds demonstrated similar potency with near-maximal activation of CFTR in vivo. CFTR activators produced sustained large ocular surface PD hyperpolarizations of 10-15 mV under low Cl^- conditions in both mice and rats. This effect was not seen in CFTR-deficient mice, and was largely reversed by a CFTR-selective inhibitor. Single-dose topical administration of the most potent CFTR activators increased basal tear secretion by >30% for over 30 minutes relative to saline vehicle-treated rats. These compounds have favorable aqueous solubility and lack significant acute cellular or ocular toxicities.

Conclusions: We discovered 6 classes of small-molecule activators of human and rodent CFTR in a target-based screen. These compounds enhance ocular surface Cl^--driven fluid secretion into the tear film, supporting their promise as topical dry eye therapeutics. Our CFTR activators have been prioritized for testing in established dry eye models for further pre-clinical validation.