Development of therapeutics as modulators of CFTR took a great leap forward with the identification of the potentiator VX-770 (Ivacaftor/Kalydeco). However, VX-770 does not appear to stimulate all variants of CFTR, and adversely impacts chemical correction of misfolded CFTR proteins; we think we can do better. Other compounds with structures distinctively different from VX-770 can also potentiate CFTR, suggesting that there are features of the protein and its gating mechanism that may be targeted in order to achieve allosteric stimulation of channel activity. However, neither the mechanisms by which these compounds (including Kalydeco) potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. The long-term goal of the proposed research is to determine how small molecules bind to and stimulate CFTR activity in Cystic Fibrosis (CF) patients and patients impacted by some forms of Chronic Obstructive Pulmonary Disease (COPD). This project addresses 3 main challenges regarding potentiation of CFTR:

The mechanism(s): Lack of knowledge about the mechanisms by which CFTR potentiators exert their effect has led to incorrect interpretation of drug efficacy in drug screening campaigns, denying access to potentially life-saving drugs for some patients. We have recently found that the degree of potentiation of CFTR by some compounds exhibits a dependence upon the degree of channel phosphorylation. At the level of mechanism, this observation suggests that at least some of the CFTR potentiators may function as allosteric modulators. At the practical level this observation may require a revision of screening protocols.

The site(s) of action: Unlike some classes of correctors, most CFTR potentiators are thought to act directly on CFTR, based upon mechanistic studies like ours.However, we do not yet know where they bind. A consequence of this knowledge gap is that even now, after the publication of the CFTR structure, we cannot undertake target-based approaches to drug discovery that could identify novel candidates that may bear therapeutic value. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites.

The drugs: Many different compounds with diverse chemical structures have been found capable of potentiating CFTR. However, these have not been systematically analyzed in a search for common denominators in terms of functional groups. This analysis will allow for the development of pharmacophore models that could be used for ligand-based virtual screening in a search for new, more effective potentiators.

CFTR is a large, polytopic integral membrane protein with multiple functions, most often studied as a chloride ion channel. Loss of CFTR function has been shown by several labs to alter plasma membrane lipids in ways that tie to both increased airway inflammation and decreased innate defenses against bacterial infection. However, the inverse relationship, i.e., the role of lipids in regulating CFTR channel function, has barely been explored. This project focuses on regulation of CFTR by sphingolipids and sphingolipid-mediated signaling. Several CF-relevant bacteria including S. aureus and P. aeruginosa produce virulence factors such as sphingomyelinase (SMase), which breaks down sphingomyelin to ceramide and phosphocholine and has been shown previously to inhibit CFTR by an unknown mechanism. Based on our strong preliminary data (Stauffer et al., 2017), we hypothesize that SMase inhibits CFTR channel activity via a novel mechanism that relies upon lipid-mediated signaling. This may limit the therapeutic potential of CFTR modulators. Our goal is to understand how SMase regulates CFTR activity.