Small molecule channels increase anion secretion and host defenses in cystic fibrosis airway epithelia: mechanistic studies and initial clinical translation

Chorghade, Rajeev Shankar

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https://hdl.handle.net/2142/108654 Copy

Description

Title

Small molecule channels increase anion secretion and host defenses in cystic fibrosis airway epithelia: mechanistic studies and initial clinical translation

Author(s)

Chorghade, Rajeev Shankar

Issue Date

2020-07-10

Director of Research (if dissertation) or Advisor (if thesis)

Burke, Martin D.

Doctoral Committee Chair(s)

Burke, Martin D.

Committee Member(s)

• Mitchell, Douglas A.
• Hergenrother, Paul J.
• Grosman, Claudio F.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Cystic Fibrosis
• Amphotericin B
• Molecular Prosthetics
• Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

Cystic fibrosis (CF) is caused by more than 1,900 loss-of-function mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Loss of CFTR-mediated HCO3- and Cl- secretion to the airway surface liquid (ASL) compromises airway host defenses in people with CF and contributes to the chronic bacterial infection and airway inflammation that characterize CF lung disease. Major clinical advances have been achieved with small molecule CFTR modulators which partially increase the activity of certain CFTR mutants. However, ~10% of people with CF have nonsense mutations and/or splice defects which result in little to no CFTR production, or they cannot tolerate CFTR modulators, and thus cannot benefit from this pharmacological approach. In the case of protein ion channel deficiencies, because the endogenous protein network upstream of the missing or dysfunctional channel remains active and may even be upregulated, we hypothesized that ion channel-forming small molecules could harness the selective buildup of ion gradients generated in this context and restore site- and direction-selective transport of the dysregulated ion. We therefore set out to test the hypothesis that because living systems are robust, akin to how imperfect prosthetic limbs can restore substantial function on the macroscopic scale, small molecules that imperfectly replicate the function of missing or dysfunctional protein ion channels may be sufficient to restore physiology. Amphotericin B (AmB) is an FDA-approved ion channel-forming small molecule which has been used clinically for over six decades as an antifungal. In a collaboration with the lab of Dr. Michael Welsh at the University of Iowa, we found that AmB increased ASL HCO3- secretion and ASL pH in cultured CF airway epithelia. Though AmB channels are permeable to a range of physiologically relevant monovalent anions and cations, ASL concentrations of Na+, K+, Mg2+, and Ca2+ were unchanged relative to vehicle-treated controls. Chemical inhibition of basolateral proteins which drive anion secretion abolished the AmB-mediated effects, suggesting that these phenomena depend upon the activity of the endogenous protein network. AmB increased ASL pH, decreased ASL viscosity, and increased ASL antibacterial activity, key components of airway host defenses, in primary cultures of CF airway epithelia. AmBisome, a clinical AmB formulation safely used to treat and prevent pulmonary fungal infections in >1,600 people, increased ASL pH in four CFTR-/- pigs and increased ASL pH and ASL bacterial killing in primary cultures of CF airway epithelia with two nonsense mutations. As a first step towards evaluating the therapeutic potential of this pharmacological approach in people with CF, we asked whether AmB could improve nasal potential difference (NPD), a key clinical biomarker used to first evaluate the efficacy of the CFTR modulator ivacaftor. Fungizone, a clinical AmB formulation safely used to treat intranasal fungal infections, caused electrophysiological changes consistent with increased anion secretion in primary cultures of CF bronchus and nasal epithelia from four people with nonsense mutations. Fungizone hyperpolarized NPD in eight people with CF not on CFTR modulators, providing the first clinical evidence that small molecule channels can impact physiology in people with CF. With the goal of determining whether AmB can increase intranasal ASL pH in people with CF, we developed a new clinical experiment involving perfusion of a mildly acidic solution (pH 5.0) through nasal epithelia and measuring the pH of the collected solution. In 24 non-CF volunteers, intranasal perfusion of a zero Cl- solution pH 5.0 caused an average increase in the pH of the collected solution of 0.077 pH units. The CFTR activator terbutaline increased the pH of the collected perfusion solution relative to the zero Cl- solution alone in 8 out of 11 nostrils tested, suggesting that this effect is due to CFTR-mediated HCO3- secretion. This non-invasive and rapid clinical experiment enables intranasal ASL pH measurement, and can be used as a diagnostic aid and as a method to evaluate the efficacy of new therapeutic strategies for CF. Finally, we found that AmB causes electrophysiological changes and increases ASL pH in cultures of CF airway epithelia at concentrations that have been safely achieved in the lungs, suggesting that aerosolized AmB may have the potential to benefit people with CF. Collectively, these results suggest that though AmB is an imperfect substitute for the CFTR anion channel, the endogenous protein network that drives anion secretion in airway epithelia establishes an environment in which a non-selective channel is sufficient to restore anion secretion and thereby increase host defenses in CF airway epithelia. Because networks of active pumps and passive transporters similarly underlie the movement of other ions in living systems, these findings suggest that small molecule surrogates may have further potential to restore site- and direction-selective mobilization and restore physiology in the case of diseases caused by dysfunctional protein ion channels. Further, these findings provide the first clinical evidence that small molecules that autonomously perform protein-like function can impact human physiology, and encourage the pursuit of this approach as a mechanistic framework to address a range of human diseases caused by a deficiency of protein function.

Graduation Semester

2020-08

Type of Resource

Thesis

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Copyright and License Information

Copyright 2020 Rajeev S. Chorghade

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