(607) A Rationally Designed Molecular Prosthetic for Cystic Fibrosis

Background: Small molecule-based ion channels formed by the antifungal natural product amphotericin B (AmB) can act as functional replacements for CFTR in cultured cystic fibrosis (CF) airway epithelia, CFTR null pigs, and the noses of people with CF [1,2]. This “molecular prosthetics” (MP) approach is independent of the CFTR protein and thus might be effective for everyone with CF – including those who cannot benefit from CFTR modulators. Previous studies have shown that FDA-approved AmB is well-tolerated in the lungs. We found that combining AmB with Chol extends the range of effective concentrations over which AmB can restore airway host defenses in human CF airway epithelia [1]. Building on this finding and additional structural [3], mechanistic, biophysical, and nanoparticle engineering studies, we have generated a new dry powder inhaler (Amphotericin B Cystetic for Inhalation - ABCI) for delivering AmB and Chol as a MP to the airways of people with CF.

Methods: We used established assays to study the structure of AmB aggregates and the capacity of ABCI to restore host defenses in CF airway epithelia:
1. Solid State NMR (SSNMR), structure calculations [3], and microcrystal electron diffraction methods.
2. Airway surface liquid (ASL) pH in cell line and primary cultures of airway epithelia [1].
3. H¹⁴CO₃⁻ transport across NuLi and CuFi monolayers [1].
4. ASL viscosity of AmB-treated primary cultures of airway epithelia [1].
5. Antibacterial activity within the ASL of ABCI-treated primary cultures of airway epithelia [1].

Results: 1. AmB aggregates consist of asymmetric homodimers arranged in a clathrate-like lattice encasing void volumes similar in size to Chol. These clathrates undergo a structural change to bind sterol guests. These data provide insight into how precomplexing Chol with AmB extends the concentration range over which AmB functions as an effective MP.
2. Based on biophysical and nanoparticle engineering studies (see complementary abstract), we have generated a new dry powder inhaler, ABCI, for delivering AmB as a MP to the airways of people with CF.
3. In immortalized cells and primary human airway epithelia, ABCI increases ASL pH at both low and high AmB concentrations. ASL pH increases within the first 6 hours post-treatment with ABCI and is sustained for at least 2 weeks.
4. ABCI is well tolerated in rats and dogs at concentrations above those predicted to be efficacious in people with CF.

Conclusions: Key structural features of AmB aggregates reveal how this molecule binds Chol and suggests a dynamic equilibrium between cell surface clathrates and transmembrane ion channels. Insights from structural, mechanistic, biophysical, and nanoparticle engineering studies allowed us to rationally design a new AmB and Chol-containing dry powder inhaler, ABCI, that can increase host defenses in CF airway epithelia over a wide range of concentrations. These preclinical studies suggest that ABCI may serve as a novel treatment for people with CF, including those who cannot benefit from CFTR modulators.

Acknowledgements: Funded by cystetic Medicines

References: [1] Muraglia KA, et al. Small-molecule ion channels increase host defenses in cystic fibrosis airway epithelia. Nature 2019;567:405–8.
[2] Chorghade RS, et al. Amphotericin B induces epithelial voltage responses in people with cystic fibrosis. J Cyst Fibros 2021;20:540–50.
[3] Lewandowska A, et al. Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes. Nat Struct Mol Biol 2021;28:972–81.