C70-36 Discovery of a Novel Gamma-Secretase Small Molecule Inhibitor for the Treatment of Cystic Fibrosis

Introduction

People with cystic fibrosis (pwCF) have deficits in mucociliary clearance (MCC) that for eligible mutations, are addressed by CFTR-targeted highly efficient modulator therapy, or HEMT. There is significant medical need for pwCF ineligible for HEMT or those who experience significant adverse effects. The CF airway presents hypertrophic goblet cells, mucus hypersecretion, and reduced airway surface liquid (ASL) contributing to a characteristic impaired MCC. It has been proposed that the Notch pathway mediates maladaptive tissue remodeling and goblet cell hyperplasia, indicating the potential of modulation through gamma-secretase inhibitors (GSIs) to restore tissue homeostasis and MCC. We evaluated multiple GSI small molecule chemotypes and initiated a targeted medicinal chemistry effort to develop a tailored GSI for the treatment of CF airway disease.

Methods

HEK293 cells expressing reporter constructs for amyloid precursor protein (APP) or Notch were employed to test the inhibitory activity of novel synthesized analogs by detection of APP/Notch cleavage via high content microscopy. Selected hits were profiled in CF donor cells (ΔF508 homozygous) grown at an air-liquid interface (ALI) by quantifying hypertrophic goblet and multiciliate cell counts vs compound concentration. We chose one compound (Compound A) for further in vitro functional profiling employing as readouts ASL reabsorption and particle transport velocity as a surrogate measure for MCC. We also performed bulk and single cell expression profiling (RNAseq) of treated cultures. Finally, as a way of confirming translation of ALI observations to tissue, we monitored selected biomarkers in the airway of rats dosed orally with Compound A via bulk RNAseq.

Results

Multiple novel GSIs dramatically decreased hypertrophic goblet cell counts in ALI and did not appear to be toxic even at the highest compound concentrations tested. Compound A was very potent, inducing >90% inhibition at concentrations as low as 1 nM. This molecule promoted ASL retention and had a remarkable effect on MCC velocity. Gene expression analysis of ALI treated with Compound A confirmed reductions in goblet cells and mRNAs associated with mucus obstruction such as Muc5b and Muc5ac. Those reductions were also observed in the airway tissues of adult rats dosed daily with Compound A.

Conclusion

Our results suggest that our novel GSIs exemplified by Compound A appear to reprogram the airway epithelium, in vitro in human CF ALI and in vivo in the airway of dosed rats, to restore fluid homeostasis and mucociliary clearance, offering a potential therapeutic option for patients ineligible or inadequately responding to HEMT.

This abstract is funded by: Internal company funds