The discovery of novel regulators of airway anion secretion, oral communication at the UK Cystic Fibrosis Trust Conference, Nottingham, UK, September 2017

The discovery of potentiators of the calcium activated chloride channel, TMEM16A, oral communication at the Cystic Fibrosis Foundation Research Conference, Stevenson, USA, June 2017

Enterprise Therapeutics receives funding from Cystic Fibrosis Trust

Award will support research into the use of bronchosphere technology as innovative treatment

Brighton, UK – 05 April, 2017: Enterprise Therapeutics Ltd, a drug discovery company dedicated to the research and development of novel therapies for the treatment of respiratory diseases, has won funding from the Cystic Fibrosis Trust to identify new drug mechanisms for the treatment of cystic fibrosis (CF).

The funding will be used to support pioneering research, leveraging Enterprise Therapeutics’ bronchosphere technology platform. Bronchospheres are a miniaturised model of the human airway. This innovative model can be used to support high-throughput drug and target discovery and will be used to facilitate the development of new classes of therapeutics for the treatment of cystic fibrosis.

Over 10,500 people are currently living with CF in the UK. This genetic condition causes the lungs to become clogged with mucus, making it difficult to breathe. People living with CF have a significantly reduced life-expectancy; median age of death is just 28 years. Quality of life is also extremely poor due to high treatment burden and susceptibility to chronic lung infections that result in frequent hospitalisations.

Commenting on this new partnership, Dr John Ford, CEO, Enterprise Therapeutics, said: “We are delighted to have the opportunity to collaborate with the Cystic Fibrosis Trust on this important project. The Trust’s funding will enable critical research to be undertaken to drive a greater scientific understanding of CF and support development of innovative treatments for this challenging genetic disease.”

Dr Janet Allen, Director of Strategic Innovation at the Cystic Fibrosis Trust commented: “This exciting approach will bring hope to the many people living with CF in the UK. We look forward to working with Enterprise Therapeutics and believe that their technology will bring us a step closer to identifying effective new medicines that may improve the lives of people with CF and those who care for them.”

The discovery of novel TMEM16A potentiator compounds, presented at the Gordon research Conference, “Mucus & Mucociliary interactions”, Galveston, USA, February 2017

It is widely accepted that the composition of mucus in the CF airway, and in particular the hydration status, significantly affects its clearance and thereby the potential for form plugs, restrict airflow and create a nidus for chronic microbial colonisation. A variety of largely ion channel-based strategies are being employed to promote mucosal hydration e.g. CFTR repair, ENaC blockers and TMEM16A potentiators. An alternative, but complimentary approach would be to reduce the excessive production and secretion of the mucin proteins that contribute to the solids component of the mucus gel.

One approach to reduce excessive mucus production in the diseased lung is to reduce the number of mucus producing goblet cells in the airways. To identify drug targets that could be regulated to achieve this, we have utilised a 3D culture model of the human airway epithelium, ‘bronchospheres’. Bronchospheres are derived from primary human airway basal cells, and can be cultured to form a well-differentiated mucociliary epithelium without the need for an air-liquid interface. As such, bronchospheres are cultured in a 384 well assay format that makes them amenable to medium throughput compound screening. Treating bronchospheres with inflammatory mediators such as IL-13 induces a mucus hypersecretory phenotype with increased numbers of goblet cells and reduced numbers of ciliated cells. Our hypothesis was that the co-administration of test compounds together with IL-13 would identify compounds capable of preventing goblet cell formation with the opportunity to seed future drug discovery programs.

Bronchospheres were cultured as previously described (Danahay et al., Cell Rep. (2015) 10(2):239). On day 2 after seeding primary human airway basal cells, treatment with IL-13 ± test compounds was initiated. A library of approximately 2,000 pharmacologically active LMW compounds was used, each with a well-annotated mechanism of action. On day 8, media and treatments were topped-up and on day 14 bronchospheres were lysed and RNA isolated. QPCR was then used to assess the expression of cell-specific markers: MUC5AC (goblet cells) and FOXJ1 (ciliated cells). Compounds that induced a ≥2-fold reduction in expression of MUC5AC were classified as hits. This hit list was then refined by checking the expression of FOXJ1. Compounds that had likewise attenuated FOXJ1 expression by ≥2-fold were deprioritised as these likely represented a non-specific effect on epithelial differentiation. Compounds that either maintained or enhanced FOXJ1 expression in addition to repressing MUC5AC gene expression were prioritised for validation using traditional air-liquid interface cultures.

In total, 92 hit compounds from the bronchosphere screen were tested for effects on goblet and ciliated cell numbers in ALI HBE cultures using quantitative immunohistochemistry. Of these, 38 (41%) significantly attenuated the MUC5AC+ stained area in IL-13 treated HBE and either maintained or increased the FOXJ1+ stained area. Validated hits were then aligned based on their previously reported pharmacological activity to enable common pathways to be identified and to refine our hypotheses through further exemplification of pathway regulators.

Based on this screen, we have identified pathways that regulates the induction and maintenance of a goblet cell metaplasia in vitro and are progressing a lead optimisation program for eventual therapy in respiratory diseases associated with mucus obstruction.