The Development of inhaled small molecules for the pharmacological inhibition of cathepsin S for the treatment of inflammatory lung diseases

Abstract

Cathepsin S (CatS), a lysosomal protease, has a restricted tissue expression where it is mainly found in antigen presenting cells. This indicates that targeting CatS in diseases potentially should have minimal side effects. In addition, Cathepsin S increased expression and secretion into the extracellular milieu was linked to a plethora of diseases with inflammatory and autoimmune components, including cancers, rheumatoid arthritis, cardiovascular and inflammatory lung
diseases.

Increasing evidence suggests the importance of CatS in the pathogenesis of multiple lung diseases including both acute and chronic diseases and in lung cancers. Research at Queen’s University, confirmed CatS role in lung diseases such as asthma, acute respiratory distress syndrome, COPD and cystic fibrosis in murine models.Understanding the clinical setting and history of disease progression is important in validating a target. For example, cystic fibrosis is a multi-organ genetic disease that affects the lungs in which mucostasis, inflammation and chronic infection are the main hallmarks of the disease. Today, cystic fibrosis is diagnosed early in newborns allowing for early intervention. Therefore, the fact that CatS is involved in the early stages of disease development makes it an attractive target in lung CF. Early inhibition of CatS holds potential benefits to preserve lung structure in CF patients. Delivering the drug directly to lungs via inhalation to treat pulmonary diseases provides an opportunity to overcome efficacy issues that plagued previous CatS inhibitors. To date there is no CatS inhibitor in the market. To our knowledge, the work presented in this thesis is a part of the first medicinal chemistry effort to identify CatS inhibitors with physiochemical properties suitable for inhaled route. The first part of this work discusses the SAR around CatS inhibitors with novel P3 group. The aim is to improve solubility of compounds from a previous series developed in our lab. While lowering solubility can increase lung retention time of inhaled drugs’ a very insoluble compounds are associated with airway irritation and may compromise target engagement. The new compounds have lower number of aromatic rings to decrease lipophilicity and subsequently to improve solubility. More than 50 compounds were generated with IC50 in nanomolar range and good Abstract xii selectivity profile. 19 compounds were identified with IC50 of less than 10 nM and were taken forward to assess their activities in cells. CLIP assay is wildly used to assess CatS inhibition in vitro. Raji cells, B-cells derived cell line, are usually incubated for 24h with CatS inhibitor. Inhibition of CatS in Raji cells results in accumulation of small protein (Lip 10) that can be detected in western blot. Five compounds showed cellular activities at 24h. As these compounds are designed as reversible covalent inhibitors, their binding kinetics were anticipated to affects the results. For example, a compound with a fast cellular offset can lose activity before 24h, consequently, disappearance of Lip 10. Therefore, selected compounds were screened and incubated for different time points. The results showed different pattern of time-dependent accumulation of Lip 10. For example, two compounds that showed no activity at the initial 24h screening showed activity at earlier time
points. These results also indicate that previous compounds with faster cellular offset, tested only at 24h and showed no activity, could mistakenly labelled as cell impermeable.

Thesis is embargoed until 31 December 2025.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Hashemite University
Supervisor	Richard Williams (Supervisor) & Roberta Burden (Supervisor)