Serine hydrolases in pancreatic cancer: target validation using activity-based protein profiling

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate out of all common cancers due to a lack of biomarkers and treatments. The location of the pancreas in the body makes early diagnosis and surgery difficult. This is exacerbated by the stromal tissue surrounding the cancer, which shields the malignancy from detection and treatment. The crosstalk between the cancer and stroma is known to promote cancer progression and metastasis. Thus, further understanding of this tumour microenvironment is key to reducing disease burden for this cancer. This thesis focuses on investigating serine hydrolase activity in pancreatic cancer using activity-based probes. A preliminary 3D co-culture model of the tumour microenvironment of PDAC was developed and its serine hydrolase activity was profiled using chemical proteomics. This model has the potential to investigate the crosstalk between PDAC and its surrounding stroma. However, this project was halted prematurely due to lockdown. Kallikrein-related peptidases (KLK) are a family of fifteen serine proteases with important roles in homeostasis. Dysregulation of KLKs have been shown to relate to cancer progression. However, the lack of chemical tools that can selectively detect KLK activity has hindered the validation of these proteases as targets or biomarkers. The focus of this work is to develop and apply novel activity-based probes for KLKs in the context of pancreatic cancer. Selective probes have been developed for KLK4, 6, 7 and 8. Probes were designed using a substrate scanning library, which gave the substrate specificity of each protease. The optimal substrate sequences including unnatural amino acids were grafted into a peptidyl diphenyl phosphonate scaffold, giving highly potent and selective probes that bind irreversibly to their target. The KLK6 probe was further validated in cellular systems using chemical proteomics, which showed it was highly selective for KLK6 in pancreatic cancer cells. A co-crystal structure between the probe and KLK6 shows its binding mode. Using the probe, we have unlocked new insights on the mechanisms of KLK6 mediated invasion and migration in PDAC. The probe was able to inhibit both invasion and migration in PDAC cells. N-terminomic analysis allowed us to identify the putative substrates of KLK6 in PDAC cells. This work adds to an existing toolbox of KLK activity-based probes, which will be shared with the rest of the scientific community for studying other KLK associated diseases such as neurodegenerative diseases, skin diseases and other cancers.Open Acces