Profiling of proteases as potential therapeutic targets

Subtitle:focus on inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS)

Proteases are potential therapeutic targets in several diseases such as gastrointestinal disorders and lung infections. However, the role of proteases in the pathophysiology of these diseases is often not fully elucidated yet. Therefore, this doctoral research explored the role of proteases in biological tissues and investigated these enzymes as therapeutic targets in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Insight into the involvement of proteases in IBD and IBS was first gained by assessing the proteolytic activity using fluorogenic substrates in colonic tissue and fecal samples from a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in the acute (IBD) and post-inflammatory (IBS) phase. Obtained results showed an elevated trypsin-like activity in colonic tissue from rats in the post-inflammatory phase of colitis. Furthermore, intracolonic treatment of these rats with the broad-spectrum serine protease inhibitor UAMC-00050 decreased this trypsin-like activity. Therefore, these results highlighted trypsin-like proteases as important enzymes and potential therapeutic targets in IBS. Next to the fluorogenic assays, a peptide-based mass spectrometry assay was used to further identify potential therapeutic targets in the same animal models for IBD and IBS. A set of peptides relevant in pain and inflammation was incubated with a protease source for fragmentation and the cleavage products were identified with mass spectrometry to obtain specific cleavage patterns. First, differential cleavage patterns were observed for a set of trypsin- and elastase-like proteases present in the human colon. These patterns could be used to understand which trypsin-like or elastase-like enzymes are active in a given biological sample. Second, the processing of the same peptides by colon samples from the TNBS-induced colitis model was investigated and pointed to a set of proteases as potential targets for therapy. An increased cleavage of several peptides was observed in the colon from acute colitis rats and suggested thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD. Finally, the value of the peptide-based mass spectrometry assay was proven in a different sample type by applying it to lung tissue of mice infected with Pseudomonas aeruginosa. In conclusion, this doctoral research contributed to the unraveling of the role of proteases in the pathophysiology of IBD and IBS and identified a set of proteases for which further investigation as potential therapeutic targets is worthwhile. However, further research is required to elucidate the exact mechanisms underlying IBD and IBS and the function of proteases therein.

Publication year:2022

Keywords:Doctoral thesis

Accessibility:Open