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Design, Synthesis and Biochemical evaluation of novel serine protease activity-based probes.

Serine proteases are a subgroup of the protease family involved in several physiological processes, including immune response, cell death and tissue healing. The upregulation of these proteases can increase inflammatory cytokines, degradation of extracellular matrix components, activation of PAR2 or MMP-9. We previously obtained an in vivo proof of concept with a multi-target serine protease inhibitor (UAMC-00050) in Dry Eye Disease (DED). Topical application of this compound in the eye of a tear-deficient dry eye rat animal model reduced both tissue damage and inflammatory parameters. Moreover, UAMC-00050 also cause a decrease in visceral hypersensitivity in a rat model of post-inflammatory visceral hypersensitivity. Therefore, we hypothesized that serine proteases play an essential role in both DED and Irritable Bowel Syndrome (IBS). The focus of this project is to characterize the proteases involved in DED and IBS. We are using activity-based protein profiling (ABPP), a proteomic technique where chemical probes are used for target identification and drug discovery research. Activity-based probes (ABPs) are small molecules that react covalently with the enzyme active site and facilitate the labelling of target proteins. Up-to-date, a series of 17 new ABPs, analogues from our inhibitor, were synthesized. The challenging synthesis of ABPs required extensive optimization of the synthetic pathways. The phosphonate warhead is crucial to target and bind irreversibly to trypsin-like serine proteases. All ABPs have been biochemically characterized by determining their IC50 in a panel of different serine proteases involved in immune responses. Several potent ABPs have been synthesised and characterized so far. Since ABPs need to bind covalently to the target protein efforts will now be made to describe the most potent probes' kinetic profile by progress curve assays. In addition jump dilution experiments will be performed to discriminate between inhibition mechanisms. In a last stage, ABPs that are potent against the serine proteases of interest and that undergo an irreversible inhibition mechanism will be used to label and identify upregulated proteases in biological samples of DED and IBS patients. Mass spectrometry and gel electrophoresis will be used to detect the ABPs. Identifying the upregulated proteases will allow the design and synthesis of more selective and potent compounds to treat DED and IBS.
Date:1 Oct 2021 →  Today
Disciplines:Drug discovery and development not elsewhere classified, Medicinal chemistry