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Title: Structural insight into the development of novel allosteric Calpain-1 inhibitors
Author: Cresser-Brown, Joel
ISNI:       0000 0004 7973 0531
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2019
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Calpain-1 is a calcium activated cysteine protease involved in a diverse range of physiological processes, such as enabling the rapid cell spreading of neutrophils during the chemotactic response to tissue damage. Rheumatoid arthritis is a pathological condition associated with the neutrophil spreading process, rendering calpain-1 a prized therapeutic target. Small molecule α-mercaptoacrylic acid inhibitors exhibit selectivity towards calpain-1, however differences in potency were reported for the compounds in different redox states. The dihedral bond geometry of the more potent oxidised disulfide form enabled an increased binding interaction with the calcium binding domain, PEF(S), however the less potent thiol form would be predominantly present in the reducing environment of the cell. In this project, diselenide analogues of the α-mercaptoacrylic acid inhibitors were synthesised and evaluated as allosteric, redox stable calpain-1 inhibitors. They were shown to inhibit human calpain-1 with moderate potency, binding to the calcium binding domains PEF(S) and PEF(L) with similar potencies to the sulfur counterparts, whilst X-ray co-crystal structures of PEF(S) with the diselenides showed that the dihedral bond geometry was analogous to the disulfides. The calpain-1 active site, CysPC, was expressed and purified as a truncated catalytic protein. The diselenides inhibited CysPC with a greater potency than the full length enzyme, suggesting they are not allosteric in their mode of action. CysPC was crystallised in the presence of a diselenide inhibitor, the electron density map suggested the presence of adducts in the active site cleft was responsible for the inhibition formed by selenyl-sulfide bonds with surface exposed cysteine residues. Computational docking experiments using the calcium binding domain PEF(S), identified ten compounds that were shown to bind to PEF(S) by a fluorescence assay. Of these compounds, three displayed an apparent allosteric mode of action by inhibiting the calpain-1 full-length enzyme but not the active site. The allosteric inhibitors did not inhibit calpain-2 up to a concentration of 100 μM indicating moderate isoform selectivity which was attributed to an interaction with the calcium binding domain PEF(L). This finding may serve as a novel target in the development of isoform selective, allosteric inhibitors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry