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Title: The synthesis and evaluation of novel calpain-I inhibitors
Author: Adams, Sarah Elizabeth
ISNI:       0000 0004 2752 3143
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2013
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The calcium activated cysteine protease calpain-I has a pivotal role in a variety of physiological processes within the human body. In particular calpain-I enables the cell spreading and subsequent chemotaxis behaviour of neutrophils in response to tissue damage. Neutrophils are linked to the pathological condition rheumatoid arthritis and so calpain-I is considered be a valuable therapeutic target. Many inhibitors of calpain-I are highly non-selective with the exception of two small molecule synthetic inhibitors. A phenyl and an indole-based α-mercaptoacrylic acid have shown a slight selectivity towards calpain-I over other cysteine proteases. In this work 24 novel monohalogenated α-mercaptoacrylic acid inhibitors were prepared based on these lead structures using Vilsmeier-Haack chemistry followed by Knoevenagel condensation of the resulting aromatic aldehydes as key steps. The thiols within the α-mercaptoacrylic acid moiety demonstrated a tendency to form disulfide bridges in solution. Analysis of this disulfide formation through 1H NMR spectroscopy, UV-Vis spectrophotometry and HPLC showed that the monomeric form was active under the reducing conditions used in subsequent assays. The analogues were tested as inhibitors of calpain-I revealing that bromoindole based inhibitors were the most potent. Selected compounds showed ~10 fold selectivity towards calpain-I versus calpain-II. In live neutrophils they were capable of slowing the cell spreading process by up to 70%. When live neutrophils containing the inhibitor were irradiated with 410 nm light, the cells completely lost the ability to spread. To show that these compounds were allosteric inhibitors the calcium binding domain PEF(S) was expressed in E. coli and purified using anion exchange chromatography and size exclusion chromatography. Solution of X-ray co-crystal structures of the calpain PEF(S) domain with two different inhibitors revealed that they bind to the protein in a similar fashion as an α-helical domain of calpastatin, the endogenous inhibitor of calpain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry