Show simple item record

dc.contributor.authorLow, Kristinen
dc.date2014-11-05 14:53:49.229
dc.date2014-11-07 14:42:07.019
dc.date.accessioned2014-11-07T20:05:33Z
dc.date.available2014-11-07T20:05:33Z
dc.date.issued2014-11-07
dc.identifier.urihttp://hdl.handle.net/1974/12611
dc.descriptionThesis (Ph.D, Biochemistry) -- Queen's University, 2014-11-07 14:42:07.019en
dc.description.abstractCalpains are cysteine proteases that depend on Ca2+ for activation. They have many roles in the cell; hence modifications of calpains or changes in their levels of activity can lead to altered physiological states. Thus, it is of great interest and medical relevance to design inhibitors that can specifically target calpain without interfering with the other numerous cellular cysteine proteases. The cytosolic and ubiquitous calpains-1 and -2 have a naturally occurring and specific inhibitor, calpastatin, which binds with high affinity to the active site but in such a way that the calpastatin itself is not cleaved. Crystal structures of the full-length calpain-2 in complex with calpastatin have aided the design of novel calpain-specific inhibitors. I have helped design cyclic peptides to mimic important conserved structural elements of calpastatin and have investigated their inhibition potency against calpain. Cyclic peptides and derivative peptidomimetics were able to inhibit cysteine proteases including calpain. Macrocycle and peptidomimetic products showed competitive, non-competitive, and mixed inhibition of calpain-2 with Ki values in the micromolar range while conferring some specificity. In a second strategy inhibitors mimicking the calpastatin subdomain B α-helical region were designed to be calpain-specific by binding outside of the common cysteine protease active site cleft but extend into it. Indeed, stabilized α-helices synthesized from conserved calpastatin residues were found to inhibit calpain specifically and potently, with potential use as fluorescent activity-based probes. Previously reported allosteric inhibitors of calpain were investigated, and their ability to inhibit the calpain enzyme through binding to the PEF domain questioned. The compound PD150606 was able to inhibit the protease core of calpain, which lacks the PEF domain of the full enzyme, and the penta-peptide LSEAL was found to have no effect at all on calpain in vitro. Thus it is now clear that binding to hydrophobic grooves in the calpain PEF domains does not confer inhibition and the mechanism of non-competitive calpain inhibition is still unknown.en
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectinhibitoren
dc.subjectcalcium-activated proteaseen
dc.subjectmacrocycleen
dc.subjectproteaseen
dc.subjectallosteric inhibitoren
dc.subjectcalpastatinen
dc.subjectcalpainen
dc.subjectenzyme kineticsen
dc.titleCalpain inhibitor design inspired by the natural inhibitor calpastatinen
dc.typethesisen
dc.description.degreePhDen
dc.contributor.supervisorDavies, Peter L.en
dc.contributor.departmentBiochemistryen
dc.degree.grantorQueen's University at Kingstonen


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record