There is accumulating evidence that cysteine proteinase activity plays an important role in cancer cell invasion and metastasis. Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. As such, increasing efforts are directed toward the development of inhibitors that are specific to the enzyme cathepsin B in order to both dissect out the role of the enzyme in these pathological processes and as potential therapeutic agents. / Unlike other members of the papain superfamily of enzymes, cathepsin B has a unique 18-residue insertion loop termed the "occluding loop" which sits over the primed subsites of the enzyme. It has now been well established that this loop is responsible for cathepsin B's unique dipeptidylcarboxypeptidase activity due to the presence of two positively charged residues, His110 and His111, that anchor the C-terminus of peptide substrates and allow the enzyme to carry out its carboxypeptidase activity. It has been demonstrated that this loop is a flexible segment that can move up and out of the way in order to accommodate binding of larger molecules such as the propeptide. Close examination of the x-ray crystal structure of the mature enzyme indicates the presence of an electrostatic interaction between the side chain of the main chain residue Asp22 and the imidazole ring of the occluding loop residue His110. This salt bridge acts as latch between the loop "open" and loop "closed" conformations of the enzyme. / It has been postulated that the development of inhibitors which interact with the "occluding loop" might provide highly selective inhibitors for cathepsin B and in fact cathepsin B-specific epoxysuccinyl inhibitors have been made which exploit the interaction of between a negatively charged carboxylate at P2' and the two positively charged histidine residues of the occluding loop. Using site-directed mutagenesis, we have dissected out the individual contributions of the two occluding loop histidine residues (His110 and His111). The effect of pH on these interactions has also been evaluated and it has been shown that increasing pH results in increased loop flexibility and diminished inhibitor potency. / Further structure-activity relationships for epoxysuccinyl inhibitors were also established by varying our inhibitor design template both in the P1' and P2' positions using known substrate specificities in these positions. Extension of the inhibitor template into the unprimed subsite region yielded the most potent epoxysuccinyl inhibitor to cathepsin B reported to date (BzlNH-Phe-NH-(2 S,3S)-tEps-Leu-Pro-OH: k2/Ki 2 900 000 +/- 300 000 M-1s-1).
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37782 |
| Date | January 2000 |
| Creators | Michaud, Stephanie. |
| Contributors | Chan, Tak Hang (advisor), Gour, Barbara J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001810796, proquestno: NQ70100, Theses scanned by UMI/ProQuest. |
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