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Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

This thesis reports the development of potent and selective inhibitors of m-calpain for the treatment of cataract. SJA6017 has been proven to prevent lens opacity in rat and has been our lead compound. A series of Val-Leu peptidyl aldehyde inhibitors (33a-e, 33g, 33i and 35) have been designed, synthesized, and tested for therapeutic potential as cataract inhibitors. Chapter 1 is an introduction to calpain and the diseases associated with it's over activation. A review of the literature on calpain inhibition is given. Structure activity relationship (SAR) theory is presented. The techniques that have been applied in our research group to drug design include molecular modeling, synthesis, assay and animal studies which are all briefly discussed. The importance of a -strand conformation for an inhibitor to bind to calpain is discussed. Chapter 2 describes the synthesis of m-calpain inhibitors. This comprises the preparation of the Val-Leu dipeptide core 29, Val-Leu dipeptidyl alcohols 31a-g and 31i, and the synthesis of dipeptidyl aldehydes 33a-e, 33g, 33i and 35. The choice of coupling regents and conditions in the coupling reactions is investigated. Sulfur trioxide pyridine oxidation for the conversion of Val-Leu dipeptidyl alcohols to aldehydes is discussed. The molecular modeling and biological assay results are presented.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (M.S. )--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on July 28, 2008). Research advisor: Dr. Isaac O. Donkor, Ph.D. Document formatted into pages (xii, 80 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 65-80).

Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 50-55). Also available on the World Wide Web.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models. / Medicine, Faculty of / Graduate