Apoptosis, also known as programmed cell death, is a normal physiological cell death process, essential for morphogenesis, homeostasis, and defense of multicellular organisms. Excessive apoptosis is associated with numerous disease states including neurodegenerative disorders, stroke, ischemic injuries, acquired immunodeficiency syndrome (AIDS), osteoporosis, and amyotrophic lateral sclerosis (ALS0. Initiation of apoptosis results upon binding of TNF or FAS ligand to various cell surface receptors. Signal transduction across the cell membrane results in the activation of caspases from their pro-forms. Caspases are cysteine proteases with specificity for Asp residues in their natural substrates. Activation of one procaspase by another caspase results in a proteolytic cascade, which eventually causes proteolysis of key proteins that are known to be selectively cleaved in apoptosis. The goal of this research is the design and synthesis of transition-state reversible and mechanism-based irreversible inhibitors for caspases, particularly caspase-3, 6, and 8. Peptide alpha-ketoamide inhibitors are transition-state inhibitors, which inhibit cysteine proteases by forming a tetrahedral, adduct with the active-site cysteine of the enzyme. We propose to synthesize specific tetrapeptide inhibitors for the caspases. A new class if irreversible inhibitors will be designed and synthesized with specificity for individual caspases. We propose to develop methods for preparing libraries of irreversible caspase inhibitors. All new inhibitors will be assayed for potency and specificity with all available caspases. The most potent and specific inhibitors for each caspase will be tested in apoptosis assays. It is likely that caspase inhibitors will have therapeutic potential for the treatment of stroke and other neurodegenerative diseases. This research should provide information of the active site structure of the various caspases and lead to the design of better drugs.
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