An important role for apoptosis has been established in various pathological processes, particularly in nervous tissues. Apoptosis essentially contributes to neuronal loss during normal development, after acute neuronal injury, and in chronic degenerative conditions such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. A major function in initiation and progression of apoptosis has been attributed to members of the caspase family. Of the 14 caspases identified in mammals, caspase-3 is a key effector of neuronal apoptosis triggered by various stimuli. Our previous studies and numerous subsequent reports from other laboratories established a central role for this caspase in execution of neuronal loss in vivo and in vitro. We have shown that blockade of caspase-3 using commercially available peptide-based reagents leads to reduction of cell death and associated neurological dysfunction. Hence, results suggest that previously developed tetrapeptide caspase inhibitors can provide promising targets for therapeutic development. However, all currently available caspase inhibitors have serious limitations for use as potential antiapoptotic drugs. Largely these limitations include insufficient specificity against individual members of the caspase family and associated toxicity at higher effective doses. Our recent research was focused on identification of more promising compounds. As a result, we have identified a tetrapeptide analog containing a 1,4-benzodiazepine ring system (compound 1) characterized by the remarkable inhibitory specificity against caspase-3 activity. A major goal of this proposal is to complete development of a more potent and specific, less toxic and cell-permeable caspase-3 inhibitor that would serve as a prototype for future therapeutic development to treat acute neurodegenerative conditions. Accordingly, we have four closely related specific aims, focused towards achievement of this aim: 1). To design and synthesize an extended library of derivatives of the identified caspase-3 inhibitor (the lead compound) using available to us modern molecular modeling tools as well as our previous experience in drug design and synthesis; 2). To evaluate and to measure inhibitory activity and relative selectivity of developed derivatives against all members of human caspase family, as well as against recombinant active caspase-3 of rat and mouse origin; 3). To estimate target selectivity of selected in Aim 2 caspase-3 inhibitors against available non-caspase CNS targets, including cell receptors, ion channels, transporters, and proteases; and 4). To evaluate cell permeability and toxicity of selected compounds in cultures of human and rat neural cells.
