This research started with a basic science question: Are there any differences in the second sphere of amino acids in the active site of the isozymes of NOS that could be identified for nNOS-selective inhibitor design? From this study, the first class of dual nNOS-selective inhibitors was identified. The moieties of the best lead compound that were important for selectivity were determined by structure modification; then the potency and selectivity were enhanced to provide highly potent and nNOS-selective dipeptide amides and peptidomi-metics, which were active in a rabbit model for fetal neurodegeneration. Crystal structures of these compounds bound to NOS isozymes showed that there was a one amino acid difference between nNOS and eNOS in the second sphere of amino acids; this was the difference that we were searching for from the beginning. With the aid of these crystal structures, a new fragment-based de novo design method was developed, called fragment hopping, which allowed the design of a new class of nonpeptide nNOS-selective inhibitors. These compounds have been modified to give low nanomolar, highly dual selective nNOS inhibitors, which were active in a rabbit model for the prevention of neurobehavioral symptoms of cerebral palsy. These compounds could have general application in neurodegenerative diseases because excessive NO leads to many of these diseases. However, these compounds are still too polar for good blood-brain barrier penetration, so future efforts are directed at increasing the bioavailability of these compounds, a common problem in drug design, which we are trying to resolve from basic principles important to bioavailability.
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