This proposal outlines a multidisciplinary research effort by four principal investigators to continue development of a series of conformationally and topographically constrained peptide analogues of dynorphin that have high receptor potency and selectivity for the kappa opioid receptor and its subtypes, potent agonist or antagonist in vivo activities, high stability in vivo, prolonged biological effects at kappa opioid receptors, and unique biological activities. This approach combines aspects of computer assisted molecular design, synthetic organic amino acid and peptide chemistry, conformational analysis and molecular mechanics and molecular dynamics calculations, and biochemical, biophysical, pharmacological and physiological studies. The specific aims of this research include: 1) continued developments in the design, synthesis and evaluation of novel conformationally and topographically constrained peptide analogues of dynorphin targeted for kappa opioid receptor types and subtypes; 2) to utilize topographically fixed or biased amino acid residues in conjunction with computer aided drug design methods to develop kappa receptor selective ligands; 3) to comprehensively examine the potency and selectivity of all new ligands for kappa vs mu and delta receptors using radioligand binding assays - highly selective and potent analogues for kappa receptors will be radiolabeled to high specific activity; 4) comprehensive examination of kappa vs. mu vs. delta binding distribution in the brain using autoradiography; 5) evaluation of opioid agonist and antagonist activities in vitro using especially the GPI and MVD assays to establish receptor selectivity and potency; 6) evaluation of in vivo kappa receptor properties of ligands highly selective for kappa receptors and kappa receptor subtypes using such tests as in vivo analgesic assays including the tail flick and hot plate assays, and the acid induced mouse abdominal constriction test; 7) careful evaluation of the enzymatic stability of key compounds and their serum half-lives, so as to develop analogues that are stable with good biodistribution; and 8) to carefully examine the conformational of properties the most potent and selective analogues by 1D and 2D nuclear magnetic resonance spectroscopy, molecular mechanics calculations, and other biophysical methods. The long-term goal of this research is to develop an understanding of the physiological roles of kappa opioid receptor types and subtypes, and to develop specific ligands so that they can be used for the treatment of pain and disease.