The overall goal of our continuing interdisciplinary studies of opioids is the design of behaviorally selective analgesics with diminished respiratory depression and physical dependence liability. The working hypothesis underlying all the proposed studies is that ligands that bind with significant affinity to the three cloned opioid receptors (mu, delta and kappa) but with different combinations of activation and inhibition properties at the three receptors could be promising behaviorally selective agents. To identify such candidates, two major themes will be pursued: 1) continued in vitro and in vivo characterization of activation selective ligands. The goal of these studies is to identify the specific combinations of agonist and antagonist activity at the three cloned receptors that lead to analgesia with diminished respiratory depression and reward response. 2) continued characterization of the requirements of recognition and activation at each of the cloned opioid receptors (mu, delta and kappa). The goal of these studies is to design new therapeutic agents with the desired combinations of activation and inhibition properties at the three receptors. In these interdisciplinary efforts, all in vitro assessment of affinities and activation will be performed in transfected cells containing cloned mouse, mu, delta and kappa receptors. All in vivo studies will be carried out using wild type and the corresponding mu, delta and kappa """"""""knockout"""""""" mice with dysfunctinal genes for each of the three opioid receptors. Ligands selected for the in vitro assessment will be those with existing evidence for activation selectivity. Ligands selected for in vivo assessment will those for which activation selectivity has been verified in the transfected cells studies. All molecular requirements for recognition and activation of receptors developed by computational methods will be made for the cloned mouse mu, delta and kappa receptors. Specifically, common molecular determinants for recognition of all three receptors and possibly unique determinants of activation of each of them will be obtained. The mechanistic implication of the activation requirements will be probed by simulating ligand receptor interactions with 3D models of the opioid receptors constructed in our laboratory. Mutation of residues identified from these studies to be important in activation will be carried out and their pharmacological effects determined. Use of the 3D models for explicit characterization of ligand receptor complexes will bridge the gap between structure and function that cannot be filled by studies of the ligands alone. Focusing on the mouse and its three cloned receptors will maximize the consistency of the results obtained and the mechanistic and therapeutic inferences draw from them.
