NEW DIRECTIONS IN RECEPTOR RESEARCH; PROMISCUITY VERSUS SPECIFICITY, AND ESTABLISHMENT OF A NEW MODEL OF OPIOID TOLERANCE/DEPENDENCE

My laboratory is predominantly interested in two research areas: one of them is to reveal the molecular mechanism of receptor functioning from the molecular level analysis of ligand-receptor interactions to in vivo studies; the other is to understand the molecular basis of drug tolerance/dependence. Evidence is accumulating to show that receptors do not always function as monomers, but may also interact with each other either via cross-talk at the signaling level, or by forming homo- and hetero-oligomers. These molecular complexes may have altered binding and signaling properties. Understanding their interactions may provide new targets for drug discovery with less side-effects. In recent years, we have been focusing on two systems, the opioid and cannabinoid receptors, both of which play key roles in analgesia and drug addiction. The ligand-specific nature of the molecular changes underlying opioid analgesic tolerance is being explored. These studies contribute to our understanding of drug addiction, thus may be useful in developing potent analgesic drugs with less side-effects.

GPCRs are the largest class of cell-surface receptors and are encoded by >1,000 genes in the human genome. More than 50% of all current drugs regulate GPCR function, and some 30% of these drugs directly target GPCRs. Currently, our laboratory is focusing on the opioid and cannabinoid receptor families and their interactions. We have demonstrated cross-antagonism between G-protein signaling of GABA_B and cannabinoid CB₁ receptors, which altered the pharmacology of the individual receptors. The interaction was tissue-specific and only manifested in membranes of hippocampus, but not in cerebral cortex or spinal cord. The cross-talk of the two receptors is being investigated by confocal microscopy and BRET studies in cell lines co-transfected with GABA_B and CB₁ receptors in Hungarian and US collaborations.

Many GPCRs exhibit constitutive activity in the absence of agonists that can be selectively blocked by ligands that is referred to as inverse agonists. The CB₁ receptors display a significant level of constitutive activity. We have shown that the CB₁ specific antagonist rimonabant (SR141716) displays CB₁ receptor-independent inverse agonist properties at high concentrations. It was found to be able to block G-protein signaling of the highly mu-opioid receptor specific agonist, DAMGO in CHO cells expressing recombinant mu-opioid receptor (MOR-CHO). Co-application of SR141716 with DAMGO unmasked a PTX-insensitive, novel, MOR-mediated G-protein signaling. Notably, the novel signaling was desensitized by chronic morphine treatment. These results provide new directions to understand drug tolerance/dependence.

The new model of tolerance, to which our previous results has contributed suggests that there is an inverse relationship between the extent of receptor internalization and the abuse potential of the ligand. To test this hypothesis, rats are treated acutely or chronically with various ligands, followed by pharmacological, receptor binding and signaling as well as autoradiography measurements. A wide spectrum of ligands is being used to test the correlation between the extent of receptor internalization and the chemical structure, the efficacy and the abuse potential of the ligand. Morphine, the most widely used opioid analgesic, which has high abuse potential and is a partial agonist in rat brain, did not internalize surface MORs, but elicited significant intracellular changes and altered signaling. All the full agonists tested, namely DAMGO, endomorphins and etorphine decreased the density of the surface MORs. It is concluded that analgesic tolerance/dependence is a complex phenomenon that cannot be described by a single molecular mechanism. The changes observed are ligand- and tissue-specific, and are further investigated.

To develop new opioid ligands with improved properties (higher specificity, selectivity or longer half-life), new peptides synthesized by the Neuropeptide Laboratory of the BRC are being studied in receptor binding assays. A new, conformationally constrained analog of the well-known delta-opioid antagonist TIPP was radiolabeled and the resulting ligand, [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH characterized in binding assays. The new ligand was 4x more potent delta-antagonist than the parent ligand. Based on structure-activity relationship studies performed with 16 new endomorphin peptidomimetics, two ‘lead’ molecules have been selected. [³H]cisACPC-endomorphin-2 and [³H]cisACHC-endomorphin-2 [results to be published] have been prepared and characterized in binding and functional assays. They were found to be high affinity and selectivity MOR agonists with long half-lives. These two new ligands may be helpful tools in mapping the bioactive conformation of MORs. These new ligands will also be evaluated in future in vivo chronic studies.