The proposed research involves a multi-disciplinary approach to develop novel psychotherapeutic agents that pass the blood-brain barrier to stimulate the human neurotensin receptor. These agents may be potential drugs for treatment of the diverse neuropsychiatric disorders of schizophrenia and Alzheimer's type dementia. To achieve this goal, we plan to use the cloned human neurotensin receptor stably expressed in a transfected cell line to continue our work on two novel neurotensin receptor agonists that we have synthesized. One compound represent an intermediate step toward the synthesis of non-peptide neurotensin agonists and was designed on the basis of our molecular modeling by computer of neurotensin (8-13). The second, is an adamantane derivative of one of our novel neurotensin (8-13) peptide analogs and appears to penetrate the brain of mice. To learn more about the binding site(s) for neurotensin and these novel compounds, we plan studies involving site- directed mutagenesis of the human neurotensin receptor. These studies will likely be feasible since, with DNA sequence information from the cloned rat receptor, we have obtained to date by polymerase chain reaction (PCR) (from a CDNA library of human substantia nigra, a rich source of neurotensin receptors) and CDNA screening of a human library, about 90% of the human neurotensin receptor open reading frame sequence. At the amino acid level, there is about 87% identify in a 377 amino acid overlap between the rat and human receptors. After obtaining the entire sequence in a clone, we plan, as we have done with the cloned rat receptor, to study the human neurotensin receptor in a transfected cell line. In addition, with our excellent probe for the human neurotensin receptor, we plan to study by in situ hybridization the expression of the receptor in normal human brain and in Alzheimer's disease brains. To characterize the expressed cloned human neurotensin receptor, we will do radioligand binding studies with [3H]neurotensin and membranal preparations from cells and measure neurotensin's effects on second messenger (inositol phosphates and CAMP) synthesis by intact cells. Our novel neurotensin receptor agonists will be tested in these assays with the expressed human neurotensin receptor. Finally, we will test in mice the effects of these compounds on sleep time induced by pentobarbital, on body temperature, and on analgesia to characterize their in vivo effects.
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