Our general goal is to investigate the mechanism by which selected bioactive peptides [corticotropin releasing factor (CRF), growth hormone releasing factor (GRF), and melanin concentrating hormone (MCH)] act on their receptors to produce a specific biological/physiological effect. We plan to: (a) define structural motifs and the involvement of specific amino acids in binding and transduction and use this information for the design of potent antagonists (pharmacological studies); (b) investigate the secondary and tertiary structures of such analogs as superagonists and antagonists as well as model template assembled proteins using spectroscopic and computational approaches in order to understand peptide/receptor interactions (structural studies); and (c) investigate the usefulness of CRF, GRF and some selected analogs as potential drugs or tools to define and understand related pathophysiological states (clinical studies). A major effort will be directed toward the design of constrained analogs of CRF and GRF. Using in vitro and in vivo biological tests, binding assays, spectroscopic measurements such as circular dichroism (CD), nuclear magnetic resonance (NMR), and computer simulations, we hope to design and synthesize long-acting analogs that have agonistic and antagonistic properties. In order to achieve this goal we will introduce intramolecular bridges and unnatural amino acids in ways that were proven successful in previous studies. In addition, in order to gain an appreciation of the bioactive conformation of CRF/GRF, we will characterize, using computer simulation, the accessible conformations of these CRF/GRF analogs in vacuo, in water, and in a membrane model. We will examine the stability of the putative alpha-helical structures, determine the effects of amino acid substitutions on their relative stability, and compare these results with those suggested by spectroscopic measurements. These results will be used, together with those derived from structure activity relationships, to predict sequences with desired features as a rational approach to drug design. Miscellaneous peptides and DNA probes to be used by other members of this program project will also be synthesized.
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