Our general goal is to investigate the mechanism by which selected bioactive peptides [corticotropin releasing factor (CRF) and growth hormone releasing factor (GRF) act on their receptors to produce a specific biological/physiological effect; for CRF, interactions with its binding protein will also be examined. 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 using both spectroscopic and computational approaches in order to understand peptide/receptor/binding protein 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, dimers, truncated dimers and chimeras of CRF (also see Project I which proposes to express receptor chimeras). Using in vitro and in vivo biological tests, binding assays, spectroscopic measurements { such as circular dichroism (CD)} and computer simulations, we hope to design and synthesize low molecular weight, long-acting analogs that have agonistic or antagonistic properties. In order to achieve this goal we will introduce intramolecular bridges (i, i+3, Glu-Lys lactam scan), unnatural and structurally constrained amino acids (beta-methyl amino acids and betidamino acid scan: betidamino acids are mono-acylated aminoglycine derivatives where each acyl group mimics an amino acid side chain) and alpha-helical templates (to be developed) in ways that were proven successful in previous studies. Additionally, in order to gain an appreciation of the bioactive conformation of CRF/GRF, we will examine, using computer simulation, the accessible conformations of these CRF/GRF analogs in vacuo , in water, and in a membrane model. We will examine the putative alpha-helical structures, determine the effects of amino acid substitutions and compare these results with those suggested by spectroscopic measurements. As a rational approach to drug design, these results will be used together with those derived rom structure activity relationships to predict new sequences with desired features. Bioactive peptides characterized by Project I and Core C will also be synthesized and preliminary SAR (such as determination of bioactive core) will be carried out.
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