Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an a-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist Nle21'38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Giu-Xaa-Xa:-Lys scaffold. We have found astressin {cyclo (30)(31)(32)(33) Nle2l,38,Glu30,Lys33] Corticotropin-releasing factor (CRF; corticoliberin) is a 41-residue peptide amide which stimulates the release of corticotropin (ACTH) (1, 2) and acts within the brain to mediate a wide range of stress responses (3). The actions of CRF are mediated through binding to CRF receptors, several of which have been characterized recently (4-10). These receptors, like those for growth hormone-releasing factor, calcitonin, and vasoactive intestinal peptide, are coupled via G proteins and have seven putative transmembrane domains. The actions of CRF can also be modulated by a 37-kDa CRF-binding protein (CRF-BP) (11). To probe the physiological role of CRF, we have developed competitive antagonists that are particularly potent when administered in the central nervous system; however, these same analogs bind pituitary receptors with lower affinity than does CRF, and their peripheral administration results in weak and short-lived effects in vivo (12). Synthetic CRF antagonists such as the a-helical CRF-(9-41)

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians.

In an earlier report we identified specific modifications and substitutions of corticotropin releasing factor (CRF) that led to the discovery of antagonists with extended duration of action as compared to that of astressin {cyclo (30)(31)(32)(33) , which was found to be longer acting than astressin (Rivier, J.; et al. J. Med. Chem. 1998, 41, 5012-5019). To further increase the efficiency (potency, duration of action, and bioavailability) of this family of antagonists, we introduced two or more CRMe-leucine residues at positions shown in earlier studies to be favorable (Hernandez, J.-F.; et al.

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Hypothesis driven and systematic structure-activity relationship (SAR) investigations have resulted in the development of effective central nervous system (CNS) antagonists of corticotropin (ACTH)-releasing factor (CRF) such as alpha-helical CRF(9-41) and analogues of our assay standard [DPhe12,Nle21,38]hCRF(12-41). On the other hand, equally potent CRF antagonists that block the hypothalamic/pituitary/adrenal (HPA) axis had not been described until recently. Predictive methods, physicochemical measurements (nuclear magnetic resonance spectrometry and circular dichroism spectroscopy), and SAR studies suggest that CRF and its family members (urotensins and sauvagine) assume an alpha-helical conformation when interacting with CRF receptors. To further test this hypothesis, we have systematically scanned the hCRF(9-41) or hCRF(12-41) sequences with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown could maintain or enhance alpha-helical structure. From this series we have identified seven analogues that are either equipotent to, or 3 times more potent than, the assay standard; in addition, as presented earlier cyclo(30-33)[DPhe12,-Nle21,38,Glu30, Lys33]hCRF(12-41) (astressin) is 32 times more potent than the assay standard in blocking ACTH secretion in vitro (rat pituitary cell culture assay). In vivo, astressin is also significantly more potent than earlier antagonists at reducing hypophysial ACTH secretion in intact stressed or adrenalectomized rats. Since the corresponding linear analogues that were tested are significantly less potent, our interpretation of the increased potency of the cyclic analogues is that the introduction of the side chain to side chain bridging element (Glu30-Lys33, and to a lesser extent that of Glu14-Lys17, Glu20-Lys23, Glu23-Lys26, Glu26-Lys29, Glu28-Lys31, Glu29-Lys32, and Glu33-Lys36) induces and stabilizes in the receptor environment a putative alpha-helical bioactive conformation of the fragment that is not otherwise heavily represented. The effect of the introduction of two favored substitutions [(cyclo(20-23) and cyclo(30-33)] yielded 37 with a potency 8 times that of the assay standard but actually 12 times less than expected if the effect of the two cycles had been multiplicative. These results suggest that the pituitary CRF receptor can discriminate between slightly different identifiable conformations, dramatically illustrating the role that secondary and tertiary structures play in modulating biological signaling through specific protein-ligand interactions.