a-Melanocyte-stimulating hormone (ai-melanotropin; a-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-MetGlu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that reversibly darkens amphibian skins by stimulating melanosome (pigment granule) dispersion within melanophores. By using a number of in vitro melanocyte assays, we have examined the conformational requirements for a-MSH activity. Synthesis of [half-Cys4,half-Cys 0.l-a MSH, a cyclic, conformationally restricted, "isosteric" analogue of a-MSH, provided a melanotropin with a potency > 10,000 times that of the native hormone in stimulating frog (Rana pipen) skin darkening. The cyclic analogue also showed substantially prolonged activity relative to the native hormone. Cys'l0-a-MSH was =30 times more potent than a-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. By using a cell-free Cloudman S-91 mouse melanoma plasma membrane preparation, we found the cyclic analogue to be "'3 times as potent as the native hormone in stimulating adenylate cyclase activity. These results provide insight into the conformational requirements for biological activity of a-MSH, and the comparative conformational requirements of a-MSH at a number of pigment cell receptors.Peptide hormones and their analogues provide useful molecular probes for investigating the chemical-physical basis ofbiological information transfer at specific target tissues. a-Melanocytestimulating hormone (a-MSH; a-melanotropin) is a linear tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-GlyLys-Pro-Val-NH2, that is synthesized and secreted by the pars intermedia of the vertebrate pituitary (1). The hormone may have important physiological roles in the control of vertebrate pigment cell melanogenesis (2), neural functioning related to learning and behavior (3, 4), and fetal development (4).a-Melanotropin structure-function relationships have been studied by analysis of the biological activities of a number of structurally or stereochemically modified a-MSH analogues and fragments in amphibian melanophores (5-8) and, more recently, on mammalian melanoma cells (9-11). The amino acid residues believed to be important in the expression of melanotropic activity of a-MSH have been systematically examined and, based on these studies, a-MSH apparently contains two message sequences, (Glu)-His-Phe-Arg-Trp and Gly-Lys-ProVal-NH2. Each ofthese sequences can independently stimulate melanosome dispersion in amphibian melanophores in vitro (7). It has been reported (12) that a-MSH, like other so-called sychnologically organized hormones [e.g., corticotropin (ACTH), f3-lipotropin, cholecystokinin] is a conformationally flexible molecule in aqueous solution. Perhaps because of the postulated inherent conformational flexibility of this peptide, the threedimensional topochemical requirements of a-MSH, which may be important for its biologically active conformation § at the melanotropin receptor, have not been experimentally examined.The various conformational requirements in a peptide hormone that ...

Here we report, for the first time, the formation of a biomimetic covalently imprinted polymeric sensor for a target ligand, the delta-opioid G-protein coupled receptor agonist DPDPE, which reproducibly exhibits subpicomolar binding affinity in an aqueous environment. In addition to having a well-defined and homogeneous binding site, the imprinted polymer template is quite stable to storage in both the dry and wet states and has at least 6 orders of magnitude higher affinities than exhibited by similar peptide-based molecular-imprinted polymers (MIPs) thus far. A highly sensitive optical detection methodology, plasmon-waveguide resonance spectroscopy, was employed, capable of measuring binding in real time and discriminating between ligand molecules, without requiring labeling protocols (fluorophores or radioisotopes). The DPDPE-imprinted polymer showed a broad structure-activity relationship profile, not unlike that found for protein receptors. Such sensitivity and robustness of MIPs suggests potential applications ranging from biowarfare agent detection to pharmaceutical screening.

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SynopsisEfforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen']. oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure-activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide a-melanotropin. Some of these peptides have exceptionally prolonged in uiuo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).

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