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Melanocortins play a key role in appetite regulation across species. One such melanocortin, beta-melanocyte stimulating hormone (beta-MSH) is receiving increasing attention for its anorexigenic effects. In chicks selected for low (LWS) and high (HWS) juvenile body weight, beta-MSH differentially decreased food intake and HWS chicks may be more sensitive to its effects. Both lines responded similarly to beta-MSH with decreased water intake. While whole blood glucose concentrations and ingestive and non-ingestive behaviors (sit, stand, preen, perch, deep rest, jumps, escape attempts, feed pecks, defecations, and total distance traveled) were not affected in either line, beta-MSH increased corticosterone in LWS chicks but not HWS chicks. However, despite the increase in corticosterone concentration in LWS, astressin, a corticotrophin releasing hormone (CRH) receptor antagonist, did not attenuate the effects of beta-MSH in either line suggesting that the altered stress response may not be acting via CRH receptors. When beta-MSH was co-administered with HS014, a highly selective antagonist for the melanocortin 4 receptor, only LWS responded with an attenuated response to beta-MSH suggesting that the differential response may in part be due to altered receptor affinity or binding resulting from the selection process. To investigate the roles of the hypothalamus and hindbrain in the differential food intake response, an experiment was designed where chicks were injected targeting either the lateral or 4th ventricle utilizing a novel freehand injection procedure. Chicks from both lines responded similarly to beta-MSH following both lateral and 4th ventricle injections. Together, these data suggest that alterations in the b-melanocortinergic appetite regulation system may be in part responsible for the differential body weights of the LWS and HWS lines. [Adaptations of chapters II, III, and IV have been published in Neuroscience Letters, Journal of Neuroendocrinology, and Behavioural Brain Research, respectively] / Ph. D.

Thesis (Ph. D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 202 pages. Includes Vita. Includes bibliographical references.

The melanocortin system regulates various physiological processes including feeding behavior, sexual function, skin pigmentation and photoprotection via five G-protein coupled receptors and several endogenous ligands. There is a need for selective and potent ligands to the human melanocortin receptors (hMCRs) that can chemically resolve these various functions. This thesis presents three studies aimed at refining the understanding of the structural differences between binding pockets of the hMCR subtypes. In the first study α-N-methylated analogues of the non-selective agonist, MT-II, are evaluated for their in vitro function. This study produced the most potent hMC1R selective agonist to date. The following two studies examine the effects of mutations on the biological activity of melanocortin receptor subtypes 1 and 4. Much of the mutation study data is preliminary and requires a demonstration of reproducibility.

Zugl.: Giessen, Univ., Diss., 2005

Universiẗat, Diss., 2005--Giessen.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

The melanocortin 3 and 4 receptors share 58% overall amino acid identity and 76% similarity. This high level of similarity between the MC3R and the MC4R underscores the difficulties associated with developing MC3R selective ligands, and as a consequence little is known of the physiological functions of the melanocortin 3 receptor. Previous research showing the differences between endogenous non-selective ligands and melanocortin 3 receptor selective ligands are mainly within the C-terminus of the melanocortin peptide. These findings have been exploited in this research using known melanocortin 3 and 4 selective ligands modified at their respective C-termini to develop some very promising melancortin 3 selective antagonists and agonists, analog 5 ([CO(CH₂)₂CO-DNal(2')-Arg-Trp-Lys]-Gly-Lys-Pro-Val-NH₂) and analog 20 ((H-DNal(2')-c[Asp-Pr6-DPhe-Arg-Trp-Lys]-Ala-Gly-Pro-Val-NH₂) respectively. Additional studies using molecular modeling have produced further insights into the structural basis for selectivity. Finally, we have been developing a new scaffold for the melanocortin receptor using cyclic dipeptide derivatives.

The melanocortin-1 receptor (MC1R) is a G-protein coupled receptor (GPCR) expressed on the surface of the melanocyte. MC1R activation after UV exposure results in the production of the dark eumelanin pigment and the tanning process in humans, providing protection from UV induced DNA damage. MC1R activation has also recently been linked to DNA repair. The MC1R gene is highly polymorphic in Caucasian populations with a number of MC1R variant alleles associated with red hair, fair skin, poor tanning and increased risk of melanoma and non-melanoma skin cancer. These MC1R variant receptors were thought to be loss of function, however the type of defect and the extent of the loss of function for individual variants was relatively unknown before the commencement of this PhD project. Many GPCR mutant proteins are intracellularly retained, resulting in a loss of signalling ability. To determine if this was the case for MC1R variant receptors, the localisation of the wild type and variant MC1R protein was investigated using immunofluorescence and radio-ligand binding on transfected melanocytic cells as well as primary melanocyte strains. For the first time, several MC1R variants including V60L, R151C, I155T, R160W and R163Q, were shown to have reduced cell surface expression compared to wild type MC1R. cAMP assays were used to determine the signalling ability of activated wild type and variant MC1R, importantly, variant receptors with reduced cell surface expression showed corresponding impairment in cAMP signalling. In contrast, the R142H and D294H variants, which have normal cell surface expression but significantly impaired cAMP signalling, are thought to have a defect in G-protein coupling. Some MC1R variants were found to have dominant negative activity on the wild type receptor in co-expression studies, this result may explain the MC1R heterozygote effect on human pigmentation phenotypes. This dominant negative effect resulted in either reduced wild type cell surface expression or reduced G-protein coupling and may be mediated by receptor dimerisation. In order to validate the in vitro studies, comparison of variant receptor characteristics with skin and hair colour data of individuals both homozygous and heterozygous for MC1R variant alleles was performed. This revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and the strength of the effects of variant alleles on human pigmentation. From the in vitro functional studies, it was clear that most variant receptors retained some signaling ability, although the relative abilities varied. An important unanswered question in the literature was whether the phenotype of carriers of the high penetrance MC1R variant alleles was actually representative of complete loss of function for MC1R. Due to the rarity of MC1R null alleles they had only previously been found in the heterozygous state, however we described the phenotype of one individual compound heterozygous for two frameshift mutations resulting in an individual unable to produce any functional MC1R protein. Phenotypic analysis indicated that red hair and fair skin is found in the absence of MC1R. Finally, preliminary studies using low temperature, chemical or pharmacological chaperones indicated that the cell surface expression of some MC1R variants could be rescued in cell transfection experiments. This resulted in a restoration of signaling ability after stimulation with agonist. These studies into the localization and function of MC1R variants have contributed to a greater understanding of the molecular mechanism underlying the association of MC1R with skin cancer risk phenotypes, and may lead to future drug based therapies that are able to rescue the function of MC1R variants that are intracellularly retained.

Peptidergic G Protein-Coupled Receptors (GPCRs) play a role in many of the most important biological functions, and the ability to modulate the activity of these critical proteins has tremendous potential to increase our understanding of biology and allow the development of new therapeutics. In some cases this knowledge will point towards the importance of interconnected proteins of the same or different classes, such as kinases, which interact in a complex and dynamic network in vivo. Understanding these systems will be crucial for addressing unmet therapeutic needs, and new chemical structures may be important at every step of the process.Our contribution to this pursuit includes the development of new ligands for the melanocortin receptors based on a bicyclic or tricyclic core structure. These were designed to be peptidomimetics, built from amino acids to leverage the accumulated knowledge of the group but with properties that complement those of peptides. Most of the molecules in this series bind to the melanocortin receptors, and many with significant selectivity. Some are selective for the MC5R, which may allow further study of this widely distributed but largely unexplored subtype. Others bind preferentially to the MC1R, a property which may be useful in the development of imaging agents targeting melanoma.Imaging using fluorescent probes can provide a tremendous amount of information in studies of receptor biology. With this in mind, we have developed new fluorescent ligands which bind to melanocortin receptors. These compounds use the previously discovered bicyclic template and incorporate the small organic fluorophores anthranilate and N-methylanthranilate.While these structures are in a sense bifunctional, as they exhibit both pharmacologic and fluorescent activity, other molecules may instead incorporate two different pharmacophores. We have synthesized designed multiple ligands (DMLs) of this type for the opioid and neurokinin receptors, as well as molecules which target both the opioid receptors and p38 MAP kinase. These structures merged known active ligands, such as fentanyl for the opioid activity, into one bifunctional molecule. In addition we have used our newly developed template to create a novel NK1R antagonist which may be part of the next generation of bifunctional ligands.