Biologically many disease states lead to changes in expressed proteins. Therefore, "system changes" that occur must be considered in any treatment for the disease. This new approach to drug design and discovery would be particularly applicable to the diseases that involve adaptive changes in the central nervous system, such as neuropathic pain. There is growing evidence that drugs behave differently in pathological states than in normal states, thus preventing their effectiveness in pathological disease states. Therefore, a new paradigm for drug design is needed. In recent years, the melanocortin-4 receptor (MC4R) found in the spinal cord and CNS has received growing attention as a therapeutic target. MC4R based agonist ligands produce anti-opioid effects, and researchers have shown that an antagonist of the MC4R can produce pronounced anti-allodynic effect. Opioid receptors have been the central and most efficacious targets sought after for relieving neuropathic pain. In our new approach, single peptide molecules are designed to interact with opioid receptors as an agonist, and as an antagonist at the MC4 receptor. For the treatment of pain, a series of linear and cyclic peptides based on the overlapping pharmacophores of endogenous melanocyte stimulating hormone and opioid ligands are designed through computational aided molecular modeling and synthesized. Throughout the studies the opioid pharmacophore is maintained towards the N-terminal while melanocortin pharmacophore is maintained towards the C-terminal. Cyclization of peptides has been the central synthetic feature in designing the bifunctional ligands. The use of microwave has been shown to be very efficient in cyclizing the peptides. Solvent, reagent, power and temperature conditions are established for the microwave application in aiding the macromolecules for cyclizing their side chain termini.

Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.

The human melanocortin receptors (hMCRs) play a fundamental role in human behavior such as satiety, feeding, sexual and more. A set of SHU9119 peptide derivatives were studied for their structure-activity relationships. These peptides contained a sequential a-N-methylation amino acid scan.A second set of peptide derivatives intended to be used to create TNF-a; inhibition, via the melanocortin receptors. These peptides were shown to bind to all of the hMCR receptors, and only exhibit cAMP stimulation at hMC1R/hMC5R.The data from both of the sets of compounds illustrate that small changes in the stereochemistry of the SH9119 and TNF-a; derivatives cause drastic changes in the binding and the agonistic/antagonist properties of the compounds.This thesis determined the effect that hMC1R mutations have on the binding and cAMP response of well characterized ligands. This study ruled out 9 different residues for being the required for the cAMP response of the hMC1R.

Oxytocin release within the brain modulates several social behaviours in animals and humans. Moreover, low central oxytocin content has been linked to neuropsychiatric disorders, such as anxiety and autism. The exogenous administration of oxytocin has been proposed for therapeutic treatment, but oxytocin does not cross the blood-brain barrier (BBB) in physiologically significant amounts. An alternative approach to oxytocin administration is to stimulate central oxytocin release using melanocortins. Central administration of the naturally occurring melanocortin, α-MSH, has been shown to trigger somatodendritic oxytocin release in vitro. Unfortunately, endogenous melanocortins also do not penetrate the BBB in neuroactive amounts. In this study, I investigated whether systemic administration of synthetic melanocortin receptor 3/4 (MC3/4) agonist, Melanotan-II (MT-II), affects oxytocin neuronal activity and secretion in anaesthetised rats. I hypothesised that systemic administration of MT-II directly (centrally) acts on magnocellular oxytocin neurones to trigger somatodendritic oxytocin release from neurones of the supraoptic nucleus (SON) of the hypothalamus in vivo. Firstly, using double immunohistochemistry against Fos protein, a widely used marker for neural activity, and oxytocin, I showed that intravenous (i.v.; 1 mg/kg), but not intranasal (1 and 30 μg rat), administration of MT-II markedly induced Fos expression in magnocellular oxytocin neurones of the SON and paraventricular nuclei (PVN) of the hypothalamus, and this response was prevented by prior intracerebroventricular (i.c.v.) administration of the melanocortin antagonist, SHU-9119 (1 μg rat). In addition, brain areas receiving peripheral inputs which are involved in the regulation of oxytocin and vasopressin release were also analysed, showing that i.v. MT-II significantly increased Fos expression in the nucleus tractus solitarii (NTS), but not in circumventricular organs of the anteroventral third ventricle (AV3V) region. MT-II-induced Fos in the NTS was not prevented by the i.c.v. melanocortin antagonist. Then, using in vivo electrophysiology, I investigated whether i.v. administration of MT-II affects the electrical activity of SON neurones. Extracellular single-unit recordings from identified magnocellular neurones of the SON showed that MT-II significantly increased the firing rate in oxytocin neurones, however, no significant changes in firing rate were detected in vasopressin neurones. Finally, in vivo oxytocin release experiments showed that i.v. administration of MT-II did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis and subsequent radioimmunoassay. Interestingly, the i.c.v. administration of MT-II (1 μg rat) also failed to trigger oxytocin release within the SON. The analysis of oxytocin content in plasma revealed that the change in oxytocin concentration was significantly greater in i.v. MT-II injected rats compared to vehicle-injected rats. Taken together, these results show that after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. As previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated, at least in part, indirectly by activation of inputs from the caudal brainstem.

No description available.

Biomarkers are biological characteristics that can be observed or measured during disease conditions, and compared to the healthy state. Biomarkers have been used in medical history to study disease progression, to develop drugs, or to predict drug efficacy. However, in complex diseases such as in cancer, biomarkers vary tremendously among patients and disease stages. Cell surface receptors, proteins that are located at the cell surface and deliver external signals into the cell, are a significant group of easily-detectable biomarkers. Along with the detection of particular biomarkers related to a disease, extensive characterization of expression patterns is necessary to optimize their application. Therefore, we designed a technique to imprint or capture the expression pattern of these receptors on silver nanoparticles. We incorporated branched molecules that can simultaneously bind to the target receptors and the nanoparticle surface. To develop the technique, we used melanocortin receptor 1 (MC1R), a receptor present at high levels on the surface of melanoma cells, as a test system. We determined optimum binding of this molecule in an established melanoma cell line, WM-266-4. We also synthesized a labeled molecule that was used to estimate the number of MC1R proteins on these cells. These studies indicate that this might be a promising approach for developing sensitive and cost-effective tools to characterize cell surface receptors in studying complex diseases and cell mechanisms. / MS / Biomarkers are biological characteristics that can be observed or measured during disease conditions, and compared to the healthy state (e.g. grades of fever during infection). Biomarkers have been used in medical history to study disease progression, to develop drugs, or to predict drug efficacy. However, in complex diseases such as in cancer, biomarkers vary tremendously among patients and disease stages. Cell surface receptors, proteins that are located at the cell surface and deliver external signals into the cell, are a significant group of easily-detectable biomarkers. Along with the detection of particular biomarkers related to a disease, extensive characterization of expression patterns is necessary to optimize their application. Therefore, we designed a technique to imprint or capture the expression pattern of these receptors on silver nanoparticles. We incorporated branched molecules that can simultaneously bind to the target receptors and the nanoparticle surface. To develop the technique, we used melanocortin receptor 1 (MC1R), a receptor present at high levels on the surface of melanoma cells, as a test system. We determined optimum binding of this molecule in an established melanoma cell line, WM-266-4. We also synthesized a labeled molecule that was used to estimate the number of MC1R proteins on these cells. These studies indicate that this might be a promising approach for developing sensitive and cost-effective tools to characterize cell surface receptors in studying complex diseases and cell mechanisms.

Prolylcarboxypeptidase (PRCP, EC3.4.16.2) ist ein ubiquitär exprimiertes Enzym, mit höchster Expression im Maushirn. Es spaltet spezifisch die letzte carboxyterminale Aminosäure von Substraten, deren vorletzte Aminosäure ein Prolin ist. Seine bisher publizierten Substrate Angiotensin II (AngII) und alpha Melanocortin Stimulierendes Hormone (alphaMSH) legen eine Rolle von PRCP in der Entwicklung von kardiovaskulären und metabolischen Krankheiten nahe. Um die in vivo Funktion von PRCP zu studieren, wurde eine Knockout Maus generiert (PRCP-/-). Metabolischer Phänotyp: PRCP-/- Mäuse zeigten generell ein reduziertes Körpergewicht, selbst wenn sie über Monate mit einer Hochfettdiät versorgt wurden. Erhöhte Plasmaleptin Werte und Proopiomelanocortin (pomc) Expression in knockout Hypothalami wiesen auf eine wichtige Rolle von PRCP in der Regulation von Futteraufnahme und Energiehomöostase hin. Eines der Genprodukte von pomc ist alphaMSH, welches im Hypothalamus die Futteraufnahme terminiert. Die carboxyterminale Struktur dieses Neuropeptids erfüllt alle Voraussetzungen, um von PRCP gespalten zu werden. Zudem konnte prcp Promotoraktivität in den selben Hirnstrukturen gezeigt werden, in denen auch alphaMSH-Wirkung beschrieben wurde. Eine mögliche Funktion von PRCP wäre somit die Inaktivierung des Appetitzüglers alphaMSH im Hypothalamus. Kardiovasculärer Phänotyp: Zunächst erwiesen sich zirkulierende Ang-Peptide in PRCP-/- Mäusen als unverändert. Jedoch konnte ein erhöhtes Niveau des Degradationsproduktes Ang1-7 in der Niere gezeigt werden. Die Entdeckung einer erhöhten Enzymaktivität von Angiotensin Converting Enzyme 2 (ACE2) in PRCP-/- Nieren, wurde als Kompensation der fehlenden PRCP Aktivität in PRCP-/- Nieren interpretiert. bot einen Erklärungsansatz für dieses Ergebnisse. Es wird davon ausgegangen, daß ACE2 die fehlende PRCP Aktivität in knockout Mäusen kompensiert. Das es sich hierbei um eine lokale begrenzte Kompensation handeln muß, zeigten der erhöhte Blutdruck und Herzrate, sowie die milde Herzhypertrophie. Da spezifische prcp Promotoraktivität in Hirnnuclei gefunden wurde, die in die Kontrolle der Herzfrequenz und des Blutdrucks involviert sind, wird eine regulatorische Funktion von Hirnstamm-PRCP auf Herzrhytmus und Blutdruck vermutet. / Prolylcarboxypeptidase (PRCP, EC3.4.16.2) is an enzyme specifically cleaving the last carboxy-terminal amino acid from substrates containing a penultimate proline. Its known potential substrates are linked to cardiovascular and metabolic phenomenon. To analyse the in vivo function of this enzyme a PRCP knockout mouse was generated. Homozygous knockout mice are viable but show tendency of decreased life span. In mice prcp expression is present in all tissues tested with very specific localizations of prcp promotor activity to distinct brain areas within the cortex, hippocampus, hypothalamus and the brain stem. The metabolic phenotype of PRCP deficient mice is characterized by low body weight even when feeding the animals a high fat diet. The increased plasma leptin levels and elevated expression of proopiomelanocortin gene (pomc) found in knockout hypothalami suggests an involvement of PRCP in the regulation of food intake and energy homeostasis. One of the gene products of pomc is alpha-melanocortin stimulating hormone (alphaMSH) that is terminating feeding when released from hypothalamic POMC neurons. Its carboxy-terminal structure is fitting the cleavage preferences of PRCP. Prcp promotor activities are localized in arcuate nucleus and paraventricular nucleus, brain areas of known alphaMSH signalling, supporting a role of PRCP in the degradation of central alphaMSH. The impact of PRCP on angiotensin II (AngII) metabolism was studied by determining the level of AngII and its degradation product Ang1-7 in blood and tissues. But instead of increased AngII levels due to the missing degradation enzyme in knockout mice, Ang1-7 levels were increased in kidney. These results were explainable by the increased activity of angiotensin converting enzyme 2 (ACE2) found in kidney. Probably ACE2 is compensating the lack of PRCP in the knockout mouse. Nevertheless, blood pressure and heart rate of PRCP knockout mice was increased. The mild hypertension was accompanied by mild hypertrophy of the hearts. Prcp promotor activity was found in brain stem an area important for regulation of blood pressure and heart rate suggesting that central PRCP regulates blood pressure.

No description available.

Domestication in chickens has made feed-restriction a necessity if broiler breeder hens should reach sexual maturity and be fertile. This is claimed to cause chronic hunger. To measure hunger the gene expression of the appetite regulators agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and adenosine monophosphate-activated protein kinase (AMPK) of the melanocortin system was quantified with qPCR. This was done in feed-restricted Red Junglefowl and compared with the gene expression of two strains of feed-restricted broilers, Ross 308 and Rowan Ranger, to detect possible effects on domestication on appetite regulation. POMC-expression was upregulated 2-fold in the feed-restricted Red Junglefowl. POMC-expression was downregulated by half in the feed-restricted Ross 308. AgRP/NPY-expression was upregulated 4-fold in feed-restricted Rowan Rangers. A comparison between the control groups (ad libitum fed) of the breeds showed that the NPY-expression was lower in Ross 308 and Rowan Ranger compared with the ancestor. Results show no difference in body weight of ad libitum fed and feed-restricted Red Junglefowl. Conclusions were that the feed-restricted Red Junglefowl was not properly restricted in food supply since no difference in body weight between the treatment groups was detected. The upregulation of POMC in the feed-restricted Red Junglefowl could be stress-linked influenced by the feeding type (scattering of food in litter). No conclusions of the impact of domestication on chicken’s appetite could be drawn. Domestication has probably had its impact by altering other signaling pathways of the melanocortin system than in the arcuate nucleus.

The grey squirrel (Sciurus carolinensis) has wildtype and melanic (dark) colour morphs. Melanism is associated with variations in the melanocortin-1 receptor (MC1R) gene in a number of species. The MC1R protein is a G-protein coupled receptor, predominantly expressed in melanocytes, where it is a key regulator of pigment production. To investigate the genetic and molecular basis of melanism, the MC1R genes of the wildtype and melanic grey squirrel were sequenced. The wildtype (MC1R-wt) and melanic (MC1RΔ24) variants of the MC1R were then functionally characterised in a cell-based assay. The MC1R gene of the grey squirrel was found to have a 24 base pair (bp) deletion associated with melanism. The MC1R is typically activated by its agonist, the alpha-melanocyte stimulating hormone (α-MSH), which stimulates dark pigment production by raising intracellular cAMP levels. Conversely, the MC1R is inactivated by its inverse agonist, the agouti signalling protein (ASIP), which stops dark pigment production by lowering intracellular cAMP levels. To investigate the effects that the 24 bp deletion have on receptor function, MC1R-wt and MC1RΔ24 genes were transfected into HEK293 cells. Cells expressing either MC1R-wt or MC1RΔ24 were stimulated with α-MSH or ASIP and intracellular cAMP levels were measured. Unstimulated MC1RΔ24 cells showed higher basal activity than the MC1R-wt cells. Both MC1R-wt and MC1RΔ24 cells responded to α-MSH with a concentration-dependent increase in intracellular cAMP. However, while the MC1Rwt cells responded to ASIP with a concentration-dependent decrease in intracellular cAMP, MC1RΔ24 cells responded with an increase in cAMP. Melanism in the grey squirrel is associated with a 24 bp deletion in the MC1R. Cells expressing MC1RΔ24 have higher basal levels of cAMP than MC1R-wt cells. ASIP acts as an inverse agonist to the MC1R-wt but as an agonist to the MC1RΔ24. As MC1RΔ24 cells have higher levels of cAMP, and higher levels of cAMP lead to dark pigment production, the 24 bp deletion is the likely molecular cause of melanism in the grey squirrel.