Design, Synthesis And Activity Evaluation Of Novel Melanotropin Analogues

Obesity and sexual dysfunction have emerged as major public health problems throughout the world, but existing medicines to combat the problems are disappointingly limited in number and effectiveness.Adrenocorticotrophic hormone (ACTH1–39) and the melanocortins (α,βandγ-melanocyte-stimulating hormone [MSH]) are derived from a precursor molecule known as the pro-opiomelanocortin (POMC) protein. They exert their numerous biological effects by activating 7 transmembrane G-protein coupled receptors (GPCR), leading to adenylyl cyclase activation and subsequent cAMP accumulation within the target cell. To date, 5 melanocortin receptors (MCRs) have been identified and termed MC1R to MC5R, they have been shown to have a wide and varied distribution throughout the body, being found in the central nervous system (CNS), periphery and immune cells. Pharmacological studies of the melanocortin receptors have revealed very broad biological effects including pigmentation, steroidogenesis, energy homeostasis, thermoregulation, anti-inflammation, nerve regeneration, sexual behavior, feeding behavior and memory, and others. The melanocortin subtype 4 receptor (MC4R) is expressed in various regions of the brain, and preclinical studies have proven that selective MC4 receptor agonists reduce food intake and body weight and increase erectile activity. Despite a number of functional studies, the precise roles of MC3R and MC4R signaling and ligand binding remain largely unknown. The potent and enzymatically stable anaogues MT-II and PT-141 are important ligands of melanocortin receptors but are relatively nonselective. To differentiate between the physiological role of melanocortin receptor 4 from those of melanocortin receptors 3 and 5, potent and selective agonists of melanocortin subtype 4 receptor are needed. Development of selective ligands with an appropriate pharmacokinetic profile will enable a pharmacological evaluation of the potential beneficial effects of the melanocortins. In addition, highly potent and selective ligands further facilitates idenfification of the 3D relationships of key pharmcophores, and contributes greatly to a better understanding for design and synthesis of peptides and peptide mimetics. Unnatural amino acids are effective as building blocks to design functional peptides from the following two points: (1) utilization of rigid unnatural amino acids for the incorporated peptides to control the conformation to appear the function, and (2) incorporation of functional and unnatural amino acids into peptides resulting in appearance of the inherent functions. Some natural amino acids through replacement with unnatural amino acids in biologically active peptides may also lead to beneficial changes in biological properties such as enhanced potency and duration of action or conversion of an agonist to an antagonist.Peptides have been discounted as drug candidates due to a perceived instability to hydrolytic enzymes and pH extremes and due to the blood-brain barrier(BBB), which blocks their entry into the central nervous system. Glycosylation has been as a method to penetrate the BBB, and enkephalin analogues glycosylated at or near the C-terminus were shown to elict prolonged and profound analgesia in mice. Similar results have been obainted with glycosylated vasopressin analogues and with deltorphin and dermorphin glycopeptides. The incorporation of carbohydrates may improve the peptides'stability, membrane permeability and bioavailability. Accordingly, glycosylatedα-MSH analogues may show potential to increase proteolytic stability and promote BBB permeability.In this thesis, 11 building blocks included 9 protected unnatural amino acids and two sugar conjugates were prepared. The structure of these compounds was confirmed by 1H NMR and ESI-MS.Referring to plenty of information onα-MSH and its analogues, we chose PT-141 as the control. To develop potent and selective agonist ligands for the melanocortin receptors, we designed a series of novelα-MSH analogues with unnatural amino acids in the linear peptide template and cyclic MT-II template.An efficient method was developed for the synthesis of cyclic peptides on solid-phase, by a combination of weak acid and base. Four MT-II analogues were synthesized by this method with relatively high purity. Some unnatural amino acids were incorporated into the MT-II at the His position through solid-synthesis on the resin.In this work, 43α-MSH analogues were synthesized and purified by solid-phase synthesis, including 4 cyclic heptapeptides, 4 linear heptapeptieds, 27 linear tetrapeptides, 2 linear tridecapeptides, and 6 glycolatedα-MSH analogues with glucose or galactose.36 novelα-MSH analogues and PT-141 at concentration of 100 nM were evaluated for their binding affinities to membrane proteins enriching MC3R and MC4R which were isolated from hypothalamus and its adjacent tissue of rats. Binding was determined by measuring competition with I125-labeled NDP-α-MSH. Inhibiting efficiency refers to the degree of displacement of the radiolabeled ligand by the compound being assayed. 7α-MSH analogues showed good affinity for the receptors compared to the control.Some conclusions were drawn from the binging activity results. 1) Linear peptides exhibit weaker affinity than corresponding cyclic peptides analogues. 2) These studies provide further experimental evidence that the His residue did not participate in binding to MC receptor and that chemically nonreactive side chains may be substituted for the imidazole ring in the design of MC4R-selective agonists. The His position could play an important role in activity as well as in selectivity toward the melanocortin receptors. 3) The introduction of hydrophilic group to the N terminal may not weaken the binding activity in cylic MT-II template.