The Melanocortin System: Structure Activity Relationships of Alpha-N-Methylated MT-II Analogues and Mutation Studies of Human Melanocortin Receptor Subtypes 1 and 4
AuthorDedek, Matthew Milan
Keywordsstructure activity relationship
melanocortin 1 receptor
melanocortin 4 receptor
AdvisorHruby, Victor J.
Committee ChairHruby, Victor J.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe 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.
Degree ProgramMedical Pharmacology
Degree GrantorUniversity of Arizona
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Synthesis of Novel Amino Acids and Use of Peptides & Peptidomimetics Containing Unnatural Amino Acids for the Development of Selective Melanocortin Peptide Antagonists and for the Study of Melanocortin Receptor SignalingHruby, Victor J; Qu, Hongchang; Mash, Eugene A.; Pyun, Jeffrey; Walker, F. Ann (The University of Arizona., 2007)Unnatural amino acids are indispensible tools, not only for the elucidation of molecular mechanisms during the study of the complicated biological system, but also for the development of novel peptide and protein drugs with better efficacy and lower toxicity. Beta-substituted gamma,delta-unsaturated amino acids have been shown to be an important type of novel amino acid because of the terminal double bond which can be converted to many other functionalities. The methodology for the synthesis of syn-beta-substituted gamma,delta-unsaturated amino acids has been developed. However, there is no satisfactory general method for the synthesis of anti-beta-substituted gamma,delta-unsaturated amino acids. Therefore, a general methodology was developed by using the Eschenmoser-Claisen rearrangement for the synthesis of both racemic and optically active anti-beta-substituted gamma,delta-unsaturated amino acids. This rearrangement is highly diastereoselective and good asymmetric induction was obtained with a relatively small C2-symmetric chiral auxiliary (2R,5R)-dimethylpyrrolidine. In an effort to design peptide antagonists that are selective for human melanocortin 4 receptor, highly constrained trans and cis 4-guanidinium proline derivatives were synthesized and incorporated in various melanotropin analogues designed to mimic the endogenous hMC1,4R selective antagonist hASIP (Agouti Signaling Protein) central loop. Biological assays show that some of these analogues are highly selective for hMC1R and/or hMC4R with partial agonist or antagonist activities due to a new beta-turn structure induced by the presence of the constrained amino acids. According to molecular modeling studies, the lowest energy conformations of these selective analogues resemble the NMR solution structure of the hASIP central loop. Therefore, a new template was developed for the rational design of novel selective melanotropin analogues that may have therapeutic potential. To further understand the molecular mechanisms of hMC4R signaling upon agonist activation, an hMC4R selective nonpeptide agonist THIQ and its fluorescent dye labeled derivatives were needed to compare to peptide agonist MTII with regard to receptor phosphorylation, internalization, etc. An improved synthetic method was developed for the efficient synthesis of THIQ. A method for the synthesis of TRITC labeled THIQ derivatives was also developed.
Developing Melanocortin 3 Selective Ligands through C-Terminal Modification of Melanocortin PeptidesHruby, Victor J.; Nyberg, Joel Benjamin; Ghosh, Indraneel; Jewett, John; Mash, Eugene; Brown, Michael; Hruby, Victor J. (The University of Arizona., 2013)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.
Exploring the stereostructural requirements of peptide ligands for the melanocortin receptors and molecular mechanism study of GPCR based drugsHruby, Victor J.; Cai, Minying (The University of Arizona., 2004)A central goal of modern biology is to develop a detailed, predictive understanding of the relationships of three-dimensional structure and biological function. We are attempting build this relationship by combining interdisciplinary work. The dissertation is divided into two parts. In the first part of work, numerous structure-activitiy relationships (SAR) studies of different conformationally constrained peptides and peptide mimetics of human melanotropins have been accomplished and discussed. Through this very tedious hard work, selective agonists and antagonists for each subtype of melanocortin receptors have been obtained. We first started investigating the NMR 3D pharmacophore of agonist and antagonist of human melanotropin based on the NMR structure of AGRP ( PDB: 1HYK) and MTII. After a long struggle with appropriate force fields and calculation methodologies, almost identical structures were obtained for MTII as well as SHU-9119 by employing two different techniques of LLMOD (Large scale Lower Modes of Modeling) and NMR. Combining the existing SAR data with this new modeling approach, a series of linear and cyclized peptides (hybridization of the pharmacophore of AGRP and MTII) have been designed, synthesized and identified. We have been successful in obtaining selective agonists and antagonists of melanocortin receptors and these new discoveries shed new insight into peptide or nonpeptide selective drug design for the future. The second part of the dissertation mainly covers human melanocortin receptor (hMCRs) studies. For the purpose of screening the novel peptides and nonpeptides of melanotropins, a series of human melanocortin receptors have been stably transfected into HEK 293 cell line, and new high throughput screening methodologies have been set up. For the purpose of purification of the melanocortin receptors, hMC4R and hMC1R, -His-tag-flag stably transfected into HEK293 cell lines have been designed and applied in receptor purification. We also further studied the mechanism of selective pathway of melanotropin in the HEK293 cells. It was found that agonist mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin mediated clathrin coated pits, and on the contrary, beta-Arrestin2-GFP recruitment is not dependent on PKA activation. The two-photon fluorescence laser scanning microscopy is a fast, powerful method to study the molecular mechanisms of G protein coupled receptor regulation. In addition, this technique also can serve as a rapid, real-time screening method to differentiate between agonists and antagonists irrespective of any knowledge of their intracellular functional properties (orphan receptors).