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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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.
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.
Design, Synthesis, And Testing Of Multivalent Compounds Targeted To Melanocortin ReceptorsMash, Eugene A.; Dehigaspitiya, Dilani Chathurika; Mash, Eugene A.; Hruby, Victor J.; Polt, Robin; Lynch, Ronald M. (The University of Arizona., 2014)The early detection and successful treatment of many human cancers would be facilitated by the availability of reagents that could seek out and selectively bind to cancer cells and report their existence and location by non-invasive molecular imaging. Our focus is on developing such reagents, which target human cancers that presently are difficult to detect, such as melanoma. We wish to apply the multivalency concept to differentiate between healthy cells and melanoma cells. Melanoma cells are known to over-express α-melanocyte stimulating hormone receptors. A successful multivalent construct should show greater avidity towards melanoma cells than healthy cells due to the synergistic effects arising from multivalency. Both oligomeric and shorter linear constructs bearing the minimum active sequence of melanocyte stimulating hormone, His-DPhe-Arg-Trp-NH₂ (MSH4), which binds with low micromolar affinity to α-melanocyte stimulating hormone receptors, were synthesized. Binding affinities of these constructs were evaluated in a competitive binding assay by competing with labeled ligands, Eu-DTPA-PEGO-MSH7 and/or Eu-DTPA-PEGO-NDP-α-MSH. The engineered cell line HEK293 CCK2R/hMC4R, which is genetically modified to over-express both the cholecystokinin 2 receptor (CCK2R) and human melanocortin 4 receptor (hMC4R), was used to test the multivalent constructs. The oligomers were rapidly assembled using microwave-assisted copper catalyzed azide-alkyne cycloaddition between a dialkyne derivative of MSH4 (both protected and deprotected forms) and a diazide derivative of (Pro-Gly)₃ as comonomers. Mixtures with up to five MSH4 residues per chain were obtained at low monomer concentrations and with up to ten MSH4 units per chain at high monomer concentrations. Three oligomer mixtures were further analyzed based on their degree of oligomerization and the route by which the MSH4 monomers were oligomerized, protected vs deprotected. All three mixtures showed evidence for at least partial cyclization. The completive binding assay against Eu-DTPA-PEGO-MSH7 showed only a statistical enhancement of binding for the three mixtures of oligomers when calculated based on the total MSH4 concentration. However, when the calculation of avidity is based on an estimation of the particles numbers, there was a seven times enhancement of binding compared to a monovalent MSH4 control. The shorter linear multivalent MSH4 constructs were synthesized using readily available ethylene glycol, glycerol, and mannitol as core scaffolds with maximum interligand distances ranging from 27 – 37 Å. The divalent construct showed nanomolar binding with 29-fold and 18-fold enhancements in potency compared to a monovalent control when competed against the probes Eu-DTPA-PEGO-MSH7 and Eu-DTPAPEGO-NDP-α-MSH, respectively. The maximum inter-ligand distance of this divalent construct was 27 Å. The trivalent and the tetravalent constructs also showed nanomolar binding with only statistical enhancement when compared to the divalent construct. The hexavalent construct showed a twelve-fold enhancement of binding compared to the monovalent construct, but was clearly less potent than the structurally related tetravalent construct. This study showed that beyond a certain ligand density, the binding towards hMC4R is adversely affected. Based on the binding data for our oligomers and shorter linear constructs, we envisioned a universal oligomeric scaffold with pendant ligands. It is our hypothesis that clusters of two ligands with an inter-ligand distance of about 27 Å distributed along an oligomeric backbone would have high potency towards melanocortin receptors. This scaffold could be modified to vary inter-ligand distances and densities of ligand presentation as necessary for a given receptor/ligand combination.