Design, Synthesis, And Testing Of Multivalent Compounds Targeted To Melanocortin Receptors

Persistent Link:
http://hdl.handle.net/10150/332660
Title:
Design, Synthesis, And Testing Of Multivalent Compounds Targeted To Melanocortin Receptors
Author:
Dehigaspitiya, Dilani Chathurika
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © 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.
Abstract:
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.
Type:
text; Electronic Dissertation
Keywords:
Chemistry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Mash, Eugene A.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleDesign, Synthesis, And Testing Of Multivalent Compounds Targeted To Melanocortin Receptorsen_US
dc.creatorDehigaspitiya, Dilani Chathurikaen_US
dc.contributor.authorDehigaspitiya, Dilani Chathurikaen_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.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.en_US
dc.description.abstractThe 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.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectChemistryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMash, Eugene A.en_US
dc.contributor.committeememberMash, Eugene A.en_US
dc.contributor.committeememberHruby, Victor J.en_US
dc.contributor.committeememberPolt, Robinen_US
dc.contributor.committeememberLynch, Ronald M.en_US
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