Design, synthesis, and testing of multivalent compounds targeted to melanocortin receptors

Our focus is on developing non-invasive molecular imaging 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 alpha 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 alpha 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-alpha-MSH on 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).;The oligomers were rapidly assembled using microwave-assisted copper catalyzed azide-alkyne cycloaddition between a dialkyne derivative of MSH4 and a diazide derivative of (Pro-Gly)3 as co-monomers. 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. Completive binding assay against Eu-DTPA-PEGO-MSH7 showed only a statistical enhancement of binding 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 ethylene glycol, glycerol, and mannitol as core scaffolds with maximum inter-ligand distances ranging from 27 - 37 A. The divalent construct with maximum inter-ligand distance of 27 A 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-DTPA-PEGO-NDP-alpha-MSH, respectively. The trivalent and the tetravalent constructs showed only statistical enhancement when compared to the divalent construct. It is our hypothesis that clusters of two ligands with an inter-ligand distance of about 27 A distributed along an oligomeric backbone would have high potency towards melanocortin receptors.