TY - JOUR
T1 - A sucrose-derived scaffold for multimerization of bioactive peptides
AU - Rao, Venkataramanarao
AU - Alleti, Ramesh
AU - Xu, Liping
AU - Tafreshi, Narges K.
AU - Morse, David L.
AU - Gillies, Robert J.
AU - Mash, Eugene A.
N1 - Funding Information:
This work was supported by grants R33 CA 95944 , RO1 CA 97360 , RO1 CA 123547 , and P30 CA 23074 from the National Cancer Institute .
PY - 2011/11/1
Y1 - 2011/11/1
N2 - A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N3(CH2)5(C=O)-His-DPhe-Arg-Trp- NH2 (MSH4) or N3(CH2)5(C=O)-Trp-Met- Asp-Phe-NH2 (CCK4), were attached to the scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-a-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) were used to study the interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited binding comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate for multivalent binding, or MSH4 is too weak a binder for a second 'anchoring' binding event to occur before the monovalently-bound construct is released from the cell surface. In contrast with this behavior, monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multivalent binding, which may be due to increased residence times for monovalently bound CCK4 constructs on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the establishment of multivalent binding.
AB - A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N3(CH2)5(C=O)-His-DPhe-Arg-Trp- NH2 (MSH4) or N3(CH2)5(C=O)-Trp-Met- Asp-Phe-NH2 (CCK4), were attached to the scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-a-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) were used to study the interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited binding comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate for multivalent binding, or MSH4 is too weak a binder for a second 'anchoring' binding event to occur before the monovalently-bound construct is released from the cell surface. In contrast with this behavior, monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multivalent binding, which may be due to increased residence times for monovalently bound CCK4 constructs on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the establishment of multivalent binding.
KW - Cholecystokinin 2 receptor ligands
KW - Melanocortin 4 receptor ligands
KW - Multimeric ligands
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U2 - 10.1016/j.bmc.2011.08.053
DO - 10.1016/j.bmc.2011.08.053
M3 - Article
C2 - 21940174
AN - SCOPUS:80054955306
SN - 0968-0896
VL - 19
SP - 6474
EP - 6482
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
IS - 21
ER -