Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors

Abstract

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low µM affinity, was prepared by solid phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-DPhe-Arg-Trp-NH 2 , exhibited a K d for hMC4R of 9.1±1.4 µM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.Early detection of many human cancers would be facilitated by the availability of imaging agents that selectively bind to cancer cells and report their existence and location by noninvasive techniques. 2-5 Development of such imaging agents involves covalently linking reporter moieties and multiple copies of ligands to produce "multivalent molecules" that will cooperatively bind to receptors expressed on the surface of cancer cells. [6][7][8][9][10][11][12] Multivalent molecules constructed from weakly binding ligands should display avidity for such cells when compared with the affinity of a single copy of the parental ligand. [6][7][8][9][10][11][12][13][14][15][16][17] The affinity of a molecule for binding to a receptor is often quantified by a competitive binding assay against a labeled ligand of known potency. For example, labeled forms of NDP-α-MSH, a superpotent ligand that binds to human melanocortin receptors,18 , 19 have been used for this purpose.20 ,21 In such assays one often assumes thermodynamic control, i.e., that the respective on-rates and off-rates of the competing ligands are similar. If this is not the case, details of how the assay is carried out (order and timing of reagent addition, © 2010 Elsevier Ltd. All rights reserved.Correspondence to: Eugene A. Mash. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. timing of measurements taken) can affect the outcome. Determination of on-rates and offrates for binding of multivalent molecules to living cells is difficult since labeled probes may be taken up by the cells and receptors may aggregate and cycle to and from the cell surface. In the absence of knowledge of ligand on-rates and off-rates, a close match between the affinity of the ligands used in constructio...

A Solanesol-Derived Scaffold for Multimerization of Bioactive Peptides

“…The K i for the monovalent control compound 14 was 1.4 times the value for the parental ligand, indicating that attachment of the triazole-containing “spacer” to the N-terminus of MSH(4) has a modest detrimental effect on ligand binding to hMC4R. In keeping with prior results obtained with the squalene-derived monovalent and divalent MSH(4) constructs 1 and 2 ,11 multimer 16e was shown to be nearly as potent as MSH(4), but not more so, in Assay A. This result suggests monovalent binding of 16e at available hMC4R.…”

“…Hek293 cells overexpressing hMC4R were used to assess ligand binding22 using two previously described europium-based competitive binding assays 11,23. Assay A23a employed Eu-DTPA-NDP-α-MSH-NH 2 ( 17 ) as the labeled probe, and Assay B11 employed Eu-DTPA-PEGO-MSH(4)-NH 2 ( 18 ) as the labeled probe.…”

“…R(CO)-MSH(4)-NH 2 was selected for this work because synergistic effects are more easily detected for multivalent constructs of low-affinity ligands 3c. Mixture 2 was found to be about 18 times more potent than mixture 1 in a competitive binding assay against a europium-labeled derivative of MSH(4) 11. The synthetic and biological results with 1 and 2 were encouraging, and work toward a polyisoprene-derived polyol scaffold was begun.…”

A sucrose-derived scaffold for multimerization of bioactive peptides

“…While MALDI-TOF analysis was reasonably consistent with statistical attachment of ligands in the case of 14c , the mass spectra for 14d and 14e suggested that ligand attachment was not statistical (see Figure 3 and Figures S4–S6 in the Supplementary Data). MALDI-TOF analysis of a 1:1 mixture of monovalent and divalent MSH4 constructs on a squalene scaffold 17,27 demonstrated a highly attenuated sensitivity for detection of the divalent construct. Thus, MALDI-TOF analysis of multimers 14c–14e is qualitative, with actual distributions of MSH4 ligands somewhere between the MALDI-TOF limit of analysis and the expected statistical distributions.…”

“…22 In our prior work, the azide N 3 (CH 2 ) 5 (C=O)-His- d Phe-Arg-Trp-NH 2 ( 1 , Scheme 1) was attached to a linear scaffold (e.g., 2 ) bearing terminal alkyne groups using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), 23–26 producing triazole-containing multivalent constructs (e.g., 3 ). 18 The abilities of 3 and of similar monovalent and multivalent constructs based on MSH4 to bind to MC4R were tested in competitive binding assays against probes 4 11 and 5 27 using Hek293 cells engineered to overexpress this receptor. Probe 4 is based on the superpotent ligand Ser-Tyr-Ser-Nle-Glu-His- d Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 (NDP-α-MSH), 28,29 while the probe 5 is based on MSH4.…”