We demonstrate the potential utility of multivalent ligands as targeting agents for cancer imaging or therapy by determining the binding of homobivalent ligands to their corresponding receptors. This manuscript details the synthesis and evaluation of a series of bivalent ligands containing two copies of the truncated heptapeptide version of [Nle 4 -D-Phe 7 ]-α-melanocyte stimulating hormone (NDP-α-MSH), referred to as MSH (7). These were connected with various semirigid linkers containing Pro-Gly repeats, with or without flexible poly(ethylene glycol) (PEGO) moieties at their termini. Modeling data suggest a distance of 20-50 Å between the ligand binding sites of two adjacent G-protein coupled receptors, GPCRs. These bivalent ligands were observed to bind with higher affinity compared to their monovalent counterparts. Data suggest these ligands may be capable of cross-linking adjacent receptors. An optimal linker length of 25 ± 10 Å, inferred from these ligands, correlated well with the inter-receptor distance estimated through modeling. Although there was no difference in maximal binding affinities between the ligands constructed with the Pro-Gly repeats versus those constructed with the PEGO inserts, the PEGO-containing ligands bound with high affinities over a greater range of linker lengths.
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.
Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20–50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors.
A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-α-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using Nhydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via coppercatalyzed "click" 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-α-MSH constructs, presumably due to slow off rates for this high affinity ligand.Early detection 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. 1 Our strategy for development of such reagents involves linking reporter moieties to multivalent ligands that contain multiple copies of individual binding units that hence cooperatively bind to cell surface receptors that are overexpressed in cancer cells. 2 Multivalent molecules should display enhanced affinity and selectivity for such cells. 2a,3The foundation for ligand-guided multivalent attachment of reporter moieties to cell surfaces bearing targeted receptors was laid in part by studies that employed a poly(vinyl alcohol) (PVA) scaffold decorated with fluorescein and NDP-α-MSH ligands. 4 Such molecules bound specifically and irreversibly to mouse and human melanoma cells that expressed and displayed melanocortin receptors. The PVA-based system was not extended to other peptide hormone/ receptor systems due to problems with the attachment chemistry and the insolubility of PVA. Recent advances in polymer-supported multivalent binding 3b,5 have prompted a reexamination of this earlier approach with the intent of developing a more soluble biocompatible polymer scaffold and more efficient ligand attachment chemistry. Herein we present model synthetic and in vitro biological studies relevant to these goals.The copper-catalyzed 3+2 cyclization of azide and alkyne moieties to generate triazole products 6 was an obvious choice to replace the maleimide electrophile/thiol nucleophile and thiol/disulfide redox attachment chemistries used previously with PVA. 4 † This paper is dedicated to Professor C. Dale Poulter on the occasion of his 65 th birthday.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 m...
A branched flexible linker that incorporates a fluorescent dansyl moiety was synthesized and used to connect two high affinity NDP-α-MSH ligands or two low affinity MSH(4) ligands. The linker was incorporated into the conjugate by solid-phase synthesis. In vitro biological evaluations showed that potency of binding to the human melanocortin 4 receptor was not diminished for linker-ligand combinations relative to the corresponding ligand alone.
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