Jatinder S. Josan

Heterobivalent Ligands Crosslink Multiple Cell-Surface Receptors: The Human Melanocortin-4 and δ-Opioid Receptors†

Cell-surface receptor mediated signaling is mechanistically complex. Hetero- and homo-multimerization of receptors appears to occur naturally and is a significant regulatory component of signal transduction.[1] Additionally, exogenous entities, such as cells and viruses, can interact with multiple heterologous receptors and induce clustering.[2] Such multivalent interactions are characterized by enhanced affinities (avidities) relative to monovalent binding and enhanced specificities with heteromultivalent interactions. For example, polymers containing α-MSH (α-melanocyte stimulating hormone) ligands bind with higher affinity to melanoma cells compared to monovalent α-MSH ligands.[3–5] Recapitulation of these natural phenomena using synthetic multivalent agents has been proposed for many years.[3–9] Although homomultivalent agents are known, there is little precedent for synthetic heteromultivalent targeting of cell-surface receptors. Herein we detail the synthesis and bioevaluation of heterobivalent ligands (htBVLs) targeted to two heterologous cell-surface receptors.

Enhanced targeting with heterobivalent ligands

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human δ-opioid receptor (δOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-α-MSH, and Eu-CCK8 for the δOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a δOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers. [Mol Cancer Ther 2009;8(8):2356–65]

Heterobivalent ligands target cell-surface receptor combinations in vivo

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.

Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand.

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH(2)) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening -studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.

Solid-phase synthetic strategy and bioevaluation of a labeled δ-opioid receptor ligand Dmt-Tic-Lys for In Vivo imaging

A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the delta-opioid receptor (deltaOR) ligand H-Dmt-Tic-Lys(R)-OH. The high delta-OR affinity (K(i) = 3 nM) and desirable in vivo characteristics of the Cy5 derivative 1 suggest its usefulness for structure-function studies and receptor localization and as a high-contrast noninvasive molecular marker for live imaging ex vivo or in vivo.

Designer antiandrogens join the race against drug resistance

By using in silico models of the complexes formed by analogues of a cancer drug and its receptor, it may be possible to strategically redesign existing drugs and win the race against mutations that lead to drug resistance in prostate cancer.

Heterobivalent Ligands Crosslink Multiple Cell-Surface Receptors ― A Step Towards Personal Medicine

Introduction Effective treatment of tumor malignancies depends upon identifying targets – molecular markers that differentiate cancer cells from healthy cells. Current cancer therapies involve targeting either differential metabolism, especially nucleic acid biosynthesis pathways, or overexpressed specific gene products. We propose an alternative approach – to specifically target combinations of cell-surface receptors using heteromultivalent ligands (htMVLs). We envision that a three cell-surface protein combination that is expressed on a cancer cell but not on a normal cell could be targeted with heteromultivalent ligands displaying cognate binding motifs of weak affinities. These constructs should bind with high avidity and specificity to cancer population in vivo [1, 2]. As a proof-of-concept, we have synthesized series of heterobivalent constructs of MSH, CCK and -opioid receptors (Fig. 1). The constituent binding motifs in these constructs were moderately potent (see Fig. 1 legend) and were connected via a semi-rigid poly(Pro-Gly) linker flanked by flexible polyethylene glycol based PEGO chains.