Lanthanide-based time-resolved fluorescence of in cyto ligand–receptor interactions

Handl, Heather L.; Vagner, Josef; Yamamura, Henry I.; Hruby, Victor J.; Gillies, Robert J.

doi:10.1016/j.ab.2004.04.012

Cited by 65 publications

(81 citation statements)

References 16 publications

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“…Ligand binding was evaluated via the lanthanide-based binding assay that has been described previously (20). In all cases, the bivalent constructs of MSH (7) bound with more than 10-fold higher affinities compared to the ligands composed of monovalent MSH (7) plus the linker ( Figure 4A).…”

Section: Homobivalent Ligands With Longer Linkers Bind With An Increamentioning

confidence: 99%

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Synthesis and Evaluation of Bivalent NDP-α-MSH(7) Peptide Ligands for Binding to the Human Melanocortin Receptor 4 (hMC4R)

et al. 2007

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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.

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Section: Homobivalent Ligands With Longer Linkers Bind With An Increamentioning

confidence: 99%

“…Lanthanide-based competitive binding assays were conducted according to the method which has been previously described (20). In brief, HEK293/hMC4R cells were plated in black and white 96-well isoplates (Wallac, 1450-584) at a density of 12 000 cells/well and were allowed to grow for 3 days.…”

Section: Lanthanide-based Binding Assaysmentioning

confidence: 99%

Synthesis and Evaluation of Bivalent NDP-α-MSH(7) Peptide Ligands for Binding to the Human Melanocortin Receptor 4 (hMC4R)

et al. 2007

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“…Briefly, the CCK(26-33) peptide was derivatized with our red d2 dye (red-CCK (26)(27)(28)(29)(30)(31)(32)(33)). This fluorescent ligand was expected to display a good affinity for both SNAP-tagfused CCK1 and CCK2 receptors.…”

Section: Cck1 and Cck2 Binding Assays Using Adherent Cellsmentioning

confidence: 99%

A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

et al. 2010

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G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescencebased technology named Tag

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“…Saturation studies using Europium-labeled ligands detected by timeresolved fluorescence [18] determined that the d-OR and the hMC4R were present on the cell at a ratio of 6:1 (104 000 AE 8600 d-OR and 18 000 AE 3300 MC4R per cell). [19] Using these cells, monovalent ligand binding to hMC4R was determined by a competitive binding assay for the heptapeptide Ac-MSH (7), revealing an IC 50 = 39 AE 4.1 nm.…”

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confidence: 99%

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

Vagner¹,

Xu²,

Handl³

et al. 2008

Angewandte Chemie

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Lanthanide-based time-resolved fluorescence of in cyto ligand–receptor interactions

Cited by 65 publications

References 16 publications

Synthesis and Evaluation of Bivalent NDP-α-MSH(7) Peptide Ligands for Binding to the Human Melanocortin Receptor 4 (hMC4R)

Synthesis and Evaluation of Bivalent NDP-α-MSH(7) Peptide Ligands for Binding to the Human Melanocortin Receptor 4 (hMC4R)

A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

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

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