Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses

Veikšina, Santa; Tahk, Maris‐Johanna; Laasfeld, Tõnis; Link, Reet; Kopanchuk, Sergei; Rinken, Ago

doi:10.1007/978-1-0716-1221-7_8

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…Fluorescence anisotropy (FA) saturation binding experiments were performed as previously described [30,31]. The experiments were carried out in PBS with the addition of 0.1% Pluronic F-127 (Sigma-Aldrich, Cat#P2443) in a final volume of 100 µL using a 96-well half area flat-bottom polystyrene NBS multiwell plates (Corning, cat #3686).…”

Section: Assessment Of Binding Affinity By Fluorescence Anisotropy In...mentioning

confidence: 99%

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

et al. 2021

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Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.

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Section: Assessment Of Binding Affinity By Fluorescence Anisotropy In...mentioning

confidence: 99%

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

et al. 2021

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“…Fluorescence anisotropy (FA) saturation binding experiments were performed as previously described [30,31]. The experiments were carried out in PBS with the addition of 0.1% Pluronic F-127 (Sigma-Aldrich, Cat#P2443) in a final volume of 100 μl using a 96-well half area flat- of the data as in [32].…”

Section: Assessment Of Binding Affinity By Fluorescence Anisotropy In a Multiwell Microplatementioning

confidence: 99%

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

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Põšnograjeva

Kopanchuk

et al. 2021

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Tumor extracellular matrix (ECM) is a high-capacity and genetically stable target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity and specificity for precision targeting of the tumor ECM and malignant cells.

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“…The human M 4 receptor in pcDNA3.1+ was purchased from cDNA Resource Center (www.cdna.org), and manufacturing and production of BBV containing human M 4 receptor were performed as described in [25] with some modification. For cloning M 4 into pFastBac vector, BamHI and XbaI sites were used with enzymes from (Thermo Fisher Scientific,Schwerte, Germany).…”

Section: Preparation Of Budded Baculovirus Particlesmentioning

confidence: 99%

“…FA measurements were performed with multi-mode plate reader Synergy NEO (BioTek Instruments, Winooski, USA), which is equipped with a polarizing 530 (25) nm excitation filter and 590 (35) nm emission filter allowing simultaneous parallelly and perpendicularly polarized fluorescence detection. At least three individual experiments were carried out in duplicate.…”

Section: Fluorescence Anisotropy Experimentsmentioning

confidence: 99%

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Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M ₄ muscarinic receptors?

et al. 2022

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M 4 muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer's disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M 4 receptor. Using budded baculovirus particles as M 4 receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM 4 R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.

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Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses

Cited by 8 publications

References 25 publications

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M ₄ muscarinic receptors?

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Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses

Cited by 8 publications

References 25 publications

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M 4 muscarinic receptors?

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Product

Resources

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Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M ₄ muscarinic receptors?