Ligand-directed covalent labelling of a GPCR with a fluorescent tag

Stoddart, Leigh A.; Kindon, Nicholas D.; Otun, Omolade; Harwood, Clare R.; Patera, Foteini; Veprintsev, Dmitry B.; Woolard, Jeanette; Briddon, Stephen J.; Franks, Hester; Hill, Stephen J.; Kellam, Barrie

doi:10.1101/2020.04.21.053405

Cited by 4 publications

(7 citation statements)

References 15 publications

(15 reference statements)

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“…[20] For many other protein modification approaches, an additional element is used to achieve a higher level of site selectivity. This element, which is often a known ligand or inhibitor for the protein of interest, [21][22][23][24] displays affinity for the protein and directs the reactive moiety -whether reactive by itself or activated by a catalyst-to a specific site on the protein. Of the ligands that have been applied, those that are based on biological entities have shown tremendous promise.…”

mentioning

confidence: 99%

DNA‐assisted site‐selective protein modification

Keijzer

Albada

2021

Biopolymers

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Protein modification is important for various types of biomedical research, including proteomics and therapeutics. Many methodologies for protein modification exist, but not all possess the required level of efficiency and site selectivity. This review focuses on the use of DNA to achieve the desired conversions and levels of accuracy in protein modification by using DNA (i) as a template to help concentrate dilute reactants, (ii) as a guidance system to achieve selectivity by binding specific proteins, and (iii) even as catalytic entity or construct to enhance protein modification reactions.

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mentioning

confidence: 99%

DNA‐assisted site‐selective protein modification

Keijzer

Albada

2021

Biopolymers

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“…The use of UV-Vis spectroscopy can inform on the type of modified residue, however, its use is limited to reactions wherein covalent transfer leads to changes in spectral properties. The TR-FRET experiments as applied previously 75 do not give realtime insight into the rate of covalent labeling nor suggest the amino acid residue being modified.…”

Section: Tr-fret Based Assay To Assess Covalent Nbd Transfer To Cb2rmentioning

confidence: 95%

“…In previous work on ligand directed chemistry, covalent labeling was validated by Western blotting, 53 mass spectrometry, 44,73,74 UV-Vis spectroscopy 63 or time-resolved Förster resonance energy transfer (TR-FRET). 75 All of these approaches suffer from disadvantages.…”

Section: Tr-fret Based Assay To Assess Covalent Nbd Transfer To Cb2rmentioning

confidence: 99%

Platform Reagents Enable Synthesis of Ligand-Directed Cova-lent Probes: Study of Cannabinoid Receptor 2 in Live Cells

Carreira

Kosar

Sykes

et al. 2022

Preprint

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Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer’s disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific con-texts. Ligand-directed covalent (LDC) labeling enables the study of endogenously expressed proteins in living cells, tissues, and animals without impairment of native protein function. Herein, we employed in silico docking and molecular dynamics simulations to evaluate feasibility of LDC labeling of CB2R and guide design of LDC probes. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD) functionalized probes in a TR-FRET as-say. The rapid proof-of-concept verification with O-NBD probes inspired incorporation of advanced elec-trophiles suitable for experiments in live cells. To this end, novel synthetic strategies towards N-sulfonyl pyridone and N-acyl-N-alkyl sulfonamide LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular experiments. The LDC probes were characterized in vitro by a radi-oligand binding assay and TR-FRET experiments. Application of the LDC probes in flow cytometry, imag-ing flow cytometry, and confocal fluorescence microscopy confirmed specific labeling of CB2R in live cells.

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“…Western blotting with an anti-biotin antibody showed that copper(II) is necessary for N-terminal modification, which is consistent with the peptide study (Figure 5C, lanes 3 and 4). The tryptic digestion of biotinmyoglobin revealed that the aldol reaction exclusively occurred on the N-terminus-containing fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] (Figure S58 and S59). The modification reaction of myoglobin with 23 reached a 77 % yield after 3 h (Figure S60).…”

Section: Chemistry-a European Journalmentioning

confidence: 99%

“…Bioconjugation reactions for the chemical modification of proteins have been rapidly developed to meet the growing demand for applications in activity-based protein profiling, [1,2] studies on cellular processes, [3][4][5] the construction of protein chimeras [6,7] and biopolymer-based materials, [8,9] and the development of biopharmaceuticals. [10][11][12] These reactions enable the effortless attachment of synthetic molecules and/or biopolymers to proteins, thereby allowing the creation of new functionalized proteins that do not naturally exist.…”

Section: Introductionmentioning

confidence: 99%

Single‐Step N‐Terminal Modification of Proteins via a Bio‐Inspired Copper(II)‐Mediated Aldol Reaction

Hanaya

Yamoto

Taguchi

et al. 2022

Chemistry A European J

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The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The Nterminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the Nterminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.

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Ligand-directed covalent labelling of a GPCR with a fluorescent tag

Cited by 4 publications

References 15 publications

DNA‐assisted site‐selective protein modification

DNA‐assisted site‐selective protein modification

Platform Reagents Enable Synthesis of Ligand-Directed Cova-lent Probes: Study of Cannabinoid Receptor 2 in Live Cells

Single‐Step N‐Terminal Modification of Proteins via a Bio‐Inspired Copper(II)‐Mediated Aldol Reaction

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