Europium-labeled synthetic C3a protein as a novel fluorescent probe for human complement C3a receptor

Dantas de Araujo, Aline, Wu, Chongyang, Wu, Kai-Chen, Reid, Robert C., Durek, Thomas, Lim, Junxian and Fairlie, David P. (2017) Europium-labeled synthetic C3a protein as a novel fluorescent probe for human complement C3a receptor. Bioconjugate Chemistry, 28 6: 1669-1676. doi:10.1021/acs.bioconjchem.7b00132

Author Dantas de Araujo, Aline
Wu, Chongyang
Wu, Kai-Chen
Reid, Robert C.
Durek, Thomas
Lim, Junxian
Fairlie, David P.
Title Europium-labeled synthetic C3a protein as a novel fluorescent probe for human complement C3a receptor
Journal name Bioconjugate Chemistry   Check publisher's open access policy
ISSN 1520-4812
Publication date 2017-05-31
Year available 2018
Sub-type Article (original research)
DOI 10.1021/acs.bioconjchem.7b00132
Open Access Status Not yet assessed
Volume 28
Issue 6
Start page 1669
End page 1676
Total pages 8
Place of publication Washington, DC, United States
Publisher American Chemical Society
Language eng
Formatted abstract
Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu–DTPA–hC3a). Time-resolved fluorescence analysis has demonstrated that Eu–DTPA–hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (Kd) of 8.7 ± 1.4 nM for Eu–DTPA–hC3a and binding affinities for hC3a (pKi of 8.6 ± 0.2 and Ki of 2.5 nM) and C3aR ligands TR16 (pKi of 6.8 ± 0.1 and Ki of 138 nM), BR103 (pKi of 6.7 ± 0.1 and Ki of 185 nM), BR111 (pKi of 6.3 ± 0.2 and Ki of 544 nM) and SB290157 (pKi of 6.3 ± 0.1 and Ki of 517 nM) via displacement of Eu–DTPA–hC3a from hC3aR. The macromolecular conjugate Eu–DTPA–hC3a is a novel nonradioactive probe suitable for studying ligand–C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
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Institute for Molecular Bioscience - Publications
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Created: Wed, 28 Jun 2017, 23:16:40 EST by Mr James Lim on behalf of Institute for Molecular Bioscience