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
1043-1802
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
Subject 1305 Biotechnology
1502 Bioengineering
2204 Biomedical Engineering
3004 Pharmacology
3003 Pharmaceutical Science
1605 Organic Chemistry
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 (K) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pK of 8.6 ± 0.2 and K of 2.5 nM) and C3aR ligands TR16 (pK of 6.8 ± 0.1 and K of 138 nM), BR103 (pK of 6.7 ± 0.1 and K of 185 nM), BR111 (pK of 6.3 ± 0.2 and K of 544 nM) and SB290157 (pK of 6.3 ± 0.1 and K 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.
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.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
Chemistry, Multidisciplinary
Chemistry, Organic
Biochemistry & Molecular Biology
Chemistry
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 1084018
DP1030100629
CE140100011
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
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