New Fairlie Group paper: Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor

Dantas de Araujo A, Wu C, Wu KC, Reid RC, Durek T, Lim J, Fairlie DP.

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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.

Bioconjug Chem. 2017, In Press.

May 31. doi: 10.1021/acs.bioconjchem.7b00132. 

http://fairlie.imb.uq.edu.au/index.php

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NEW OBESITY PAPER PUBLISHED

Fig 3C5aR and C3aR antagonists each inhibit diet-induced
obesity, metabolic dysfunction, and adipocyte and
macrophage signaling

Junxian Lim, Abishek Iyer, Jacky Y. Suen, Vernon Seow, Robert C. Reid, Lindsay Brown, David P. Fairlie*

FASEB, 201327, 822-831.

 

Fairlie Group Home Page: http://fairlie.imb.uq.edu.au

New Fairlie Group C3aR, C5aR and obesity paper out in press

C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling.

Lim J, Iyer A, Suen JY, Seow V, Reid RC, Brown L, Fairlie DP.

FASEB J. 2012 Nov 1. [Epub ahead of print]

PMID: 23118029

http://www.ncbi.nlm.nih.gov/pubmed/23118029

Fairlie Group Home Page: http://fairlie.imb.uq.edu.au