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

New Fairlie Group Paper: Quinazolinone derivatives as inhibitors of homologous recombinase RAD51.

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51.

Ward A, Dong L, Harris JM, Khanna KK, Al-Ejeh F, Fairlie DP, Wiegmans AP, Liu L.

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Bioorg Med Chem Lett. 2017, In Press.

May 15. pii: S0960-894X(17)30520-6. doi: 10.1016/j.bmcl.2017.05.039.

Abstract

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.