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May 31, 2017 - Probe for Human Complement C3a Receptor. Aline Dantas de .... ing ligand for europium (Eu−DTPA−hC3a; Figure 1). Next, we validated ...
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Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor Aline Dantas de Araujo,†,‡,∥ Chongyang Wu,†,‡,∥ Kai-Chen Wu,†,‡,§ Robert C. Reid,†,‡,§ Thomas Durek,† Junxian Lim,*,†,‡,§ and David P. Fairlie*,†,‡,§ †

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, ‡Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, and §Centre for Inflammation Disease Research, The University of Queensland, Brisbane, Queensland 4072, Australia S Supporting Information *

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.

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INTRODUCTION The human complement anaphylatoxin C3a is a 77 residue protein derived from proteolytic cleavages of the human complement protein C3 by C3 convertases. C3a mediates a variety of proinflammatory and immunoregulatory functions through binding to its cognate G protein-coupled C3a receptor (C3aR). C3aR is ubiquitously expressed on immune cells, adipocytes, epithelial cells, kidneys, liver, spleen, heart, and brain. Due to its extensive distribution, C3aR is linked to physiological responses such as metabolism, inflammation, and immunity.1,2 Apart from modulating immunity, C3aR is implicated in the pathogenesis and progression of various inflammatory diseases including asthma, diet-induced obesity, sepsis, colitis, and arthritis.3−6 However, the biological role and interaction of C3a and C3aR-mediated disease pathologies remain to be fully elucidated. The design and development of small potent synthetic molecules that can selectively target C3aR is required to interrogate and potentially treat C3aRmediated diseases. Biophysical binding assays that interrogate C3aR-ligand interactions are essential analytical tools to discover potent C3aR modulators. We have previously employed traditional radioisotope binding assays using [125I]-C3a as a label to study C3aR binding affinity of a series of small ligands capable of mimicking C3a and acting as effective antagonists or agonists of C3aR.7,8 However, the short half-life of [125I]-C3a and © XXXX American Chemical Society

restrictions associated with manipulation of the radionuclide have limited the practicability of this assay, prompting us to pursue alternative methods. Lanthanides such as europium, samarium, and terbium are rare-earth metals regarded as attractive alternatives to organic fluorescent molecules and radioactive labeling probes. They display unique fluorescence properties, such as the long lifetime of luminescence (microseconds to milliseconds), which allows the highly sensitive detection of biological probes in complex environments by time-resolved fluorescence spectroscopy; a large Stokes shift (∼150 nm), which minimizes emission and excitation overlap; and narrow emission peaks (10−20 nm), which collectively contribute to increase signal-to-noise ratio and sensitivity.9,10 Together with the widely used dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) system, the bound lanthanide on the receptor is dissociated under low pH to give a highly stable fluorescent signal.11 On the basis of these advantageous analytical properties of lanthanide-based fluorophores, we sought to develop a novel human C3a probe labeled with a Eu3+ chelate for use in timeresolved fluorescence binding assays for the screening of C3aR interacting molecules. Herein, we apply the native chemical Received: March 9, 2017 Revised: May 4, 2017

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DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. Total chemical synthesis of Eu−DTPA−hC3a by native chemical ligation. Reaction conditions: (a)(i) Fmoc−AA−OH, HCTU, and DIPEA in DMF; (ii) 33% piperidine in DMF; several cycles. (b) DTPA anhydride, HOBt, DMSO, overnight. (c)(i) NaNO2, 6 M guanidine, 0.2 M phosphate buffer, pH 3.0, −15 °C, 20 min; (ii) MESNA, 6 M guanidine, 0.2 M phosphate buffer, pH 7.0, room temperature, 5 min. (d) 50 mM TCEP, 50 mM MPAA, 6 M guanidine, 0.2 M phosphate buffer, pH 7.0, 5−6 h. (e) Methoxyamine. HCl, pH 3.5. (f) 8 mM reduced glutathione, 1 mM oxidized glutathione, 6 M guanidine, 50 mM phosphate buffer, pH 7.5, room temperature, 2 h. (g) EuCl3, 0.1 M ammonium acetate buffer, pH 7.5, overnight. R: CH2CH2CO−Arg; R′: CH2CH2SO3H. Bottom: mass spectrum of pure synthetic Eu−DTPA−hC3a. Expected mass: 9614.9. Observed mass: 9615.5.

ligation (NCL) approach12 to synthesize full-length human C3a specifically modified at the N-terminus by appending diethylenetriaminepentaacetic acid (DTPA),13 a known chelating ligand for europium (Eu−DTPA−hC3a; Figure 1). Next, we validated its functional profile by measuring intracellular calcium mobilization and phosphorylation of extracellular signal-regulated kinase (ERK1/2) in pharmacological assays. We also demonstrated saturation and competitive binding for the novel Eu−DTPA−hC3a in competition with human C3a and known C3aR-specific small molecule ligands (TR16, BR103, BR111, and SB290157). The results establish that Eu−DTPA−hC3a retains the same pharmacological properties as native human C3a, inducing full C3aR activation, competitively binding to C3aR, and competing like C3a with binding by four small molecule agonists and antagonists.

a chelating ligand due to its high affinity for europium, excellent solubility, and compatibility with NCL methods and C3a folding steps.13 The positioning of the Eu−DTPA complex at the N-terminus of human C3a was designed so as not to interfere with the key C-terminal effector or activating region of C3a. The human C3a sequence was divided into three peptide fragments: DTPA−C3a[1−22]−NHNH2 (1), H−C3a[23− 48]−NHNH2 (2), and H−C3a[49−77]−OH (3), which were individually prepared on resin by standard Fmoc−solidphase peptide synthesis (SPPS) using a peptide synthesizer. Fragments 1 and 2 were assembled on a hydrazine-modified trityl resin,15 while fragment 3 was built on a 2-Cl-trityl solid support. DTPA was attached to the N-terminus of fragment 1 using DTPA anhydride via a previously described method.16 While fragments 1 and 3 were obtained in good yield and purity, fragment 2 could not be readily isolated after a troublesome synthesis by Fmoc−SPPS. Therefore, we instead adopted a Boc−SPPS method previously reported by us12 to build the second fragment with higher efficiency, as the thioester Thz23−C3a[24−48]−COSR (4). Before NCL, the Cterminal hydrazide of 1 was transformed into a reactive thioester upon activation with NaNO2 and reaction with sodium 2-mercaptoethanesulfonate (MESNA),14 affording DTPA−C3a[1−22]−COSCH2CH2SO3H thioester 5 quantitatively. Full assembly of reduced C3a was accomplished by consecutively joining the three polypeptides in the C- to Ndirection (Figure 1). First, thioester 4 was combined with 3 under typical NCL-based thiolysis conditions to form

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RESULTS AND DISCUSSION Chemical Synthesis of the Novel Eu−DTPA−hC3a. Human C3a is composed of 77 amino acid residues and contains three intramolecular disulfide bonds (Figure 1). According to Ghassemian et al., synthetic access to full-length linear C3a molecules is better achieved using a fragment ligation approach due to its extended sequence.12 Synthetic peptide segments can be readily prepared using an automated peptide synthesizer employing standard Fmoc-protected amino acids and established solid support detachment procedures. We therefore sought to construct synthetic human C3a using a NCL approach suitable for automated Fmoc chemistry based on the sequential ligation of C-terminally modified peptide hydrazides developed by the Liu group.14 DTPA was chosen as B

DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry intermediate 6. After the conversion of Thz23 to Cys23, C3a[23−77]−OH 6 was likewise ligated to 5, affording reduced DTPA-C3a 7. Mass spectroscopy analysis confirmed the formation of the 77 residue DTPA-capped peptide 7 (expected mass: 9469.0; observed mass: 9471.1; Figure S1). Folding of the C3a derivative was carried out in the presence of a glutathione redox system12 to give oxidized DTPA−C3a 8 (MS: expected mass: 9463.0; observed mass: 9464.8; Figure S2). Finally, Eu3+ complexation was achieved by treating peptide 8 with three equivalents of EuCl3 in ammonium acetate buffer to give pure folded Eu−DTPA−hC3a (Figure 1). Europium complexation was appropriately performed as the last step of the synthesis because europium chelation is not compatible with acidic reverse-phase high-performance liquid chromatography (RP-HPLC) conditions. Pharmacological Validation of Eu−DTPA−hC3a as a C3aR Agonist. To characterize the potency and specificity of synthetic Eu−DTPA−hC3a, concentration-dependent intracellular calcium mobilization was determined on HEK293 cells stably transfected with human Gα16 and human C3aR (HEK293 Gα16−C3aR). Gα16 belongs to the Gαq protein family, which mediates the activation of phospholipase C leading to the subsequent inositol triphosphate-stimulated mobilization of intracellular calcium from the endoplasmic reticulum.17 Gα16 proteins are widely used in high-throughput fluorometric calcium imaging assays for the screening of new GPCR ligands.18 Furthermore, we have previously characterized C3aR ligands using intracellular calcium mobilization,7,8 and previous studies have also overexpressed Gα16 to examine the functional activity of human C3aR using an intracellular calcium mobilization assay.19 Based on concentration-dependent curves, we determined that Eu−DTPA−hC3a (pEC50: 8.2 ± 0.1; EC50: 6 nM) and C3a (pEC50: 8.5 ± 0.1; EC50: 3 nM) demonstrated very similar full agonist activity and comparable potency in inducing C3aRmediated intracellular calcium mobilization (Figure 2A). Hence, the addition of Eu−DPTA to the N-terminus of C3a did not interfere with the ability of the protein to act as a full agonist at C3aR. To establish whether Eu−DTPA−hC3a was specific for C3aR, and that the intracellular calcium mobilization was not an off-target activation of other endogenous GPCRs, both Eu−DTPA−hC3a and C3a were tested on HEK293 cells transfected with empty vector (HEK293 Gα16 cells). Even at a high concentration of 300 nM, Eu−DTPA−hC3a failed to induce any intracellular calcium mobilization in HEK293 Gα16 cells (Figure 2B). These results indicate that the novel conjugate Eu−DTPA− hC3a is a full agonist in mediating intracellular calcium mobilization while retaining its native specificity for human C3aR. To further validate the retention of C3a-like signaling properties of Eu−DTPA−hC3a in mediating C3aR activation, phosphorylation of ERK1/2 in HEK293 Gα16−C3aR cells was also analyzed. A temporal profile was first established by treating HEK293 Gα16−C3aR cells with Eu−DTPA−hC3a (100 nM) or C3a (100 nM) at different time points, as ERK1/2 phosphorylation can be rapid or delayed depending on the nature of the ligand, cell type, receptor, and signaling pathway being examined.20 Both Eu−DTPA−hC3a and hC3a showed similar temporal profiles, with maximum phosphorylation of ERK1/2 at 3 min before returning to basal levels at 120 min (Figure 3A).

Figure 2. Eu−DTPA−hC3a mediates intracellular calcium mobilization via C3aR in HEK293 cells transfected with both human Gα16 and human C3aR (HEK293 Gα16−C3aR). (A) Concentration-dependent curves of intracellular calcium mobilization by Eu−DTPA−hC3a vs C3a on HEK293 Gα16−C3aR cells. Eu−DTPA−hC3a (pEC50: 8.2 ± 0.1; EC50: 6 nM) showed similar full agonist activity compared to C3a (pEC50: 8.5 ± 0.1; EC50: 3 nM). (B) Eu−DTPA−hC3a and C3a failed to induce intracellular calcium mobilization in HEK293 cells transfected with empty vector (HEK293 Gα16). Calcium responses were expressed as a percentage of the maximum response induced by 300 nM human C3a on HEK293 Gα16−C3aR cells. Error bars represent mean ± SEM of n > 3 independent experiments.

Concentration-dependent curves of Eu−DTPA−hC3a versus C3a also demonstrated similar agonist profiles in C3aRmediated ERK1/2 phosphorylation (Figure 3B). Eu−DTPA− hC3a (pEC50: 8.8 ± 0.1; EC50: 1 nM) showed similar full agonist activity as C3a (pEC50: 9.0 ± 0.1; EC50: 0.9 nM). Consistent with the results from the intracellular calcium mobilization assay (Figure 2), Eu−DTPA−hC3a exhibits similar agonist and temporal profiles as C3a in inducing phosphorylation of ERK1/2. These results further confirmed that incorporating the Eu−DPTA complex onto the Nterminus of human C3a does not interfere with potency, selectivity, or pharmacological properties mediated by C3a through its receptor, human C3aR. Saturation and Competitive Binding of C3aR Using the Novel Eu−DTPA−hC3a. Ligand affinity plays a critical role in GPCR drug design and development,21 but in the case of C3aR, ligand−receptor affinity measurements have required use of the radioligand [125I]−C3a.7,8 Lanthanide-based fluorescence studies have been developed for other GPCRs, such as CXC chemokine receptor 1, CXC chemokine receptor 2, neurokinin 1 receptor, neurokinin 2 receptor, neurotensin receptor, β2-adrenergic receptor, δ-opioid receptor, relaxin- and insulin-like family peptide receptor 2, melanocortin-4 receptor, and protease-activated receptor 2.22−26 To test the applicability of Eu−DTPA−hC3a as a labeled ligand for C3aR affinity, saturation binding experiments were performed on HEK293 Gα16-C3aR cells (Figure 4). Eu−DTPA−hC3a was found to bind in a saturable and specific manner to C3aR, with a calculated Kd of 8.7 ± 1.4 nM when fitted to a one-site binding model. The nonspecific binding was determined to be 3 independent experiments.

Figure 4. Saturation binding curves of Eu−DTPA−hC3a to HEK293 Gα16−C3aR cells. For total binding, cells were incubated with increasing concentrations of Eu−DPTA−hC3a for 60 min at room temperature with shaking. Nonspecific binding was determined in the presence of 5 μM unlabeled C3a. The calculated dissociation constant (Kd) for Eu−DTPA−hC3a was 8.7 ± 1.4 nM. Error bars represent mean ± SEM of n > 3 independent experiments.

Figure 5. Competitive binding assay of Eu−DTPA−hC3a with C3a, selective C3aR ligands (TR16, BR103, BR111, and SB290157), or selective C5aR ligand (3D53). Competitive binding experiments were performed on (A) primary human peripheral blood mononuclear cells (PBMC), (B) HEK293 Gα16−C3aR and HEK293 Gα16 cells using a single concentration of Eu−DTPA−hC3a (2 nM) in the presence of increasing concentrations of human C3a. Data for primary human PBMC are shown from one representative donor from four donors. (C) Competitive binding experiments were performed on HEK293 Gα16−C3aR cells using a single concentration of Eu−DTPA−hC3a (2 nM) in the presence of increasing concentrations of C3aR or C5aR ligand. Cells were incubated with Eu−DTPA−hC3a and C3aR or C5aR ligand for 60 min at room temperature with shaking. Binding affinities of C3a (pKi: 8.6 ± 0.2; Ki: 2.5 nM), TR16 (pEC50: 6.8 ± 0.1; Ki: 138 nM), BR103 (pKi: 6.7 ± 0.1; EC50: 185 nM), BR111 (pKi: 6.3 ± 0.2; Ki: 544 nM), SB290157 (pKi: 6.3 ± 0.1; Ki: 517 nM) and 3D53 (pKi > 4.5; Ki > 30 μM) as measured by the displacement of Eu− DTPA−hC3a (2 nM). Error bars represent mean ± SEM of n ≥ 3 independent experiments.

the total binding at the highest concentration of Eu−DTPA− hC3a (100 nM). To demonstrate Eu−DTPA−hC3a as a fluorescence nonradioactive probe for C3aR in a physiological context, primary human peripheral blood mononuclear cells (PBMC) from buffy coats were used for competitive binding assays with human C3a (IC50: 1.2− 2.7 nM) (Figures 5A and S3). The specificity of Eu−DTPA−hC3a to C3aR was again confirmed using HEK293 Gα16−C3aR and HEK293 Gα16 cells in a competitive binding assay with C3a (Figure 5B). HEK293 Gα16−C3aR cells yielded a typical sigmoidal competitive curve with Ki of 2.5 nM, while TR16 > BR103 > BR111 > SB290157) that were in the same order as found by [125I]−C3a affinity binding. It is estimated that about 30% of marketed drugs target GPCRs or GPCR-mediated mechanisms.35 The evaluation of the ligand−receptor affinity using labeled probes is therefore essential for drug development and discovery. An ideal label should not alter the intrinsic bioactivity and pharmacological properties of the ligand, should be cost-effective and easy to operate, and should offer a good signal-to-noise ratio. Radioisotopes such as 125I or 3H are widely used as labels without significantly affecting binding properties of the ligand; however, the high cost, limited shelf life due to isotope decay, limitations on how they are used, and potential health hazards are drawbacks of radiolabeled ligand binding assays. The use of lanthanide-based fluorophores, however, overcomes most of these disadvantages, offering a cost-effective and user-friendly probe with a considerably enhanced signal-to-noise ratio.36 In conclusion, this study has successfully developed a novel Eu−DTPA−hC3a bioconjugate using chemical synthesis. Human C3a protein was synthesized and specifically conjugated at the N-terminus with a Eu−DTPA complex. The DTPA chelator was unaltered during all steps of synthesis and did not interfere with oxidative protein folding, allowing the formation of a functionally active C3a protein that behaved as a full agonist at the C3a receptor. The novel Eu−DTPA−hC3a bioconjugate showed no discernible differences in potency, specificity, and pharmacological profiles compared to native C3a. The development of this nonradioisotope alternative for a C3aR-ligand binding assay can help confirm hC3aR binding of other short peptides, such as casoxin C, oryzatensin, and TLQP-21, which have been reported to be ligands for C3aR.37−40 It could also potentially accelerate the design and development of potent and selective small molecules to elucidate and modulate C3aR-mediated physiology and disease pathology.

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EXPERIMENTAL PROCEDURES Solid-Phase Peptide Synthesis. The human C3a sequence was assembled from three synthetic segments: DTPA−C3a[1−22]−NHNH 2 (1), Thz23 −C3a[24−48]− COS−CH2CH2CO−Arg (4),12 and H−C3a[49−77]−OH (3). Peptides 1 and 3 were synthesized on solid support by Fmoc-based SPPS on an automated peptide synthesizer (Symphony, Protein Technologies) using a standard HCTU/ DIPEA activation protocol and Fmoc-protected amino acids.41 Hydrazide-modified peptide 1 was assembled on a freshly prepared hydrazine−trityl resin, which was obtained from hydrazination of 2-Cl−trityl resin as previously described.15 Fragment 3 was assembled directly on a 2-Cl−trityl resin. Coupling of DTPA to the N-terminus of the C3a[1−22] segment was accomplished by treating the dry free-amine peptide-bound resin overnight with a mixture of DTPA anhydride (4 equiv) and 1-hydroxybenzotriazole (8 equiv) in anhydrous DMSO (preheated to allow reagent dissolution and allowed to react for 30 min before addition to resin).16 Peptide E

DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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trations of hC3a or C3a ligand for 60 min at room temperature with shaking. Cells were then washed thrice with PBS supplemented with 0.2% BSA, 20 μM EDTA, and 0.01% Tween-20 by repeated centrifugation. After being washed, cells were resuspended with 20 μL of DELFIA enhancement solution (PerkinElmer) and then transferred to a white 384 well ProxiPlate (PerkinElmer) to incubate for 90 min at room temperature. Time-resolved fluorescence was measured using PHERAstar plate reader (BMG Labtech) at 337 nm excitation followed by a 400 μs delay before 620 nm emission. Data Analysis. Data were plotted and analyzed using GraphPad Prism 7 for Mac OS X. EC50 and Ki values were calculated using nonlinear regression, a four-parameters dose− response curve, and one site-competitive binding model, respectively. All values of independent parameters are shown as mean ± SEM of least three independent experiments (n ≥ 3) unless otherwise stated.

Oasis HLB cartridge using the manufacturer instructions. The eluted pure Eu3+-labeled peptide was then lyophilized to yield an amorphous white powder. Full conversion to lanthanidelabeled Eu−DTPA−hC3a was confirmed by measuring the mass spectrum of an analytical sample dissolved in acid free 50% acetonitrile and directly injected into the mass spectrometer. A single mass spectrum corresponding to Eu− DTPA−hC3a was found (deconvoluted mass: 9615.5; expected, 9614.9; Figure 1). Other C3aR and C5aR Ligands. C3aR ligands (TR167, BR10327, BR11129, and SB29015728) and C5aR ligand (3D5331) were synthesized and characterized as previously described (Figure S4). Cell Culture. Cell culture reagents were purchased from Invitrogen. Human embryonic kidney (HEK)293 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 50 units/mL of penicillin-streptomycin at 37 °C in a 5% CO2 incubator. Human full-length wild-type Gα16 and C3aR plasmids were purchased from cDNA Resource Center and Sino Biological, respectively. HEK293 cells were transfected using Lipofectamine 3000 Reagent to coexpressed Gα16 and C3aR. Stable clones were selected in the presence of hygromycin B (300 μg/ mL) and Geneticin (750 μg/mL). Primary human PBMC were isolated from buffy coat (Australian Red Cross Blood Service) and used for binding experiments on the same day. PBMC were harvested and purified using Ficoll-Paque PLUS (GE Healthcare) density centrifugation. Intracellular Calcium Mobilization Assays. Intracellular calcium mobilization assays were performed as previously described.42 HEK293 Gα16−C3aR or HEK293 Gα16 cells were seeded at a density of 5000 cells per well and allowed to adhere overnight. Various concentrations of C3a or Eu−DTPA−hC3a were added via a fluorescent imaging plate reader (FLIPR, Molecular Devices), and intracellular calcium mobilization was monitored via fluorescence measurement for 300 s (excitation of 470−495 nm and emission of 515−575 nm). Phosphorylation of ERK1/2 Assays. HEK293 Gα16− C3aR cells were seeded at a density of 10 000 cells per well and allowed to adhere overnight. Cells were serum-starved for 3 h prior to the experiment, and dilution of Eu−DTPA−hC3a and C3a were performed in serum-free medium. For temporal analysis, HEK293 Gα16−C3aR cells were treated with 100 nM Eu−DTPA−hC3a or hC3a at 1, 3, 5, 10, 15, 30, 60, or 120 min for 37 °C. For concentration-dependent analysis, HEK293 Gα16−C3aR cells were treated with various concentrations of Eu−DTPA−hC3a or hC3a for 3 min at 37 °C. Phosphorylation of ERK1/2 was quantified using AlphaLISA SureFire Ultra pERK1/2 Assay Kit (PerkinElmer) according to the manufacturer’s instructions. Saturation and Competitive Binding Assays. HEK293 Gα16−C3aR or HEK293 Gα16 cells were nonenzymatically lifted using Versene Solution. Cells were then resuspended in 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and seeded at 30 000 cells per well in a round-bottom 96 well plate. All dilutions of Eu−DTPA−hC3a, hC3a, and ligands were performed in PBS supplemented with 2% BSA. Saturation binding were tested using increasing concentration of Eu− DTPA−hC3a, and nonspecific binding was performed in the presence of 5 μM C3a. For competitive binding experiments, primary human PBMC (600 000 cells per well) or HEK293 Gα16−C3aR cells (30 000 cells per well) were simultaneously treated with Eu−DTPA−hC3a (2 nM) and various concen-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00132. Figures showing characterization data of peptide fragments, intermediates, and full-length Eu−DTPA−hC3a; competitive binding data on primary human PBMC; and chemical structures of TR16, BR111, BR103, SB290157, and 3D53. A table showing amino acid sequences and MS analysis of peptide fragments, intermediates, and fulllength human C3a. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Junxian Lim: 0000-0002-8309-0704 David P. Fairlie: 0000-0002-7856-8566 Author Contributions ∥

A.D.d.A. and C.W. are co-first authors.

Notes

The authors declare the following competing financial interest(s): The small molecule agonists and antagonists of C3aR (TR16, BR103, and BR111) and the previous antagonist of C5aR (3D53, also known as PMX53) originated from our lab. They are shown in Figure S3 and have been published, patented and are owned by the University of Queensland. We have no other conflicts.

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ACKNOWLEDGMENTS This research was supported by the National Health and Medical Research Council (grant no. 1084018; Senior Principal Research Fellowships nos. 1027369 and 1117017), the Australian Research Council (grant no. DP1030100629), the Australian Research Council Centre of Excellence in Advanced Molecular Imaging (grant no. CE140100011), and the Australian Red Cross for human PBMC isolation from buffy coats. F

DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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ABBREVIATIONS BSA, bovine serum albumin; C3aR, C3a receptor; DELFIA, dissociation-enhancer lanthanide fluorescence immunoassay; DIPEA, diisopropylethylamine; DTPA, diethylenetriaminepentaacetic acid; ERK, extracellular signal-regulated kinases; Eu, europium; FLIPR, fluorescent imaging plate reader; GPCR, G protein-coupled receptor; HCTU, (2-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate); HEK, human embryonic kidney; MPAA, 50 mM 4mercaptophenylacetic acid; MESNA, sodium 2-mercaptoethanesulfonate; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; RP-HPLC, reverse-phase highperformance liquid chromatography; SEM, standard error of the mean; TFA, trifluoroacetic acid; TCEP, tris(2carboxyethyl)phosphine

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DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00132 Bioconjugate Chem. XXXX, XXX, XXX−XXX