Labeling of CC Chemokine Receptor 2 with a Versatile Intracellular Allosteric Probe

Interest in affinity-based probes (AfBPs) as novel tools to interrogate G protein-coupled receptors (GPCRs) has gained traction in recent years. AfBPs represent an interesting and more versatile alternative to antibodies. In the present study, we report the development and validation of AfBPs that target the intracellular allosteric pocket of CCR2, a GPCR of interest for the development of therapies targeting autoimmune and inflammatory diseases and also cancer. Owing to the two-step labeling process of these CCR2 AfBPs through the incorporation of a click handle, we were successful in applying our most efficient probe in a variety of in vitro experiments and making use of multiple different detection techniques, such as SDS-PAGE and LC/MS-based proteomics. Collectively, this novel probe shows high selectivity, versatility, and applicability. Hence, this is a valuable alternative for CCR2-targeting antibodies and other traditional tool compounds and could aid in target validation and engagement in drug discovery.


N-(2-(4-amino-2-(2-(prop-2-yn-1-yloxy)ethoxy)phenoxy)-5-chlorophenyl)-4-chloro-3-(trifluoromethyl)benzenesulfonamide (5b). To a roundbottom flask containing
benzenesulfonamide (0.47 g, 0.77 mmol) in ethyl acetate (10 mL) was added tin (II) chloride dihydrate (0.87 g, 3.86 mmol, 5.0 equiv.)and the solution was subsequently stirred at 40 °C for 16 hours.TLC indicated full conversion to the desired aniline so the reaction mixture was cooled to room temperature and quenched by addition of aqueous NaOH (10 mL, 3.0M).Layers were separated and the aqueous was extracted with ethyl acetate (3x).The combined organics were washed with brine, dried over MgSO4 and concentrated in vacuo.The crude was purified by column chromatography eluting with dichloromethane as eluent and affording the title compound as a brown solid.Yield: 50% (0.22 g, 0.39 mmol). 1 benzenesulfonamide (0.07 g, 0.11 mmol) in ethyl acetate (5.0 mL) was added tin (II) chloride dihydrate (0.19 g, 0.83 mmol, 7.5 equiv.)and the solution was subsequently stirred at 40 °C for 32 hours.TLC indicated full conversion to the desired aniline so the reaction mixture was cooled to room temperature and quenched by addition of aqueous NaOH (5.0 mL, 3.0M).Layers were separated and the aqueous was extracted with ethyl acetate (3x).The combined organics were washed with brine, dried over MgSO4 and concentrated in vacuo.The crude was purified by column chromatography eluting with 0-5% MeOH in dichloromethane as eluent and affording the title compound as a light brown solid.Yield: 38% (0.03 g, 0.04 mmol). 1
Transfection.HEK293T and CHO cells were transfected using the polyethylenimine (PEI) method as previously published. 1,2In short, cells were cultured so that they were ~50% confluence on the day of transfection and kept at 37 °C, 5% CO2. 5 µg purified HA-CCR2 (cDNA resource center, Bloomsburg, PA) or 10 µg FLAG-CCR2 or FLAG-CCR2-C70S/C75S/C232S 1 with 1 mg/mL polyethyleneimine (PEI, Polysciences Inc. Warrington, PA) in a mass ratio of 1:8 for HEK293T and 1:6 for CHO cells in a sterile 150 mM NaCl solution was added to each 10 or 15 cm Ø plate.After 24 hours at 37 °C, 5% CO2, sodium butyrate with a final concentration of 5 mM for HEK293T was added.After an additional 24 hours, cells were used for ELISA assays or membrane preparation.
ELISA.Transfected HEK293T cells were diluted to 100 000 cells/well in medium supplemented with 5 mM sodium butyrate and plated onto poly-D-lysine coated, tissue culture treated, clear bottomed, 96well plates.Medium was removed and cells were washed with PBS after 24 hours at 37 °C, 5% CO2 and fixed with paraformaldehyde for 10 minutes before removing and washing twice with Tris Buffered Saline (TBS).Wells were blocked with buffer containing 2% (w/v) Bovine Serum Albumin (BSA) in Tris-Buffered Saline, 0.1% Tween (TBST) for 1 hour at room temperature, while gently agitating.Cells were washed with TBST before addition of RabbitαHA (AHP1075, BioRad, Hercules, CA) in 0.1% (w/v) BSA in TBST with a dilution of 1:2500 for 1 hour at room temperature, while gently agitating.After washing 3 times with TBST, cells were incubated with HRP-conjugated GoatαRabbit antibody (111-035-003, Jackson ImmunoResearch Laboratories, Ely, UK) in 0.1% (w/v) BSA in TBST with a dilution of 1:2500 for 30 minutes at room temperature.Unbound secondary antibody was removed by washing 3 times with TBS, and 3,3′,5,5′-Tetramethylbenzidine (TMB) was added for 5-15 minutes until a clear colour change to blue was visible.H3PO4 was added to stop the reaction and the plate was measured in the EnVision multilabel plate reader (PerkinElmer, Inc, Waltham, MA).
Generation of CCR2 knock-out U266B1 cell line.The CCR2 gene deletion in U266B1 cells was generated using the Alt-R™ CRISPR-Cas9 system (IDT).The two optimal pre-designed crRNA sequences 1: GACUUCUUCACCGCUCUCGUGUUUUAGAGCUAUGCU and 2: CCUGACAAUCGAUAGAUACCGUUUUAGAGCUAUGCU according to the IDT design tool were purchased from IDT together with Alt-R® CRISPR-Cas9 tracrRNA, and Alt-R® S.p. Cas9 Nuclease V3.First, the crRNA and tracRNA oligos were combined in equimolar concentrations to a final duplex concentration of 100 µM and heated at 95 °C for 5 min.Subsequently, the formed gRNA duplexes were cooled down to RT. Next, the duplexes (120 pmol) and the Cas9 enzyme (104 pmol) were combined in PBS with a total volume of 5 µL and incubated at RT for 20 min to form the ribonucleoprotein (RNP) complexes.Then, the RNP complexes were transfected into the U266B1 cell line using the Amaxa Nucleofector™ 2b device.One day prior to transfection the cells were cultured to a cell density of 5x10 5 cells/mL.The next day, the cells were centrifuged at 200 rcf for 5 min and washed with PBS.Then, 2x10 6 cells per condition were centrifuged at 100 rcf for 10 min.The cells were resuspended in 100 µL nucleofection solution kit C and combined with 5 µL RNP complex.The cells were nucleofected using program X-005 and afterwards allowed to recover for 30 min at RT. Simultaneously, a 12-well plate with 1.5 mL/well antibiotics free medium was preincubated at 37 °C under 5% CO2.After recovery, the cells were transferred to the 12-well plate and incubated at 37 °C under 5% CO2.
Validation of CCR2 knock-out U266B1 cell line and single cell isolation.Two days after transfection, 500 µL per condition cell suspension was centrifuged at 1000 rcf for 5 min and resuspended in 30 µL lysis buffer (0.45% (v/v) Tween 20, 0.45% (v/v) Triton X -100, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3) supplemented with proteinase K (100 µg/mL).The samples were incubated at 60 °C for 10 min and at 98 °C for 5 min.The genomic DNA extracts were directly used in PCR reactions of 100 µL with forward primer: 5' TGGCACACATGCTTTCAGG 3', reverse primer: 5' ACAACAATCAAACTGCTCCTCG 3' (custom order IDT), and Phusion Hot start II DNA Polymerase (ThermoFisher F-549S).The PCR products were purified by gel electrophoresis (1% agarose) and their DNA sequences were determined by Sanger sequencing.TIDE (Tracking of Indels by Decomposition) analysis (http://shinyapps.datacurators.nl/tide/) was performed with the sequences to determine the knockout efficiency.For gRNA 1 (GACUUCUUCACCGCUCUCGUGUUUUAGAGCUAUGCU) and gRNA 2 (CCUGACAAUCGAUAGAUACCGUUUUAGAGCUAUGCU) this was 3.5% and 61.5%, respectively.Single cell cultures were obtained 7 days post transfection by dilution in culture medium and in 96 well plates.The cells were checked once a week under the microscope and after 9 weeks the cells were expanded in T25 flasks.Next, genomic DNA of the single cell cultures was extracted, PCR reactions were performed, Sanger sequencing data were obtained and TIDE analysis was performed as described before in order to find the correct CCR2 knock-out U266B1 single cell culture.

SDS-PAGE.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were performed with HEK293T membranes expressing HA-CCR2 (HEK293T_HA-CCR2).Samples were prepared under nitrous conditions to minimize oxidation of cysteines.Membranes were diluted in assaybuffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.1% CHAPS, degassed with nitrogen) to a final concentration of 1 mg/mL in 38 µL per sample. 1 µL of competitor or assaybuffer containing 1% DMSO was added to the membranes, which were incubated for 1 hour at 25 °C.If 1 µL restriction enzyme was added, samples were pre-incubated at 37 °C for one hour.1 µL probe diluted in assaybuffer with a final concentration of 1% DMSO was then added to the membranes for an additional incubation step of 4 hours at 25 °C.Click mix containing 1 µM Alexa-Fluor 647-N3 was prepared with 50 parts 100 mM CuSO4, 30 parts 1 M sodium ascorbic acid (NaAsc), 10 parts 100 mM trishydroxypropyltriazolylmethylamine (THPTA) and 10 parts dye, added in order.After allowing the click reaction to occur for 1 hour at 25 °C, membranes were denatured with 4x Laemmli (BioRad) buffer containing β-mercaptoethanol for 15 minutes at RT. 10 µL sample was loaded onto a 12.5% acrylamide gel and run at 150-180 V for approximately 90 minutes until the blue dye had run of the gels.Images were taken with ChemiDoc TM MP imaging system (BioRad) with the Cy5 (695/55 filter) and Cy3 (605/50 filter) settings.Gels were stained for at least 18 hours in Coomassie staining before destaining the gel with 50% methanol.10% acetic acid for 1 hour at RT and an additional 30-60 minutes in demineralized water before imaging with the ChemiDoc TM MP imaging system.
Immunoblotting.CCR2 expression in transiently HA-CCR2 transfected HEK293T cell membranes was determined using immunoblotting.Membranes were diluted to 1 mg/mL protein and homogenized using the Ultra Turrax homogenizer (IKA-Werke GmbH & Co.KG, Staufen, Germany).Proteins were denatured in 4x Laemmli buffer (BioRad) buffer containing β-mercaptoethanol for 15 minutes at RT. 10 µL sample was loaded onto a 12.5% acrylamide gel and run at 150-180 V for approximately 90 minutes until the blue dye had run of the gels.Proteins were transferred unto a Hybond-Enhanced Chemiluminescence (ECL) membrane using the BioRad Trans-Blot Turbo for 7 minutes at 2.5 A. The blot was blocked with 5% BSA in TBST for 1 hour at RT.The primary antibody RabbitαCCR2 (Abcam ab227236) was added with a dilution of 1:5000 in 1% BSA in TBST over night at 4 °C.The membrane was washed three times with TBST for 20 minutes, before subsequent addition of the secondary antibody Horseradish Peroxidase-(HRP) conjugated GoatαRabbit antibody (Jackson ImmunoResearch Laboratories) in 1% BSA in TBST for 1 hour at RT.The membrane was washed twice with TBST and once with TBS, before addition of the of the ECL Western Blotting reagent (GE Healthcare, the Netherlands) for 5 min at RT while agitating in the dark.Images were taken with the ChemiDoc TM MP imaging system.
Affinity-based pull down proteomics.Affinity-based pull down proteomics was performed as previously. 3In short, 2 mg/mL HEK293T_HA-CCR2 membranes suspended in assaybuffer (see [ 3 H]CCR2-RA-[R] radioligand binding assays) were incubated with 1 µM probe 6c or 1% DMSO for 4 hours at RT while shaking at 650 rpm.In the experiments of Figure S8, the samples were pre-incubated with 10 µM of compound 1 for 1 h at RT, prior to addition of probe 6c.The membrane suspension was then incubated for 1 hour at RT with click mix consisting of 35 parts 100 mM CuSO4, 21 parts 1 M NaAsc, 7 parts 100 mM THPTA and 1 parts 1 mM biotin-PEG3-Azide (Sigma Aldrich).Proteins were denatured by addition of SDS with a final concentration of 2.5% for 1 hour at RT while shaking at 650 rpm.Consecutively, MeOH, CHCl3 and demineralized water were added to the suspension to precipitate the proteins according to a modified version of the previously reported CHCl3/MeOH method. 4Samples were centrifuged for 10 minutes at 1500 rcf after which the upper (aqueous) layer was removed.After addition of MeOH, the centrifugation step was repeated.1% SDS buffer containing 25 mM NH4HCO3 was added to the pellet and samples were sonicated (Branson Sonifier; 3x5 s, 15% amplitude) to fully resuspend the pellet.Samples were reduced with 0.5 M dithiothreitol (DTT) for 15 minutes at 65 °C while shaking at 650 rpm.To alkylate samples 0.25 M iodoacetamide was used for 30 minutes at RT in the dark before quenching the excess iodoacetamide with 0.5 M DTT for 15 minutes at RT while shaking at 650 rpm.Homogenized slurry containing Avidin Agarose beads (ThermoFischer cat# 11846734) was washed three times by adding PBS followed by centrifugation for 2 min at 2500 rcf.Beads were resuspended in PBS and added to the samples for overnight incubation at 4 °C to coat the beads with protein.Samples were centrifuged for 2 minutes at 200 rcf and the pellet was resuspended in PBS containing 0.1% SDS.Samples were centrifuged (2 min at 2500 rcf) and washed three times with PBS and once with digestion buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM CaCl2 and 2% (v/v) acetonitrile) via centrifugation (2 min at 2500 rcf). 5The coated beads were resuspended in 250 µL digestion buffer with 1 µg chymotrypsin (Promega cat# V1061) and incubated overnight at 37 °C while shaking at 1000 rpm to fragmentize proteins.Next, samples were quenched by the addition of 12 µL formic acid and samples were loaden unto Bio-Spin columns (Bio-Rad cat# 7326204) to remove the beads by centrifugation (2 min, 600 rcf).Peptides were desalted using the StageTips method and concentrated with an Eppendorf concentrator. 5,6Dry peptides were stored at -20 °C until measurement.
The peptides were reconstituted in LCMS solution (H2O:ACN:Formic Acid (FA) 97:3:0.1)containing 10 fmol/µL enolase digest (Waters cat #186002325).The first pull-down samples (Exp 1; Figure 5) were measured in the following manner: The desalted peptides solution was separated on an UltiMate 3000 RSLCnano system set in a trap-elute configuration with a nanoEase M/Z Symmetry C18 100Å, 5µm, 180µm x 20 mm (Waters) trap column for peptide loading/retention and nanoEase M/Z HSS C18 T3 100Å, 1.8µm, 75 µm x 250 mm (Waters) analytical column for peptide separation.The column was kept at 40°C in a column oven.Samples were injected on the trap column at a flow rate of 15 µl/min for 2 min with 99%A, 1%B eluent.The 85 min LC method, using mobile phase A (0.1% formic acid (FA) in ULC-MS grade water (Biosolve)) and mobile phase B (0.1% FA in ULC-MS grade acetonitrile (MeCN, Biosolve)) controlled by a flow sensor at 0.3µl/min with average pressure of 400-500 bar (5500-7000 psi), was programmed as gradient with linear increment to 1% B from t0 to t2 min, 5%B at t5 min, 22%B at t55, 40%B at t64, 90%B at t65 to t74 and 1%B at t75 to t85 min.The eluent was introduced by electro-spray ionization (ESI) via the nanoESI source (Thermo) using stainless steel Nano-bore emitters (40 mm, OD 1/32", ES542, Thermo Scientific).The QExactive HF was operated in positive mode with data dependent acquisition without the use of lock mass, default charge of 2+ and external calibration with LTQ Velos ESI positive ion calibration solution (88323, Pierce, Thermo) every 5 days to less than 2 ppm.The tune file for the survey scan was set to scan range of 350 -1400 m/z, 60.000 resolution (m/z 200), 1 microscan, automatic gain control (AGC) of 1e6, max injection time of 50 ms, no sheath, aux or sweep gas, spray voltage ranging from 1.7 to 3.0 kV, capillary temp of 250°C and an S-lens value of 80.For the 10 data dependent MS/MS events the loop count was set to 10 and the general settings were resolution to 15,000, AGC target 1e5, max IT time 100 ms, isolation window of 1.6 m/z, no fixed first mass and normalized collision energy (NCE) of 28 eV.For individual peaks the data dependent settings were 5.00e4 for the minimum AGC target yielding an intensity threshold of 5.0e5 that needs to be reached prior of triggering an MS/MS event.No apex trigger was used, unassigned, +1 and charges >+8 were excluded with peptide match mode preferred, isotope exclusion on and dynamic exclusion of 20 sec.
For technical reasons, the latter experiments (Exp 2 and 3; Figure 5 and Figure S8) were measured using a different setup, in the following manner: the peptide solution was separated with a 85 min LC method using an Thermo Scientific Vanquish TM Neo system with a Double nanoViperTM PepMapTM Neo 2 µm C18 75 µm x 150 mm column.The method was controlled by a flow sensor at 0.3 µl/min with average pressure of 300-400 bar and was programmed as a gradient with linear increment from A (0.1% formic acid in H2O) to B (0.1% formic acid in MeCN:H2O 8:2), starting from 1% B from t0 to t2 min, to 5% B at t5 min, 28% B at t55 min, 50% B at t64 min, 100% B from t65 min to t74 min and 1% B from t75 min to t85 min.The eluent was introduced by electro-spray ionization (ESI) via the nanoESI source (Thermo Scientific) using stainless steel Nano-bore emitters (40 mm, OD 1/32", ES542, Thermo Scientific).The Orbitrap Exploris 240 was operated in positive mode with data dependent acquisition, without the use of lock mass and a default charge of 2+.The tune file for the survey scan was set to a scan range of 350-1400 m/z, 60.000 resolution, 1 microscan, automatic gain control (AGC) of 10 6 , a maximum injection time of 50 ms, no sheath, aux or sweep gas, spray voltage of 1.9 kV, capillary temp of 280 °C and an RFlens value of 80%.For the data dependent MS/MS events the loop count was set to 15 and the general settings were resolution to 15,000, AGC target 10 4 or 10 5 , maximal IT time 100 ms, isolation window of 1.6 m/z, no fixed first mass and higher-energy collisional dissociation (HCD) of 28%.No apex trigger was used, unassigned, +1 and charges >+7 were excluded with peptide match mode preferred, isotope exclusion on and dynamic exclusion of 20 seconds.Data analysis.Data are shown as representative or as mean ± SD of at least three individual experiments, unless otherwise specified.Data analyses were performed using GraphPad Prism 9 (GraphPad software, San Diego, CA).(p)IC50 values were determined using the non-linear regression curve fit.Using pIC50 values, (apparent) pKi values were calculated with the ChengPrusoff equation.Images of SDS-PAGE and WB experiments were analysed using the ImageLab software (6.0.1, Biorad).Pull down proteomics data was analysed using MaxQuant (version 2.4.2.0). 7A custom made FASTA file was used for peptide identification, consisting of the reviewed (Swiss-Prot) human proteome (downloaded on October 26 2021), isoform B of the human C-C chemokine receptor type 2 (uniprot code P41597-2) and the background proteins bovine serum albumin (P02767), chicken avidin (P02701), yeast enolase (P00924), bovine chymotrypsinogen (P00766) and Streptomyces streptavidin (P22629).Changes to the standard MaxQuant settings were made, including setting Oxidation (M) and Acetyl (Protein N-term) as variable and Carbamidomethyl (C) as fixed modification.The digestion enzyme was set to Chymotrypsin+ with 3 max.missed cleavages.The peptide length was set to be between 7 and 25 with a max.peptide mass of 4600 Da.Contaminants were included.A false discovery rate (FDR) of 0.01 was used for peptide-spectrum match (PSM) FDR, protein FDR and site decoy FDR and the minimum amount of peptides for protein identification was set to 3. Label-free quantification was chosen with a LFQ min ratio count of 1 and 'Fast LFQ' enabled.'Match between runs' was enabled with a match time window of 0.7 minutes and an alignment time window of 20 minutes.The output tables 'peptides.txt'and 'proteingroups.txt'files were used for further analysis.Perseus (version 2.0.11) 8 was used for data analysis of the proteingroups.txtfile.In brief, proteins only identified by site, reverse hits and potential contaminants were removed.Missing values were replaced from a normal distribution using the standard settings of Perseus.In case of the competition experiments (Figure S8), the median LFQ values of two separate experiments were calculated prior to the abovementioned further analysis with Perseus.The remaining LFQ values were used to calculate fold shifts and significance using the volcano plot function in GraphPad Prism 9. Log2(ratio) values show the ratio between the positive samples and control samples (probe/vehicle), positive samples and samples that were pre-incubated with 1 (probe/(probe+1)), and samples that were pre-incubated with 1 and control sampels ((probe+1)/vehicle).

Figure S1 .
Figure S1.Relative expression of HA-CCR2 transfected into HEK293T cells as measured with ELISA.HEK293T cells were transfected with either 5 µg pcDNA3.1 (Mock) or 5 µg pcDNA3.1_HA-CCR2(HA-CCR2) plasmids.Expression of HA-CCR2 in HEK293T cells was measured using an anti-HA antibody.Data is shown as a representative graph of at least three separate experiments performed in quintuplicates.

Figure S2 .
Figure S2.Effect of a protease-inhibitor cocktail (PIC) on HA-CCR2 visualization on SDS-PAGE.(A) HEK293T cell membranes transiently expressing HA-CCR2 were pre-incubated with a protease inhibitor cocktail (PIC) and with or without 1 µM CCR2-RA-[R] before labelling with 50 nM probe 6c.(B) corresponding Coomassie staining.SDS-PAGE and Coomassie staining images are cropped to preserve space.

Figure S3 .
Figure S3.Immunoblotting of CCR2 expression in transiently HA-CCR2 transfected HEK293T cell membranes.Experiments were performed with an anti-CCR2 primary antibody and a Horseradish Peroxidase-(HRP) conjugated secondary antibody, before addition of enhanced chemiluminescence (ECL) reagent.Specific bands were observed around ~35 and ~55 kDa.Image is a representative image of three separate experiments.Image is cropped to preserve space.

BFigure S5 .
Figure S4.Protein loading controls of the gel images shown in Figures3 and 4. The imaged gels were stained with Coomassie Brilliant Blue (CBB) overnight, de-stained using (H2O:MeOH:AcOH 5:4:1) and afterwards imaged using Coomassie settings.Shown are the gel images from Figures3 (A) and 4 (B) and the respective protein loading controls.

Figure S7 .
Figure S7.Validation of CRISPR-Cas9 generated CCR2 knock-out U266 cell line.(A) CRISPR-Cas9 aided CCR2 knock out strategy in the U266B1 cell line.The selected guide RNA targets the target site (blue line) resulting in a double-strand break three base pairs upstream of the PAM (red line) site.The corresponding protein translation is also depicted.(B) Sanger sequencing traces of WT and polyclonal

Figure S8 .
Figure S8.Competition for the CCR2 intracellular binding pocket in proteomic pull-down experiments.Pull-down samples were either pre-treated with or without 10 µM of irreversible antagonist compound 1, prior to probe incubation, click reaction and further sample preparation.(A) Comparison between the samples that were pre-treated with compound 1 to the vehicle control (1% DMSO) ((probe+1)/vehicle).No significant enrichment of the CCR2 was detected after pre-incubation with antagonist 1. (B) Comparison of the probe-treated samples to the samples that were pre-treated with compound 1 (probe/(probe+1)).There is a high enrichment of CCR2 in the positive samples, as compared to the pre-treated samples.(C) Bar graph showing the label free quantification (LFQ) intensity of the CCR2 in the three conditions: vehicle control (1% DMSO), probe-treated and pretreated with compound 1.Volcano plot data is plotted as enrichment ratio (log2(Ratio)) and probability (-log10(p))as determined in a multiple t test.Data originates from N=2 containing triplicates.The dotted lines indicate threshold values of a ratio > 2 and a p-value < 0.05.