CCR1

Researchers compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through β-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, Researchers also evaluated the compounds for their ability to alter β-arrestin translocation. Key results: There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses. Conclusions and implications: Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of β-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization [2].

Binding assay [2]
Membranes were prepared from HEK_CCR1 or RPMI 8226 cells as previously described (Gilchrist et al., 1998) and stored in aliquots at −80°C until needed. For the competition assays, membranes were suspended at 10 μg mL−1 in HEM buffer comprised of 50 mM HEPES, pH 7.5, 1 mM EDTA and 5 mM MgCl2. Compounds were serially diluted in HEM buffer to a 10× concentration and then added to the membrane mixture. A final concentration of 2 pM [125I]-CCL3 was added and the tubes were incubated at 37°C, shaking, for 2 h. Initial experiments were performed to determine that equilibrium had been reached at the 2 h time point (data not shown). The bound and free radioligands were separated by filtration through Whatman GF/C filter paper soaked in TEM2 buffer comprised of 20 mm Tris-HCl, pH 7.4, 0.5 mM EDTA and 5 mM MgCl2 supplemented with 0.3% polyethyleneimine and 20 mg·mL−1 BSA using a tissue harvester. Filters were washed twice with ice-cold TEM2 buffer and then counted (1 min per sample) using a Packard Gamma Counter. Binding assays were performed in duplicate, and non-specific binding was determined by adding cold CCL3 (100 nM) at the same time as the radioligand to some samples. Data from binding experiments were analysed by non-linear regression analysis to determine IC50 values. For saturation experiments, we used 10 μg mL−1 of membrane prepared from HEK_CCR1 or RPMI 8226 cells and increasing concentrations of [125I]-CCL3 (0–100 pM) with and without cold CCL3 (100 nM) to define the non-specific binding. Binding experiments were carried out at 37°C for 2 h and the KD and Bmax values were determined using GraphPad Prism version 6.0. Experiments were conducted in duplicate and repeated as indicated in Table 2.

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PathHunter β-arrestin translocation assay [2]
To quantitatively assess β-arrestin translocation, we utilized an enzyme fragment complementation format from DiscoveRx Corporation in which CCR1 is fused to a ProLink™ peptide derived from β-galactosidase, and β-arrestin 2 is fused to an N-terminal deletion mutant of β-galactosidase [enzyme acceptor (EA) ]. Following addition of CCL3, the β-arrestin–EA fusion protein binds activated CCR1-ProLink. PathHunter hCCR1_CHO cells were plated in 96-well half volume white plates at 1 × 104 cells per well in Optimem and allowed to attach overnight. Initial experiments established an EC50 of 200 pM for CCL3. Serial dilutions of the antagonists were made in Optimem and added to triplicate wells. The plates were incubated at 37°C with 5% CO2 for 90 min and then increasing concentrations of CCL3 were added and the plates incubated at 37°C with 5% CO2 for an additional 60 min. Detection reagent was added and the plate incubated for 60 min at room temperature. The plates were read using the luminescent setting on a DTX800 multimode plate reader. The pKB values were determined using the Gaddum/Schild equation on GraphPad Prism version 6.0. Experiments were conducted in triplicate and repeated as indicated in Table 3.

Receptor internalization assay [2]
Receptor internalization experiments were performed using flow cytometry. Staining for surface CCR1 and CCR5 was performed as recommended by the manufacturer of the PE-conjugated anti-CCR1 and FITC conjugated anti-CCR5). Briefly, 5 × 106 RPMI 8226 cells were suspended in RPMI 1640 media with 1% FBS and incubated with various concentrations of antagonist. After 15 min, the cells were activated with CCL3 (1 nM) and incubated for 2 h at 37°C. After being washed with RPMI 1640 media with 1% FBS, cells were suspended in PBS with 1% FBS and 10 μL of each mAb was added. Thereafter, cells were incubated for 30 min in the dark at 4°C. Following two washes with PBS with 1% FBS, samples were analysed on a BD FACScalibur flow cytometer using Cell Quest software. At least 10 000 events were acquired. Healthy populations were identified and gated on FITC versus PE plots. For the cell surface expression figure, the percentage of cells in the upper left quadrant (high CCR1/low CCR5) without CCL3 exposure was set to 1.0 and the fold change in fluorescence of the 1 nM CCL3 only (control), and 1 nM CCL3 with increasing concentrations of CCR1 antagonist are shown. To calculate the IC50 values (Table 3), the samples with 1 nM CCL3 (no antagonist) were set to 1.0, and a non-linear regression analysis was run using GraphPad Prism version 6.0. Experiments were repeated as indicated in Table 3.

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Chemotaxis assay [2]
MultiScreen®-Migration Invasion and Chemotaxis filter plates with 8 μm pore size were used for all chemotaxis experiments. We utilized the fluorescent dye Calcein AM to label RMPI 8226 cells. The cells were washed once with HBSS supplemented with 10 mM HEPES (HHBSS) and suspended at 4 × 106 cells mL−1. Calcein AM (2 μM) was added, and the cells were incubated for 30 min at 37°C. Cells were then washed twice with HHBSS before being suspended in HHBSS at 2 × 106 cells mL−1. Labelled cells (50 μL) were added to the top chambers along with antagonists (or vehicle controls). The bottom chambers contained 150 μL control media (HHBSS) or chemoattractant (1 nM CCL3 in control media). The plate was placed in the 37°C incubator for 3 h. After 3 h, 50 μL of the cell-containing media were removed from the bottom chamber and transferred to a 96-well black plate with a clear bottom. This plate was read using EX490/EM520 on a DTX800 multimode plate reader. For chemotaxis, each point was performed in quadruplicate and the number of cells that migrated spontaneously to the chemotaxis buffer was subtracted. The chemotactic index was determined by dividing the number of migrated cells in the presence of the antagonist by the number of cells that migrated spontaneously to CCL3. Experiments were performed in quadruplicate and IC50 values calculated using non-linear regression analysis (GraphPad Prism version 6.0). For Table 3, the chemotactic index was averaged for triplicate experiments with each compound.

[1]. AZD-4818, a chemokine CCR1 antagonist: WO2008103126 and WO2009011653. Expert Opin Ther Pat. 2009;19(11):1629-1633.

[2]. Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays. Br J Pharmacol . 2014 Nov;171(22):5127-38.

AZD4818 has been used in trials studying the treatment of Chronic Obstructive Pulmonary Disease (COPD).

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Using membranes from HEK_CCR1Gqi5 cells, we confirmed that AZD-4818, BX471, CCX354, CP481715 and PS899877 are all potent inhibitors of [125I]-CCL3 binding (Figure 1; Table 2). In the cases of BX471, CP481715 and PS899877, our findings are similar to those previously reported with HEK cells (Anders et al., 2002; Vallet et al., 2007; Merritt et al., 2010). We then examined the compounds using membranes from RPMI 8226 cells ( Figure 2; Table 2;). While two compounds were equally potent with membranes from either cell line (BX471, PS899877), the others had a bias for the recombinant HEK_CCR1 system. In the case of AZD4818, CCX354 and CP481715, the affinity is significantly better with membranes from HEK_CCR1 cells than those from RPMI 8226 cells when tested using an unpaired t-test with Welch's correction (P < 0.05). Furthermore, there was a drastic shift in the rank order of potency between the membranes tested. With HEK_CCR1 membranes we found MLN3879 > CCX354 ≥ AZD4818 > CP481715 = BX471 > PS899877 while with membranes from RPMI 8226 cells we found MLN3879 > BX471 > CP481715 ≥ PS899877 > AZD4818 > CCX354.[1]

C27H32CL2N2O7

567.46

566.159

C, 57.15; H, 5.68; Cl, 12.50; N, 4.94; O, 19.74

1003566-93-5

1003566-93-5

24955007

White to off-white solid powder

4.317

3

8

9

38

838

1

CC(C(O)=O)(OC1=C(Cl)C=C(C(NC)=O)C(OC[C@@H](O)CN2CCC3(CC4=CC(Cl)=CC=C4O3)CC2)=C1)C

HVTUHSABWJPWNK-SFHVURJKSA-N

InChI=1S/C27H32Cl2N2O7/c1-26(2,25(34)35)37-23-12-22(19(11-20(23)29)24(33)30-3)36-15-18(32)14-31-8-6-27(7-9-31)13-16-10-17(28)4-5-21(16)38-27/h4-5,10-12,18,32H,6-9,13-15H2,1-3H3,(H,30,33)(H,34,35)/t18-/m0/s1

2-[2-chloro-5-[(2S)-3-(5-chlorospiro[3H-1-benzofuran-2,4'-piperidine]-1'-yl)-2-hydroxypropoxy]-4-(methylcarbamoyl)phenoxy]-2-methylpropanoic acid

AZD4818; AZD-4818; AZD-4818; 1003566-93-5; UNII-S5UP6P540K; S5UP6P540K; 2-(2-Chloro-5-(((2S)-3-(5-chloro-2,3-dihydrospiro(benzofuran-2,4'-piperidin)-1'-yl)-2-hydroxypropyl)oxy)-4-((methylamino)carbonyl)phenoxy)-2-methylpropanoic acid; AZD-4818(AZD4818); AZD 4818

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~88.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)