Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor.

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4(+) T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4.

Introduction

The chemokines are a family of small (predominantly 8–10 kDa) proteins which act as leucocyte chemoattractants. They may be subdivided into four families based on the arrangement of the first two of four conserved cysteine residues. The largest of these families are the CC-chemokines in which the cysteine residues are adjacent and the CXC-chemokines in which the cysteines are separated by an intervening amino acid residue (Zlotnik and Yoshie 2000). The chemokine receptors are Gi-protein coupled receptors and are also divided into four families, based on their ligand specificity, for example CC-chemokines are agonists for CC-chemokine receptors while CXC-chemokine receptors respond only to CXC-chemokines (Murphy et al. 2000).

CCR4 is the receptor for the CC-chemokines CC-chemokine ligand (CCL) 17 (previously known as thymus and activation-related chemokine, TARC) and CCL22 (or macrophage-derived chemokine, MDC; chemokine and receptor nomenclature follows Alexander et al. 2011). It has also been reported that CCR4 is a receptor for chemokine-like factor 1, an immune cell chemoattractant which is not a member of the chemokine family (Wang et al. 2006). CCR4 is found on a number of cells of the haematopoietic lineage, for example T cells, platelets (Clemetson et al. 2000), and mast cells (Juremalm et al. 2002). The expression on T cells is restricted to specific subsets as CCR4 has been reported to be expressed on CD25+ regulatory T cells (Iellem et al. 2001), skin-homing (cutaneous lymphocyte antigen+) T cells (Campbell et al. 1999) and TH2 and TH17 but not TH1 helper T cells (Bonecchi et al. 1998; Lim et al. 2008). The expression of CCR4 on TH2 cells has prompted some interest in it as a therapeutic target for asthma and other allergic diseases as the cytokines produced by these cells (interleukins 4, 5, 9, and 13) are thought to induce the pathological changes associated with these diseases (Larche et al. 2003). Indeed, CCR4+ T cells have been shown to be elevated at the sites of inflammation in a number of allergic diseases (Panina-Boudignon et al. 2001; Nouri-Aria et al. 2002) and numbers are further increased after allergen challenge (Panina-Boudignon et al. 2001). There are also a number of studies in human disease which have shown that CCL17 and CCL22 are elevated in plasma, serum or at sites of inflammation in patients with a number of allergic or eosinophilic conditions (Lezcano-Meza et al. 2003; Jahnz-Rozyk et al. 2005) and that the levels are correlated with disease severity.

Several classes of low-molecular weight antagonist of CCR4 have now been identified (Purandare and Somerville 2006) and it has recently been reported that at least one of these classes of antagonist may act at an intracellular binding site on CCR4 (Andrews et al. 2008) and must therefore act as allosteric modulators of this receptor. However, in the same study it was clear that the Bristol-Myers Squibb antagonist (compound 5 in Fig. 1) did not bind to this binding site. In this report, we demonstrate that the interactions of a range of CCR4 antagonists (see Fig. 1 and Table 1), are consistent with the presence of two distinct binding sites for low-molecular weight antagonists on CCR4 and that both of these sites are distinct from the binding site for chemokines suggesting that CCR4 has three spatially distinct ligand-binding sites. Some theory required for the interpretation of the antagonist interaction studies is developed in the Appendix.

Figure 1

Chemical structures of the low-molecular weight CCR4 antagonists used in this study.

Table 1

The sources of the low-molecular weight antagonists used in this study

Compound	Source patent
1	US7144903B2 (Amgen)
2	WO2010097395A1 (GSK)
3	WO2010097395A1 (GSK)
4	WO2004020584A2 (Bristol-Myers-Squibb)
5	WO2004020584A2 (Bristol-Myers-Squibb)
6	WO2007111227A1 (Astellas)
7	WO2003051870A1 (Astra Zeneca)
8	WO2003059893A1 (Astra Zeneca)
9	US20060004010A1 (Ono)

Materials and Methods

Chemokine-stimulated increases in cellular F-actin content

Blood was taken from normal volunteers who had taken no medication within the previous 10 days and chemokine-induced increases in the filamentous (F)-actin content of CD4+ CCR4+ T cells were measured as previously described (Slack and Hall 2012). Briefly, the peripheral blood mononuclear cells (PBMC) were isolated and stained with fluorescein isothiocyanate-conjugated anti-human CD4 and phycoerythrin-conjugated anti-CCR4 antibodies. The cells were then incubated with antagonist or vehicle (0.1% dimethylsulphoxide [DMSO]) for 30 min at 37°C before stimulation with agonist for 15 sec. The assay was terminated by addition of 3% formaldehyde. The fixed cells were stained with Alexa fluor-647 phalloidin and the mean fluorescence intensity of 1000 CD4+ CCR4+ cells per sample was determined. This was expressed as a fraction of the mean intensity of the CD4+ CCR4− cells in the same sample.

Acquisition of the blood samples was approved by the Hertfordshire Research Ethics Committee and all donors gave informed consent prior to donation.

Cell culture and membrane preparation

Chinese hamster ovary (CHO)-K1 cells expressing CCR4 under Geneticin selection (CHO-CCR4) were grown in a 95% O2/5% CO2 atmosphere in Dulbecco's modified eagle medium F12 nutrient mix containing 5% heat-inactivated dialysed foetal bovine serum, 2 mmol·L−1 L-gln, and 0.5 mg·mL−1 Geneticin. Membranes were prepared from the CHO-CCR4 cells as previously described (Slack and Hall 2012).

Radioligand binding

[125I]CCL-17 binding studies

Inhibition of the binding of [125I]CCL-17 to CHO-CCR4 membranes was determined using a scintillation proximity assay (SPA) as previously described (Slack and Hall 2012). To allow quantification of the number of binding sites, saturation binding experiments were also performed by filtration. These studies were performed with 20 μg·mL−1 membrane protein at room temperature (20–22°C) in SPA-binding buffer (20 mmol·L−1 HEPES, 100 mmol·L−1 NaCl, 10 mmol·L−1 MgCl2, 10 μg·mL−1 saponin, 0.1% bovine serum albumin (BSA) adjusted to pH 7.4 with KOH) in a total volume of 500 μL. Non-specific binding (NSB) was determined in the presence of 10 nmol·L−1 CCL22. Plates were incubated with gentle agitation for 2 h and the reaction terminated by rapid filtration on a Brandel harvester (Brandel Inc. Gaithersburg, MD) through GF/C filter papers presoaked in 0.3% polyethylenimine. Samples were washed three times with ice-cold 0.5 mol·L−1 NaCl solution and filters allowed to dry before the amount of bound radioligand was measured using a Packard Cobra II Gamma Counter (PerkinElmer LAS UK Ltd., Beaconsfield, UK). All experiments were performed in the presence of 1% DMSO.

[3H]antagonist binding studies

[3H]antagonist binding experiments were performed as previously described (Slack et al. 2011) with minor modifications. Assays were performed at room temperature (20–22°C) in SPA binding buffer, without BSA, incubation was done for 2 h in a total volume of 1.4 mL. Saturation binding experiments contained 14 μg·mL−1 membrane protein, while inhibition experiments were performed at 50 μg·mL−1 protein. NSB was determined in the presence of 10 μmol·L−1 of the unlabelled compound. Inhibition curves were constructed in the presence of approximately 1 nmol·L−1 [3H]5 or approximately 0.7 nmol·L−1 [3H]8. Binding was terminated by filtration on a Brandel harvester through GF/B filter papers presoaked in 0.3% v/v polyethylenimine ([3H]5) or water ([3H]8). Filters were washed three times with ice-cold distilled water. The amount of radioligand bound was measured by liquid scintillation spectroscopy, in Ultima-Flo™M (PerkinElmer LAS UK Ltd.), using a TriCarb 2900 TR liquid scintillation counter (PerkinElmer LAS UK Ltd). All experiments were performed in the presence of 1% DMSO.

Materials

All cell culture media and reagents were purchased from Gibco (Invitrogen Ltd., Paisley, UK). DMSO was obtained from Fisher Scientific UK Ltd. (Loughborough, UK). All other chemicals were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, UK) unless otherwise stated. Chemokines were obtained from R&D systems (Abingdon, UK), Peprotech (London, UK) or ALMAC (Craigavon, UK) (CCL22). [125I]CCL-17 (specific activity 2200 Ci mmol−1) were obtained from PerkinElmer LAS UK Ltd. [3H]5 and [3H]8 (specific activity 37 and 53 Ci mmol−1 respectively) were synthesized by GE Healthcare UK Ltd. (Little Chalfont, UK). Small molecule antagonists were synthesized by Respiratory CEDD Medicinal Chemistry, GlaxoSmithKline.

Data analysis

Concentration-response curves were fitted with a Hill function of the form where, [A] is the agonist concentration, E is the response to that concentration of agonist, Emax is the maximal response to the agonist, Basal is the level of activity in the absence of agonist and n is the Hill coefficient.

To quantify the effects of antagonists in the functional assays, concentration-ratios (DR) were estimated. In cases where the antagonist caused a change in the maximal response, the DR was calculated at the response level corresponding to half the maximal response in the presence of the antagonist (this is justified in the Appendix). When the effect of a combination of antagonists was investigated, the concentration-ratio was calculated at half of the maximal response for the curve with the lowest maximal response of the set (see Appendix).

Binding inhibition curves were fitted with a Hill function of the following form where, [I] is the inhibitor concentration, B is the level of binding in the presence of that concentration of inhibitor, Imax is the maximal level of inhibition of binding, B0 is the level of radioligand binding in the absence of the inhibitor and n is the Hill coefficient. Where inhibitors reduced the binding to a level which wasn't significantly different from NSB, the affinity (K) was determined using the Cheng-Prusoff correction (Cheng and Prusoff 1973; Leff and Dougall 1993). Where specific binding was only partially inhibited and data quality allowed, the interaction was assumed to be allosteric and the data were fitted with the following equation (Ehlert 1988) where, A is the radioligand, I is the inhibitor, K is the dissociation constant of the radioligand, K is the dissociation constant of the inhibitor and α is the binding cooperativity constant.

Results

CCL17 and CCL22 induced concentration-dependent increases in the F-actin content of human CD4+ CCR4+ cells. The pEC50 of CCL17 was 9.97 ± 0.02 (n = 69) and that of CCL22 was 9.99 ± 0.04 (n = 17) (Fig. 2A). The effects of the low-molecular weight antagonists on the increase in F-actin content of the T cells induced by CCL17 are summarized in Figure 2B,C, and Table 2. The effects of the antagonistic chemokines are shown in Figure 2D. Compounds 6, 7, and 8 caused a small but statistically significant decrease in the F-actin content of the cells (P < 0.05, paired t-test) while the other low-molecular weight antagonists had no significant effect. CCL11 and CCL223-69 were also without effect on the F-actin content of the cells. With the exception of compound 3, all of the low molecular weight antagonists significantly changed the maximal response to CCL17 (P values are noted in Table 2). Compounds 4, 5, 6, 8, and 9 were insurmountable while compounds 1, 2, and 7 increased the maximal response to this agonist (for contrast, in the remainder of the text this phenomenon will be referred to as suprasurmountability), although the effect of compound 1 was relatively small compared with that of the other two compounds. The antagonistic chemokines had no significant effect on the maximal response to CCL17.

Figure 2

Effects of the antagonists on increases in the F-actin content of human CD4+ CCR4+ T cells. (A) The effects of CCL22 and CCL17 alone. (B) The effects of CCL17 alone (ctrl) or in the presence of 3 μmol·L−1 1, 10 μmol·L−1 2, 1 μmol·L−1 3 or 300 nmol·L−1 4. (C) The effects of CCL17 alone (ctrl) or in the presence of 30 nmol·L−1 5, 100 nmol·L−1 6, 3 μmol·L−1 7, 300 nmol·L−1 8 or 100 nmol·L−1 9. (D) The effects of CCL17 alone (ctrl) or in the presence of 1 μmol·L−1 CCL11, or 300 nmol·L−1 CCL223-69. Data are the mean of the replicate determinations (as specified in Table 2 or the text) and vertical bars show the SEM. Curves show the Hill function generated from the mean of the fit parameters.

Table 2

The effects of the antagonists on CCL17-induced increases in the F-actin content of human CD4+ CCR4+ T cells when used alone

Treatment (n)	Concentration	pEC50	Basal	Maximum	Log(slope)	Log(DR1)	% Inhibition2
Control (69)	–	9.97 ± 0.02	1.00 ± 0.01	1.94 ± 0.03	0.17 ± 0.01	–	–
1 (42)	3 μ mol·L−1	8.99 ± 0.04	1.00 ± 0.02	1.99 ± 0.03	0.10 ± 0.01	0.97 ± 0.03	−3.5 ± 1.4*
2 (32)	10 μ mol·L−1	9.11 ± 0.06	0.98 ± 0.02	1.96 ± 0.04	0.00 ± 0.02	0.80 ± 0.04	−8.9 ± 2.8**
3 (3)	1 μ mol·L−1	8.79 ± 0.22	1.00 ± 0.04	1.96 ± 0.07	−0.12 ± 0.05	1.16 ± 0.26	−5.3 ± 10.8
4 (21)	300 n mol·L−1	9.03 ± 0.09	0.97 ± 0.02	1.55 ± 0.04	−0.05 ± 0.02	1.12 ± 0.06	38.4 ± 2.2***
5 (19)	30 n mol·L−1	9.26 ± 0.09	0.97 ± 0.02	1.50 ± 0.05	0.01 ± 0.02	0.96 ± 0.06	42.1 ± 2.3***
6 (14)	100 n mol·L−1	9.65 ± 0.06	1.00 ± 0.02	1.69 ± 0.06	0.10 ± 0.02	0.61 ± 0.05	31.0 ± 1.9***
7 (15)	3 μ mol·L−1	9.14 ± 0.05	0.99 ± 0.02	2.11 ± 0.07	0.03 ± 0.02	0.81 ± 0.03	−15.1 ± 2.7***
8 (27)	300 n mol·L−1	9.07 ± 0.07	0.99 ± 0.02	1.61 ± 0.04	−0.06 ± 0.03	1.19 ± 0.06	40.3 ± 2.0***
9 (9)	100 n mol·L−1	8.98 ± 0.13	0.93 ± 0.02	1.36 ± 0.03	−0.08 ± 0.03	1.23 ± 0.07	49.4 ± 2.4***
CCL11 (7)	1 μ mol·L−1	9.29 ± 0.05	1.02 ± 0.07	1.90 ± 0.10	0.17 ± 0.04	0.64 ± 0.07	−2.8 ± 3.7
CCL223-69 (9)	100 n mol·L−1	8.92 ± 0.06	1.07 ± 0.04	1.97 ± 0.07	0.15 ± 0.03	0.91 ± 0.07	−2.1 ± 4.9

Concentration-ratio: calculated relative to the response at the midpoint of the curve in the presence of the inhibitor.

Percentage inhibition of the maximal response to CCL17.

P < 0.02

P < 0.005

P < 10−4 (Student's t-test).

As an initial approach to determining the minimum number of binding sites available to CCR4 antagonists, we determined the effects of combinations of the antagonists on CCL17-induced increases in CD4+ CCR4+ cell F-actin content. The effects of coincubation with compounds 1 and 2 are shown in Figure 3A. The DR of the combination (49.0 [22.9, 105], n = 4) was much greater than the sum of the DRs of the two antagonists alone (13.7) and close to their product (45.9). A similar pattern of behaviour was observed on coincubation with compounds 1 and 7 (Fig. 3B). However, in this case, the DR of the combination (90.0 [65.5, 124], n = 4) was greater than the product of the individual DRs (49.8). The sum was 14.7. Interestingly, coincubation of CCL17 with 2 and 7 (Fig. 3C) resulted in a DR of 10.8 (5.6, 21.0) (n = 3), which was similar to the sum of their individual DRs (14.0) and markedly less than their product (46.2).

Figure 3

The effects of combinations of antagonists on chemokine-induced increases in the F-actin content of human CD4+ CCR4+ T cells. (A) The effects of CCL17 alone (ctrl) or in the presence of 3 μmol·L−1 1, 10 μmol·L−1 2 or 1 and 2 at these concentrations. (B) The effects of CCL17 alone (ctrl) or in the presence of 3 μmol·L−1 1, 3 μmol·L−1 7 or 1 and 7 at these concentrations. (C) The effects of CCL17 alone (ctrl) or in the presence of 10 μmol·L−1 2, 3 μmol·L−1 7 or 2 and 7 at these concentrations. (D) The effects of CCL22 alone (ctrl) or in the presence of 3 μmol·L−1 1, 10 μmol·L−1 2 or 1 and 2 at these concentrations. Data are the mean of the replicate determinations (as specified in Table 3 or the text) and vertical bars show the SEM. Continuous curves shown the Hill function generated from the mean of the fit parameters. The dashed curves show the expected position of a concentration-response curve shifted by the sum of the DRs of the two antagonists.

Table 3

The effects of combinations of compounds 1 or 2 and the other antagonists on CCL17-induced increases in the F-actin content of human CD4+ CCR4+ T cells

Compound1	DR2 ipo1	Sum	Product	DR2 ipo2	Sum	Product
3 (3, 3)	91.1 (10.5, 790)	18.7	86.2	18.3 (5.4, 62.5)	22.4	113
4 (4, 5)	19.0 (8.6, 42.0)	19.6	95.8	30.2 (11.4, 79.9)	16.2	57.5
5 (3, 4)	12.8 (4.2, 38.7)	10.8	28.8	27.8 (15.7, 49.4)	18.4	37.9
6 (4, 3)	13.5 (8.7, 21)	13.3	37.6	14.0 (5.0, 38.9)	10.0	24.7
8 (3, 3)	83.7 (20.3, 566)	20.8	108	11.3 (6.4, 19.9)	17.7	73.5
9 (5, 5)	183.9 (91.2, 372)	24.1	146	21.1 (9.2, 48.6)	21.2	73.1
CCL11: 1 (4)	25.1 (8.5, 73.9)	10.0	24.8	ND	ND	ND
CCL223-69 (4, 5)	67.7 (46.3, 99.0)	17.3	73.6	23.4 (13.2, 41.4)	12.4	37.8

ND, not determined.

Concentrations were the same as stated in Table 2. Numbers in parentheses are the number of replicates for the determinations in the presence of 1 then 2.

Values in parentheses after the DRs show the 95% confidence interval.

This suggests that 1 binds to a site distinct from that to which 2 and 7 bind but that 2 and 7 may bind to a common site (see Appendix 1). Hence, we examined the interactions of these compounds with the other antagonists to explore their binding site specificity. The interactions of the other low-molecular weight antagonists with compounds 1 and 2 are summarized in Table 3. Coincubation with compound 1 gave a DR greater than the sum of the individual DRs with compounds 3, 8, and 9 while coincubation of these compounds with compound 2 resulted in DRs close to the sum of the individual DRs. The converse was true of compounds 4 and 5 whose effects approximately summated with compound 1 and were greater than additive with those of compound 2. Interestingly, compound 6 had an approximately additive interaction with both 1 and 2. Coincubation of CCL223-69 with either 1 or 2 resulted in a DR which was greater than the sum of the DRs for the individual antagonists and this was also true of coincubation of CCL11 with 1. This is also summarized in Table 3. Coincubation of the cells with CCL223-69 and CCL11 resulted in a DR (12.4 [4.2, 36.3], n = 3) which was very close to the sum of the DRs of the individual antagonists (13.9, product 36.2). No formal statistical analysis was performed on the interaction data as the statistical distribution of the sum of two DRs or its logarithm is not known. The effects of the combinations of antagonists on the maximal response to CCL17 are summarized in Table 4. Figures illustrating the effects of coincubation of cells with 1 or 2 and the other antagonists are provided in the Supporting information.

Table 4

The effects of combinations of compounds 1 or 2 and the other antagonists on the maximal increase in the F-actin content of human CD4+ CCR4+ T cells in response to CCL17

Compound1	i.p.o.1	Alone2	1 2	i.p.o.2	Alone2	2 2
3 (3, 3)	36.5 ± 7.1	−5.3 ± 10.8	−6.2 ± 5.2	3.6 ± 3.1	−5.3 ± 10.8	−8.6 ± 4.7
4 (4, 5)	17.6 ± 2.1	37.5 ± 13.4	−4.4 ± 3.6	31.1 ± 5.1	36.6 ± 3.6	−0.7 ± 3.5
5 (3, 4)	22.8 ± 6.5	36.0 ± 3.4	−2.5 ± 1.0	49.6 ± 4.9	54.9 ± 1.5	2.5 ± 2.4
6 (4, 3)	4.1 ± 2.5	28.9 ± 1.7	−0.1 ± 2.2	−2.4 ± 10.3	33.8 ± 3.2	−29.4 ± 9.9
7 (4, 3)	19.2 ± 5.5	−16.0 ± 1.6	−4.1 ± 3.5	−34.3 ± 10.6	−33.2 ± 4.2	−29.8 ± 9.7
8 (3, 3)	49.5 ± 6.1	40.5 ± 1.3	−1.5 ± 3.1	9.7 ± 2.9	42.5 ± 4.2	−17.6 ± 5.0
9 (5, 5)	68.1 ± 4.1	49.3 ± 3.6	−8.8 ± 1.9	32.9 ± 8.2	49.3 ± 3.6	−0.7 ± 3.5
CCL11: 1 (4)	−8.9 ± 2.8	−1.7 ± 2.5	−3.2 ± 5.2	ND	ND	ND
CCL223-69 (4, 5)	1.7 ± 3.4	7.3 ± 7.1	2.7 ± 4.2	−0.5 ± 3.7	−9.7 ± 5.1	0.9 ± 11.7

ND, not determined.

Concentrations were the same as stated in Table 2. Numbers in parentheses are the number of replicates for the determinations in the presence of 1 then 2.

The mean and sem of the percentage inhibition of the maximal response to CCL17 in the presence of the antagonists determined in the same experiments as the effects of their combination.

The effect of the combination of compounds 1 (3 μmol·L−1) and 2 (10 μmol·L−1) was also determined on CCL22-induced increases in CD4+ CCR4+ T cells (Fig. 3D). Coincubation with the two antagonists caused a shift in the CCL22 concentration-response curve (DR = 35.5 [28.0, 45.0], n = 3) that was much greater than the sum of the individual DRs (10.6). Indeed, in this case it was somewhat larger than the product of the DRs (27.8). In contrast to their effects on CCL17, neither compound alone nor their combination had a significant effect on the response to high concentrations of CCL22. Also, 1, 4, 5, 6, 7, and 8 (at the concentrations tested against CCL17) had no effect on increases in the F-actin content of this T-cell population in response to CXCL12, an agonist of CXCR4, in cells from two donors (data not shown).

Binding site interactions were further explored in radioligand binding assays. In saturation binding experiments, [125I]CCL17 bound to CHO-CCR4 membranes with affinity 0.15 nmol·L−1 (pKD = 9.82 ± 0.06, n = 4). The saturating amount of specific binding was 0.73 ± 0.06 pmol per mg membrane protein. [3H]5 bound with affinity 1.4 nmol·L−1 (pKD = 8.87 ± 0.06, n = 3) and, at saturation, labelled 10.0 ± 2.8 pmol of binding sites per mg protein while [3H]8 bound with affinity 0.28 nmol·L−1 (pKD = 9.56 ± 0.08, n = 3) and labelled 11.0 ± 1.0 pmol binding sites per mg protein at saturation. The number of binding sites labelled by the two tritiated antagonists was not significantly different (Student's t-test). None of the radioligands showed a measurable level of specific binding to membranes from CHO-K1 cells which had not been transfected with CCR4.

The binding of [125I]CCL17 was inhibited to a level which was not significantly different from NSB by all of the chemokines and all of the low-molecular weight antagonists except 3, 8, and 9 (Fig. 4, summarized in Table 5). These three compounds caused a maximum of 84.9 ± 0.7%, 93.1 ± 1.9%, and 90.5 ± 3.0% inhibition, respectively, and were analysed assuming an allosteric interaction. When effects on the binding of [3H]5 or [3H]8 were determined (Figs. 5A,B and 6A,B, Table 5), the low molecular weight antagonists clearly fell into two groups: those which displaced [3H]5 to its NSB but only partially inhibited [3H]8, if at all, (4, 5, and 6) and those which did the converse (2, 3, 7, 8, and 9). Compound 1 was of limited solubility under the conditions of the tritiated antagonist binding assays, precipitating at concentrations above 1 μmol·L−1. It was not, therefore, possible to generate complete inhibition curves for this compound. However, lower concentrations of 1 did inhibit the binding of [3H]5 while they did not inhibit the binding of [3H]8 (indeed there may have been an increase in the binding). The chemokines had only limited effects on the binding of either tritiated ligand (Figs. 5C and 6C, Table 5). Indeed, the antagonistic chemokines had no effect at concentrations below 1 μmol·L−1.

Figure 4

Inhibition of [125I]CCL17 binding to membranes from CHO-CCR4 cells by: (A) CCL22, 1, 4, 5 or 6; (B) CCL22, 2, 3, 7, 8 or 9; (C) CCL22, CCL17, CCL223-69 or CCL11. Data are the mean of at least three separate determinations and vertical bars show the SEM. Curves show the Hill function generated from the mean of the fit parameters.

Table 5

The binding affinities and cooperativity factor (α) values for unlabelled antagonists and chemokines against [125I]CCL17, [3H]compound 5 and [3H]compound 8 in CHO CCR4 membranes. Data are the mean ± SEM of at least three separate determinations with 95% confidence limits shown in parentheses where appropriate

Radioligand	[125I]CCL17		[3H]compound5	[3H]compound8
Compound	pKi	α	pKi	pKi
1	6.34 ± 0.07	–	6.21 ± 0.06	<6.00
2	7.74 ± 0.41	–	ND	8.26 ± 0.05
3	8.21 ± 0.141	0.14 (0.09, 0.21)	ND	8.22 ± 0.01
4	8.56 ± 0.08	–	8.76 ± 0.06	ND
5	9.10 ± 0.09	–	9.14 ± 0.03	ND
6	8.70 ± 0.21	–	8.66 ± 0.05	ND
7	7.53 ± 0.05	–	ND	7.56 ± 0.04
8	9.04 ± 0.171	0.03 (0.02, 0.04)	ND	9.19 ± 0.04
9	8.74 ± 0.091	0.03 (0.02, 0.05)	ND	8.73 ± 0.07
CCL17	9.64 ± 0.10	–	ND	ND
CCL22	10.2 ± 0.05	–	ND	ND
CCL223-69	8.17 ± 0.09	–	ND	ND
CCL11	6.17 ± 0.06	–	ND	ND

ND: Ehlert equation not fitted due to poor definition of the individual inhibition curves.

Data derived from fitting the equation of Ehlert (1988).

Figure 5

Inhibition of [3H]5 binding to membranes from CHO-CCR4 cells by: (A) 1, 4, 5 or 6; (B) 2, 3, 7, 8 or 9; (C) CCL22, CCL17, CCL223-69 or CCL11. Data are the mean of three separate determinations and vertical bars show the SEM. Curves show the Hill function generated from the mean of the fit parameters.

Figure 6

Inhibition of [3H]8 binding to membranes from CHO-CCR4 cells by: (A) 1, 4, 5 or 6; (B) 2, 3, 7, 8 or 9; (C) CCL22, CCL17, CCL223-69 or CCL11. Data are the mean of three separate determinations and vertical bars show the SEM. Curves show the Hill function generated from the mean of the fit parameters.

Discussion

In this report, we have studied interactions between CCR4 antagonists to investigate the number of binding sites on the receptor. To enable the interpretation of the functional experiments, theory was developed to describe the effects of combinations of allosteric modulators on the response to an agonist (Appendix). The key results of this analysis are that the DRs caused by two allosteric modulators that act at the same site are, generally, maximally additive while those of two modulators acting at distinct sites are likely to be supraadditive. These results are analogous to those describing the interaction of two competitive antagonists (Paton and Rang 1965) and an allosteric and competitive antagonist (Christopoulos and Mitchelson 1994), respectively. However, our treatment considered ligands which are not simply neutral antagonists at the allosteric site and this can modify the behaviour. For example, perfect multiplicativity can only result if at least one of the modulators is surmountable and at most one is an agonist. Furthermore, a surmountable modulator can “protect” the receptors from the effects of an insurmountable compound on the maximal response. This provides another diagnostic criterion for a noncompetitive interaction between modulators as any pair for which this protection is not observed must bind to distinct sites.

In the actin polymerization assays, CCL11 and CCL223-69 behaved as simple surmountable antagonists, consistent with them acting competitively. Of the small molecule antagonists, 4, 5, 6, 8, and 9 were insurmountable while 1, 2, and 7 were suprasurmountable. Insurmountability has two frequent explanations: noncompetitive inhibition or a pseudo-irreversible competitive interaction. Given the rather short agonist contact time in the assay the latter cannot be dismissed outright. Suprasurmountability cannot be the result of a purely competitive interaction. Allosteric ligands can increase the maximal response to an agonist (e.g., Hall (2000)) but this could also be due to an interaction further down the signal transduction cascade. However, neither 1 nor 7 had any effect on the response to CXCL12, an agonist of the related receptor CXCR4, which is also Gi-coupled (Murphy et al. 2000), suggesting an effect at the level of CCR4.

The small molecule antagonists could be classified into two groups based on their interaction profiles: those which interacted supraadditively with 1 and (sub) additively with 2 and those which did the converse. This suggests that there are two small molecule antagonist binding sites on CCR4, one which binds 1, 4, and 5 (site 1) and one which binds 2, 3, 7, 8, and 9 (site 2). The binding site of 6 is ambiguous as it had an approximately additive interaction with both 1 and 2. CCL11 and CCL223-69 showed a third profile as their effects were supraadditive with both 1 and 2 but additive with each other indicating an additional binding site for these ligands. Strictly, given the caveats noted in the Appendix, these data are consistent with a minimum of three binding sites. Strong negative-binding cooperativity is indistinguishable from competition and hence an additive interaction can also occur when ligands bind to distinct sites.

To test this hypothesis and to further probe the binding site of 6, radioligand binding experiments were performed with representative small molecule radioligands: [3H]5 for site 1 and [3H]8 for site 2. We also examined effects on [125I]CCL17 binding. In CHO-CCR4 cell membranes, all of the ligands inhibited the binding of [125I]CCL17 suggesting they do indeed interact with CCR4. Again, the small molecule antagonists divided into two groups: those which inhibited the binding of [3H]5 to NSB but only partially inhibited the binding of [3H]8 (4 and 6) and those which did the converse (2, 3, 7, 8, and 9). It was not possible to define a complete inhibition curve for 1 due to its poor solubility under the conditions of the tritiated ligand-binding assays. However, at the concentrations tested, it inhibited the binding of [3H]5 but not that of [3H]8 confirming that 1 binds to site 1. Thus, using two probes which differ from those in the functional studies, the compounds partitioned into the same two sets. This provides further evidence that there are only two binding sites available to bind these compounds as the alternative hypothesis would be quite complex.

CCL11 and CCL223-69 had limited effects on the binding of either tritiated antagonist suggesting that they bind to neither of their binding sites. However, they did inhibit [125I]CCL17 binding, suggesting that they bind to CCR4. CCL11 has previously been shown to increase the migration of CCR4-transfected 300-19 cells in response to CCL22 (Sebastiani et al. 2005). This was suggested to result from binding of CCL11 to CCL22 rather than from an interaction via the receptor. However, in our hands CCL11 behaved as a surmountable antagonist of CCL22 and CCL17 in actin polymerization assays (Nalesso et al. 2008; Fig. 2D) and inhibited the binding of [125I]CCL17. Although these effects could still be due to an interaction with the chemokines, it is noteworthy that, in Sebastiani et al., CXCL10, and by inference CCL11, did not affect the binding of [125I]CCL22. Thus, in our hands, the behaviour of CCL11 is more consistent with that of a simple competitive antagonist. This does not exclude the possibility that CCL11 also binds to CCL17 but this interaction is not apparent under our assay conditions.

One discrepancy noted by Andrews et al. and confirmed in this study was an apparent lack of reciprocity between the effects of the small molecule antagonists on the binding of [125I]CCL17 and those of CCL17 on the binding of the small molecules. In Andrews et al., the chemokine had no effect on the binding of the antagonist radioligand. In this report there was a clear inhibitory effect of CCL17 on [3H]8 and of CCL22 on [3H]5 binding, however, in both cases, the inhibition was partial and occurred at concentrations much higher than those required to inhibit the binding of [125I]CCL17. This is in contrast to close agreement of the affinities obtained for 5 and 8 in [125I]CCL17 and antagonist binding experiments. However, this apparent lack of reciprocity is not inconsistent with the ternary complex model of G-protein activation (De Léan et al. 1980; Appendix). In particular, if an allosteric ligand has no binding cooperativity with the agonist but is an inverse agonist or has negative activation cooperativity, then the ligands only interact when bound to receptor:G-protein (RG) complexes. If RG complexes represent a small proportion of the receptors (and [125I]CCL17 labelled approximately 7% as many sites as the tritiated antagonists), the effect of the agonist on labelled antagonist binding may simply not be detectable. If there is binding cooperativity, the midpoint of the agonist inhibition curve will be closer to the affinity for free receptors than that for the RG complex.

As both tritiated antagonists labelled a similar number of binding sites and only bound to membranes from cells transfected with CCR4 both binding sites appear to be present on CCR4. Andrews et al. (2008) showed that 7 and related compounds bound to a site on the intracellular surface of CCR4. Hence, site 2 must correspond to this site. This site binds to aryl sulphonamides and hence to acidic ligands. It is not clear where site 1 resides on the receptor. However, the compounds which bind to this site are all bases. Interestingly, it has been shown that a conserved glutamic acid residue in the seventh transmembrane domain of chemokine receptors (E290 in CCR4) is involved in antagonist binding (Berkhout et al. 2003; Rosenkilde and Schwartz 2006; Wise et al. 2007). It is, therefore, tempting to speculate that site 1 on CCR4 involves this residue. Wherever it is located, it must be an allosteric site given the similarity between the ability of the chemokines to inhibit binding of [3H]5 and [3H]8 and the interaction of CCL223-69 with 1 and 2. Thus, there appear to be two allosteric sites on CCR4. Hence, the insurmountability of compounds 4, 5, 6, 8, and 9 is consistent with negative activation cooperativity with CCL17. Indeed, the effects of strictly surmountable allosteric antagonists (i.e., those that only affect orthosteric ligand affinity) cannot become insurmountable when the system does not reach steady state: a finite change in the kinetic constants cannot affect a ligand's ability to saturate the receptors at some finite concentration.

Chemically, the compounds studied here fall into two fundamental classes: the site 1 compounds feature a large lipophilic moiety some distance from a basic centre (as illustrated in Fig. 7); the site 2 compounds are aryl sulphonamides. The former general pharmacophore is well represented among small molecule chemokine antagonists, in particular the CCR5 antagonist maraviroc (A) and a number of CCR2 compounds, for example B (Xia and Sui 2009) and D (Berkhout et al. 2003). Aryl sulphonamides are also found among CCR2 antagonists from Chemocentryx (C) and GlaxoSmithKline (see Xia and Sui 2009) so it is interesting to speculate that CCR2 may have two analogous binding sites.

Figure 7

A comparison of selected representative chemokine ligands demonstrating the pharmacophore features of two apparently distinct classes of small molecule. The CCR4 compounds studied here (4, 7) are compared with literature examples of the CCR5 antagonist maraviroc (A), CCR2 antagonists (B–D) and CXCR1–2 compounds (E, F). Lipophilic features are highlighted in green, basic centres in blue, and acidic features in red.

A characteristic property of the biaryl sulphonamide core is its acidity. It is noteworthy that an acidic biaryl motif has also been described in the putative intracellular antagonists of CXCR1 and CXCR2, for example E (Nicholls et al. 2008) and F (Salchow et al. 2010). Thus, it is further tempting to propose that these general pharmacophores describe the characteristics required of a small molecule to bind to, respectively, the transmembrane and intracellular sites which may be common in several chemokine receptors.

In summary, the use of antagonist interaction and radioligand binding experiments demonstrates that there are three sites on CCR4 at which antagonists can act, the orthosteric site and two allosteric sites.