Functional studies in primary human T cells indicate a second allosteric regulatory site on CCR4

Cathryn Weston, David Hall

GlaxoSmithKline, Stevenage, UK

CC-chemokine receptor 4 (CCR4) is expressed on Th2 cells which have been suggested to be a key cell type involved in the development of chronic inflammation in allergic diseases. The chemokine agonists of CCR4 are CCL22 (MDC) and CCL17 (TARC) which are chemoattractants of these cells. A recent publication (Andrews et al, 2008) has suggested that one class of CCR4 antagonist (exemplified by compound 2) binds to an intracellular allosteric regulatory site. In this report we extend these studies to other classes of CCR4 antagonist.

Agonist-induced increases in the filamentous-actin content of human CCR+ T cells were measured by a slight modification of the method of Pilette et al (2004). Compound 1 caused surmountable rightward shifts in concentration-response curves (CRCs) to TARC and MDC. Schild plots of these data were non-linear and suggested that there was an upper limit to the shift that could be caused by this antagonist. This effect is inconsistent with competition and may indicate an allosteric effect. Using the method of Ehlert (1998), the pKb of this compound was 6.46 ± 0.09 against TARC and 5.84 ± 0.09 against MDC (n = 4). The values of logB were 1.64 ± 0.19 and 1.19 ± 0.22 against the two agonists. Compound 2 also caused rightward shifts in CRCs to the two agonists. However, in the case of TARC there was also a significant increase in the maximal response (again inconsistent with simple competition). The pA2 for inhibition of MDC was 6.02 ± 0.11 (n = 3). To investigate whether these two compounds bound to the same site on CCR4, the effect of treating cells with both compounds was determined. Against TARC, the rightward displacement by 3 μM of both antagonists together was 77 fold (n = 3) compared with 10 fold for compound 1 and 7 fold for compound 2. Thus, the effect of the two compounds was multiplicative rather than additive, indicating that they bind to distinct binding sites on the receptor. Compounds 3, 4 & 5 were insurmountable antagonists making quantification of their effects difficult. Interaction studies with 3 and 4 (calculating dose-ratios at the midpoint level of the inhibited curves) showed that their effects were approximately additive with those of both compounds 1 & 2 suggesting that they may bind to both sites. Compound 5 was sub-additive with 2 and multiplicative with 1 and 3. This suggests that 5 binds to the same site as 2 and that 3 binds to a different site from 5 and hence, presumably, to the same site as 1.

In conclusion, several classes of CCR4 antagonist appear to be allosteric modulators of CCR4 and interact with a site distinct from the previously described intracellular site for allosteric antagonists.

Andrews et al (2008) Mol. Pharmacol. 73, 855 – 867

Ehlert (1998), Mol. Pharmacol., 33, 187 – 194

Pilette et al (2004) Eur. Resp. J. 23, 876 – 884