Site specific radiolabelled antagonists confirm the presence of two allosteric regulatory sites on CCR4

Robert Slack (presenter), Linda Russell, Yu Hua Chen, Christopher Browning, Kenneth Clark and David Hall. GlaxoSmithKline, Stevenage, UK.

CC-chemokine receptor 4 (CCR4) is expressed by 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 for CCR4 are CCL22 (MDC) and CCL17 (TARC) which are chemoattractants of these cells. A recent publication has suggested that one class of CCR4 antagonist (exemplified by compound 1) binds to an intracellular allosteric regulatory site (Andrews et al., 2008). Our recent data (Hall et al, Poster 007 this meeting) indicate there may be a second allosteric regulatory site on this receptor. In this report we have further explored this hypothesis using putative site specific radioligands.

Equilibrium radioligand binding assays were performed with [125I]-TARC, [3H]-compound 1 and [3H]-compound 2 in membranes from CHO cells stably transfected with human CCR4 (CHO-CCR4) using a scintillation proximity assay technology. All three radioligands bound saturably to CHO-CCR4 membranes but not to untransfected CHO membranes. The affinity of each radioligand was estimated in saturation binding experiments, these were 9.73 ± 0.08 (TARC; n=3), 8.59 ± 0.09 (1; =3) and 8.26 ± 0.02 (2; n=3). All antagonists tested inhibited [125I]-TARC binding to the non-specific binding (NSB) level (as defined by 10nM MDC). However, the binding of the small molecule radioligands was inhibited by distinct structural classes of antagonists. [3H]-1 was inhibited by 3 (pKi = 7.28 ± 0.17, n=6) and related structures. [3H]-2 was inhibited by 4 (pKi = 5.08 ± 0.16; n=5) and 5 (pKi = 8.04 ± 0.17; n=6) and related structures. This confirms the presence of two antagonist binding sites on CCR4 with distinct structure-activity relationships. Interestingly, neither TARC nor MDC were able to inhibit the binding of either [3H]-1 or [3H]-2. This appears to contradict the requirement of reciprocal effects for an allosteric interaction. However, it can be shown in a model with allosteric regulation incorporated into the ternary complex model of receptor activation that this observation is predictable when the small molecules are allosteric inverse agonists. In this case, if agonist-induced RG complex formation is only a small proportion of the total receptor density (<5%), the effect of this formation of RG on allosteric inverse agonist binding cannot be detected since it is an insignificant proportion of its total binding.

In conclusion, site specific radiolabelled CCR4 antagonist binding data are consistent with observations from functional pharmacology on human primary T-cells and consistent with the existence of two independent allosteric sites on the CCR4 receptor which can be targeted independently by different structural classes of CCR4 antagonists.

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