Abstract
A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an allosteric, intracellular site on the receptor. The existence of this site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and β-arrestin) suggests that this could be a generic allosteric site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular site.
Footnotes
-
↵1 Protein sequence identity between human CCR4 and CCR5 is ∼50% overall, approximately average across the CC chemokine receptors. If an arbitrary line were drawn through a homology model of CCR4 at the midpoint of the longitudinal axis of the predicted transmembrane domains, then extracellular and intracellular identity between the two receptors would be estimated at 45 and 53%, respectively.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; CCR, CC chemokine receptor; SCH-C (SCH 351125), (4-bromophenyl) {1′-[(2,4-dimethyl-1-oxido-pyridin-3-yl) carbonyl]-4′-methyl-1,4′-bipiperidin-4-yl} methanone O-ethyloxime; BMS-397, N-(2,4-dichlorobenzyl)-2-{4-[(2R)-piperidin-2-ylcarbonyl]piperazin-1-yl}pyrido[2,3-d]pyrimidin-4-amine; BSA, bovine serum albumin; FB, fluorometric microvolume assay technology-Blue; PCR, polymerase chain reaction; [3H]compound 1, 5-chloro-N-(5,6-dichloro-3-[1,1,1-3H]methoxy-pyrazin-2-yl)thiophene-2-sulfonamide; CCR4-5T, CCR4 with the C-terminal domain of CCR5; CCR5-4T, CCR5 with the C-terminal domain of CCR4; WT, wild type; CHO, Chinese hamster ovary; Gqi5, Gq protein α subunit with the last five C-terminal amino acids replaced with those from Gi protein α subunit; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; TM, transmembrane; FLIPR, fluorometric imaging plate reader; for, forward; rev, reverse.
- Received June 28, 2007.
- Accepted November 27, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|