An Intracellular Allosteric Site for a Specific Class of Antagonists of the CC Chemokine G Protein-Coupled Receptors CCR4 and CCR5

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 Cterminal 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 ␤-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.The superfamily of G protein-coupled receptors (GPCRs) represents a productive area for drug discovery. Worldwide sales of drugs that act via GPCRs were estimated in 2004 to be in excess of $120 billion/year (Business Insights Research, 2005). In that same year, chemical programs to target GPCRs accounted for 40% of the portfolio of the pharmaceutical industry, with nearly 40% of these in clinical development (Business Insights Research, 2005).Much of the progress in discovery of small-molecule ligands (i.e., Ͻ500 mol. wt.) for GPCRs comes from improvements in technologies to screen large numbers of compounds, where many campaigns will use functional assays with cell lines genetically engineered to express high amounts of the GPCR of interest, often coexpressed with a genetically modified G protein to improve signaling. This cell-based screening approach is supported by an understanding that the natural agonists for GPCRs interact with extracellular domains of the receptor that form a binding pocket, in analogy with the retinal-binding site of rhodopsin, and from mutagenesis studies of cloned GPCRs, which suggest that antagonists bind competitively at either the orthosteric or a syntopic site within the exofacial core of the transmembrane domains (Kristiansen, 2004). Structural modeling of GPCRs, based on homology with the resolved crystal structures of rhodopsin (Palczewski et al., 2000;Schertler, 2005), also is used to lead rational drug design, and it is ce...

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