Identification of a novel allosteric binding site in the CXCR2 chemokine receptor.

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.

Binding characterization of N-(2-chloro-5-thiomethylphenyl)-N'-(3-[3 H]3 methoxy phenyl)-N'-methylguanidine ([3 H]GMOM), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist.

Labeled with carbon-11, N-(2-chloro-5-thiomethylphenyl)-N'-(3-methoxyphenyl)-N'-methylguanidine ([11 C]GMOM) is currently the only positron emission tomography (PET) tracer that has shown selectivity for the ion-channel site of N-methyl-D-aspartate (NMDA) receptors in human imaging studies. The present study reports on the selectivity profile and in vitro binding properties of GMOM. The compound was screened on a panel of 80 targets, and labeled with tritium ([3 H]GMOM). The binding properties of [3 H]GMOM were compared to those of the reference ion-channel ligand [3 H](+)-dizocilpine maleate ([3 H]MK-801), in a set of concentration-response, homologous and heterologous inhibition, and association kinetics assays, performed with repeatedly washed rat forebrain preparations. GMOM was at least 70-fold more selective for NMDA receptors compared to all other targets examined. In homologous inhibition and concentration-response assays, the binding of [3 H]GMOM was regulated by NMDA receptor agonists, albeit in a less prominent manner compared to [3 H]MK-801. Scatchard transformation of homologous inhibition data produced concave upward curves for [3 H]GMOM and [3 H]MK-801. The radioligands showed bi-exponential association kinetics in the presence of 100 µmol L-1 l-glutamate/30 µmol L-1 glycine. [3 H]GMOM (3 nmol L-1 and 10 nmol L-1 ) was inhibited with dual affinity by (+)-MK-801, (R,S)-ketamine and memantine, in both presence and absence of agonists. [3 H]MK-801 (2 nmol L-1 ) was inhibited in a monophasic manner by GMOM under baseline and combined agonist conditions, with an IC50 value of ~19 nmol L-1 . The non-linear Scatchard plots, biphasic inhibition by open channel blockers, and bi-exponential kinetics of [3 H]GMOM indicate a complex mechanism of interaction with the NMDA receptor ionophore. The implications for quantifying the PET signal of [11 C]GMOM are discussed.