No abstract

AMD3465 is a novel, nonpeptide CXCR4 antagonist and a potent inhibitor of HIV cell entry in that one of the four-nitrogen cyclam rings of the symmetrical, prototype bicyclam antagonist AMD3100 has been replaced by a two-nitrogen N-pyridinylmethylene moiety. This substitution induced an 8-fold higher affinity as determined against 125 The CXCR4 receptor is a broadly expressed chemokine receptor, which in contrast to chemokine receptors in general, is found not only on cells within the immune system, but also, for example, in the central nervous system and gastrointestinal system (1, 2). The CXCR4 receptor is activated by a single chemokine, CXCL12 (previously called stromal-derived factor-1) in contrast to the promiscuous binding of several chemokines by other chemokine receptors. CXCR4 is involved in the migration and homing of leukocytes, and importantly it plays a central role for the anchorage of CD34ϩ stem cells in bone marrow. Yet, in contrast to most other 7TM 2 receptors, targeted deletion of either the gene for CXCR4 or for CXCL12 leads to embryologic lethality (3-5) and thereby emphasize the importance of proper CXCR4 function. In addition, CXCR4 is expressed on many different types of cancer cells where it functions as a survival factor in addition to directing cancer cell migration, for example, metastasis to the bone marrow, where its ligand CXCL12 is produced in large quantities (6).The CXCR4 receptor acts as the main co-receptor for cell entry by so-called CXCR4 using (X4) strains of HIV (human immunodeficiency virus). The prototype non-peptide antagonist for CXCR4, AMD3100, was discovered as an anti-HIV agent long before the action through CXCR4 was described (7). AMD3100 is composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) moieties connected by a conformationally constraining heteroaromatic phenylenebismethylene linker (Fig. 1). AMD3100 is highly specific for CXCR4 and inhibits the binding and function of CXCL12 and the HIV cell entry with high affinity and potency (8,9) through an interaction with three acidic residues, Asp 171 (AspIV:20), Asp 262 (AspVI:23), and Glu 288 (GluVII:06) located in the main ligand binding pocket of CXCR4 (10 -12) (Fig. 1). Based on the knowledge of the strong preference of the cyclam moiety for interactions with carboxylic acid groups (21) and on molecular modeling, we have previously suggested that one cyclam ring of AMD3100 interacts with Asp 171 in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp 262 and Glu 288 from TM-VI and -VII, respectively (11). Importantly, we were able to successfully transfer the essential components of this rather simple tridentate binding mode of the prototype CXCR4 non-peptide antagonist AMD3100 into the otherwise rather distinct CXCR3 receptor (11). Despite the fact that * This work was supported by grants from the Danish Medical Research Council, the Novo-Nordisk Foundation, the Astrid Thaysen Foundation, and the European Community's Sixth Framework Program (LSHB-CT-2005-518167). The c...

The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu(2+), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with the carboxylate group of Asp(262). It is suggested that this occurs through a strong concomitant interaction of one of the oxygen's directly with the metal ion and the other oxygen to one of the nitrogens of the cyclam ring through a hydrogen bond.

The anti-inflammatory drugs indomethacin and ramatroban, the latter showing clinical efficacy in treating allergic asthma, have been shown to act as a classic agonist and antagonist, respectively, of the G protein-coupled chemoattractant receptorhomologous molecule expressed on Th2 cells (CRTH2 receptor). Here, we report the identification of two indole derivatives 1-(4-ethoxyphenyl)-5-methoxy-2-methylindole-3-carboxylic acid and N ␣ -tosyltryptophan (hereafter referred to as 1 and 2, respectively), which are structurally related to indomethacin and ramatroban but which selectively interfere with a specific G protein-independent signaling pathway of CRTH2. In whole-cell saturation-binding assays, 1 and 2 both increase the number of [ 3 H]prostaglandin D2 (PGD2)-recognizing CRTH2 sites and the affinity of PGD2 for CRTH2. Enzyme-linked immunosorbent assays show that they do not alter the total number of CRTH2 receptors on the cell surface. Analysis of their binding mode indicates that unlike indomethacin or ramatroban, 1 and 2 can occupy CRTH2 simultaneously with PGD2. On a functional level, however, 1 and 2 do not interfere with PGD2-mediated activation of heterotrimeric G proteins by CRTH2. In contrast, both compounds inhibit PGD2-mediated arrestin translocation via a G protein-independent mechanism. In human eosinophils endogenously expressing CRTH2, 1 selectively decreases the efficacy but not the potency of PGD2-induced shape change, unlike ramatroban, which displays competitive antagonistic behavior. These data show for the first time that "antagonists" can cause markedly dissimilar degrees of inhibition for different effector pathways and suggest that it may be possible to develop novel classes of specific signal-inhibiting drugs distinct from conventional antagonists.The proximal event mediating cellular signaling by a seven-transmembrane (7TM) receptor is the binding of ligand, which causes the receptor to change its behavior toward the cell. In the past, ligands targeting 7TM receptors have been believed to cause a single type of functional response for all effectors linked to a given receptor; thus, compounds were classified as agonists or antagonists/inverse agonists, respectively, according to their intrinsic efficacy. Drugs with positive intrinsic efficacy stabilize the active receptor conformation and elicit a signaling response (agonists), whereas drugs with negative intrinsic efficacy preferentially stabilize the inactive receptor conformation and shut down a signaling response (antagonists/inverse agonists). In addition, this traditional view of either turning on or off receptor responses has been linked exclusively to signaling pathways involving the activation of heterotrimeric G pro- Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.010520.ABBREVIATIONS: 7TM, seven transmembrane; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; PGD2, prostaglandin D2; ELISA, enzyme-linked immunosorbent a...