Molecular interactions of cyclam and bicyclam non-peptide antagonists withthe CXCR4 chemokine receptor

The non-peptide CXCR4 receptor antagonist AMD3100, which is a potent blocke r of human immunodeficiency virus cell entry, is a symmetrical bicyclam com posed of two identical 1,4,8,11-tetraazacyclotetradecane (cyclam) moieties connected by a relatively rigid phenylenebismethylene linker. Based on the known strong propensity of the cyclam moiety to bind carboxylic acid groups , receptor mutagenesis identified Asp(171) and Asp(262), located in transme mbrane domain (TM) TV and TM-VI, respectively, at each end of the main liga nd-binding crevice of the CXCR4 receptor, as being essential for the abilit y of AMD3100 to block the binding of the chemokine ligand stromal cell-deri ved factor (SDF)-1 alpha as well as the binding of the receptor antibody 12 G5. The free cyclam moiety had no effect on 12G5 binding, but blocked SDF-1 alpha binding with an affinity of 3 muM through interaction with Asp(171). The effect on SDF-1 alpha binding of a series of bicyclam analogs with var iable chemical linkers was found to rely either only on Asp(171), i.e. the bicyclams acted as the isolated cyclam, or on both Asp(171) and Asp(262), i .e, they acted as AMD3100, depending on the length and the chemical nature of the linker between the two cyclam moieties. A positive correlation was f ound between the dependence of these compounds on Asp(262) for binding and their potency as anti-human immunodeficiency virus agents, It is concluded that AMD3100 acts on the CXCR4 receptor through binding to Asp(171), TM-IV and Asp(262) in TM-VI with each of its cyclam moieties, and it is suggested that part of its function is associated with a conformational constraint i mposed upon the receptor by the connecting phenylenebismethylene linker.