Molecular Interactions of Cyclam and Bicyclam Non-peptide Antagonists with the CXCR4 Chemokine Receptor*

The non-peptide CXCR4 receptor antagonist AMD3100, which is a potent blocker of human immunodeficiency virus cell entry, is a symmetrical bicyclam composed 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 Asp171 and Asp262, located in transmembrane domain (TM) IV and TM-VI, respectively, at each end of the main ligand-binding crevice of the CXCR4 receptor, as being essential for the ability of AMD3100 to block the binding of the chemokine ligand stromal cell-derived factor (SDF)-1α as well as the binding of the receptor antibody 12G5. The free cyclam moiety had no effect on 12G5 binding, but blocked SDF-1α binding with an affinity of 3 μm through interaction with Asp171. The effect on SDF-1α binding of a series of bicyclam analogs with variable chemical linkers was found to rely either only on Asp171, i.e. the bicyclams acted as the isolated cyclam, or on both Asp171 and Asp262, 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 found between the dependence of these compounds on Asp262 for binding and their potency as anti-human immunodeficiency virus agents. It is concluded that AMD3100 acts on the CXCR4 receptor through binding to Asp171 in TM-IV and Asp262 in TM-VI with each of its cyclam moieties, and it is suggested that part of its function is associated with a conformational constraint imposed upon the receptor by the connecting phenylenebismethylene linker.