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

Lars Ole Gerlach,Renato T Skerlj,Gary J Bridger,Thue W Schwartz

doi:10.1074/jbc.m010429200

Lars Ole Gerlach, Renato T Skerlj + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m010429200

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Abstract

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 Asp(171) and Asp(262), 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)-1alpha as well as the binding of the receptor antibody 12G5. The free cyclam moiety had no effect on 12G5 binding, but blocked SDF-1alpha binding with an affinity of 3 microm through interaction with Asp(171). The effect on SDF-1alpha binding of a series of bicyclam analogs with variable 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 found 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) in 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 imposed upon the receptor by the connecting phenylenebismethylene linker.

Highlights

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 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 ligandbinding 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)-1␣ as well as the binding of the receptor antibody 12G5
Initial mutagenesis studies have shown that acidic residues in the second extracellular loop and in transmembrane domain (TM) IV of the CXCR4 receptor apparently are involved in the antiviral effect of AMD3100 [14]

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—Point mutations were introduced in the receptor by the polymerase chain reaction overlap extension technique [21] using the human wild-type CXCR4 receptor cDNA as template. The wild-type and mutant CXCR4 receptors were transiently transfected into COS-7 cells by the calcium phosphate precipitation method as described previously [23]. Ligands—The human chemokine Met-SDF-1␣ was kindly provided by Michael A. Cells were assayed by competition binding performed on whole cells for 3 h at 4 °C using 12 pM 125I-Met-SDF-1␣ or 32 pM 125I-12G5 plus variable amounts of unlabeled peptide or non-peptide compounds in 400 ␮l of 50 mM HEPES (pH 7.7) supplemented with 1 mM CaCl2, 5 mM MgCl2, and 0.5% (w/v) bovine serum albumin (Sigma). Calculations—IC50 values were determined by nonlinear regression using Prism 3.0 (GraphPAD Software, San Diego, CA)

RESULTS

Low expression

DISCUSSION