Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, D-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with I-125-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their L-peptide counterparts. This was surprising because of the profoundly different side chain topologies between D- and L-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using D-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that D- and L-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than L-counterparts, the D-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the D-peptides that have high affinity and stability.