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G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target SDF-1α-CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]n ([-CH2-CH2-O-]n) linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-D-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and SDF-1α1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC50 values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG3]2 linked homodimeric DV3, was an effective CXCR4 antagonist (IC50 = 22 nM); and ligand 21, a [PEG3]2 linked heterodimeric DV3–SDF-1α1–8, was an effective CXCR4 agonist (IC50 = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate SDF-1α-CXCR4 interaction and function.