Design, synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12-CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_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 CXCL12_1-8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50nM and <800nM, 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=22nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3-CXCL12_1-8, was an effective CXCR4 agonist (IC₅₀=407nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12-CXCR4 interaction and function.