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

Abstract

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.

High affinity CXCR4 inhibitors generated by linking low affinity peptides

“…Both AR5 and AR6 exhibited very high binding affinity for CXCR4, with IC 50 values of 18 and 16 nM in the CHO-CXCR4 cells and 23 and 14 nM in the Sup-T1 cells, respectively. In contrast, DV3 showed low CXCR4 binding with IC 50 value of 1.1 μM on CHO-CXCR4 cell which is similar to the result published previously [42]. AR5r and AR6r, which are peptide fragments derived from the gp120 V3 loop [46,52], displayed very low CXCR4 affinity, with IC 50 values of more than 50 μM and 9.12 μM, in both the CHO-CXCR4 and the Sup-T1 cells.…”

“…AR5 and AR6 are designed using a fragment based combinational approach that links two low binding affinity fragments derived from viral protein ligands of CXCR4, namely HIV-1 gp120 and viral chemokine vMIP-II [7,42]. HIV-1, a highly mutated virus, is highly drug resistant.…”

“…However, it is not always the case that the larger size peptides will have higher affinity, as optimal lengths are required to produce better binding affinities. For example, (DV3-PEG3)2K has a better binding affinity than (DV3-PEG3) 2K or (DV3-PEG3) 2K [42], whereas AR3 (H2Nlgaswhrpdkcclgyqkrplp-Ahx-Kkrffdgll-CONH2) and AR4 (H2N-lgaswhrpdkcclgyqkrplp-Ahx-KGLLGLSK-CONH2) are both larger than AR5 or AR6 but they show lower binding affinities (AR3: IC50=193 nM; AR4: IC50=147 nM) when tested under the same conditions (our unpublished data).…”

“…Another bivalent analog of DV1 with DV1 linked to DV3, a shorter fragment of DV1 (residues 1-10 of the N-terminus of DV1) displayed high CXCR4 binding affinity [13]. DV3 was also used in another designed peptide SDV3 (DV3-PEG3-K-(PEG3-CXCL12 1-8 )), an effective CXCR4 agonist [42].…”

Alterations of CXCL12 in Serum of Patients with Vitiligo

“…In mouse models of psoriasis, inhibition of CXCL12 was able to reduce skin inflammatory responses (Zgraggen et al, 2014). CXCR4 antagonists have already been developed and could be an interesting treatment for several immune-mediated skin disorders (Yang et al, 2016). However, CXCL12 exerts multifaceted immunologic activities including some anti-inflammatory events, and further research into its involvement in the pathogenesis of vitiligo is required.…”