Structural analysis of a novel small molecule ligand bound to the CXCL12 chemokine

Abstract

CXCL12 binds to CXCR4, promoting both chemotaxis of lymphocytes and metastasis of cancer cells. We previously identified small molecule ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now report a 1.9 Å resolution X-ray structure of CXCL12 bound by such a molecule at a site normally bound by sY21 of CXCR4. The complex structure reveals binding hot spots for future inhibitor design and suggests a new approach to targeting CXCL12-CXCR4 signaling in drug discovery.

Figure 1

Monomeric representation of CXCR4 bound by CXCL12 through a two-step/two-site process. (A) CXCR4 has a flexible extracellular N-terminal domain. (B) In step-1/site-1, CXCL12 recognizes and binds the N-terminal domain of CXCR4 aided by sulfotyrosine recognition. (C) In step-2/site-2, the flexible N-terminal domain of CXCL12 docks into CXCR4 causing activation. Multiple lines of evidence suggest that CXCR4 can form dimers, but there is no evidence to suggest that the site 1 interface would be altered by a change in oligomeric state of the receptor.

Figure 2

A ZINC 310454 derivative binds in the sY21 binding pocket as predicted by in silico docking. (A) Fragment-based SAR analysis of ZINC 310454 led to the design and synthesis of compound 1. (B) Significant chemical shift perturbations in the presence of 1600 μM compound 1 map to the predicted binding pocket on CXCL12 (PDB: 2K05). The residues most perturbed are colored in orange.

Figure 3

Complex crystallographic structure of CXCL12 dimer (ribbon and surface model) with compound 1 (yellow) bound to the sY21-binding site.

Figure 4

Complex crystallographic structure and characterization of the sY21 binding site. (A) Stereo image depiction of the unbiased F_o−F_c map (at 3σ) of compound 1 bound to the sY21-binding site of CXCL12. (B) Complex crystal structure of CXCL12 (green) bound by compound 1 (yellow) superimposed to the apo crystal structure (PDB ID: 2J7Z) (cyan) shows conformational changes induced upon binding. (C) NMR complex structure of CXCL12 bound to the D20-sY21-D22 segment of CXCR4_1–38 (PDB ID: 2K05) outlines the sY21-binding site as well as the possible hydrogen bond interactions.

Figure 5

Heparin/sY12 vs compound 1 binding. (A) CXCL12–heparin sulfate crystal structure (PDB ID: 2NWG) positions haparin sulfate (yellow) in the region where sY12 of CXCR4_1–38 normally binds. (B) CXCL12–compound 1 crystal structure with heparin sulfate (yellow) and sY12 (purple) superimposed in sY12 site suggests that compounds specific to both sites could potentially be linked together via linkers.

Figure 6

Docking pose and chemotaxis inhibition. (A) Compound 2 is based on the compound 1 scaffold but contains an additional two-carbon linker past the urea. Docking pose prediction suggests the flexible linker may help increase hydrophobic interactions with the cleft above Val39. (B) Chemotaxis assay in THP-1 cells demontrates that 25 μM compound 1 reduces cell migration by ∼20%, while 25 μM compound 2 reduces cell migration by ∼40%.