Targeting SDF-1/CXCL12 with a ligand that prevents activation of CXCR4 through structure-based drug design

Abstract

CXCL12 is an attractive target for clinical therapy because of its involvement in autoimmune diseases, cancer growth, metastasis, and neovascularization. Tyrosine sulfation at three positions in the CXCR4 N-terminus is crucial for specific, high-affinity CXCL12 binding. An NMR structure of the complex between the CXCL12 dimer and a sulfotyrosine-containing CXCR4 fragment enabled high-throughput in silico screening for inhibitors of the chemokine-receptor interface. A total of 1.4 million compounds from the ZINC database were docked into a cleft on the CXCL12 surface normally occupied by sulfotyrosine 21 (sY21), and five were selected for experimental screening. NMR titrations with CXCL12 revealed that four of the compounds occupy the sY21 site, one of which binds with a K(d) of 64 microM. This compound selectively inhibits SDF1-induced CXCR4 signaling in THP1 cells. Our results suggest that sulfotyrosine recognition sites can be targeted for the development of novel chemokine inhibitors.

Figure 1

Correlation between chemical shift perturbations and docking of small molecules. A) CXCR4 residues D20 and sY21 occupy a cleft on the CXCL12 surface bordered by residues in the N-loop (H17 and V18) and β3-strand (V49). B) HSQC spectra of 250 µM [U-¹⁵N] CXCL12 acquired in the presence of 0, 0.5 and 1.25 mM 1709621. C) HSQC shift perturbations induced by binding of three ZINC compounds identify H17, V18 and V49 (purple bars).

Figure 2

3-(naphthalene-2-carbonylthio carbamoylamino)benzoic acid (ZINC ID 310454) binds CXCL12 and inhibits CXCR4-mediated Ca²⁺-flux. A) CXCL12 chemical shift perturbations induced by ligand binding. B) A consistent set of perturbed residues (red) surround the docked 310454 (naphthyl) and 308174 (phenyl) compounds, but R12 (blue) is shifted only when the naphthylene is present. C) Modifications to the naphthyl (308174) or benzoic acid (2827223) groups reduced CXCL12 binding affinity relative to 310454. D) Ca²⁺-flux of THP-1 cells in response to 30 nM CXCL12 followed by 10 nM of the chemokine CCL2/MCP-1 (upper panel), 10 nM CCL2/MCP-1 pre-incubated with ZINC ID 310454 followed by 30 nM CXCL12 (middle panel) and 30 nM CXCL12 pre-incubated with 100 µM ZINC ID 310454 followed by 10 nM CCL2/MCP-1.