Structural analysis of a novel small molecule ligand bound to the CXCL12 chemokine - PubMed (original) (raw)

. 2014 Nov 26;57(22):9693-9.

doi: 10.1021/jm501194p. Epub 2014 Nov 13.

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

Emmanuel W Smith et al. J Med Chem. 2014.

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.

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Figures

Figure 1

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

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

Figure 3

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

Figure 4

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 CXCR41–38 (PDB ID: 2K05) outlines the sY21-binding site as well as the possible hydrogen bond interactions.

Figure 5

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 CXCR41–38 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

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%.

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