Structural analysis of CXCR4 – Antagonist interactions using saturation-transfer double-difference NMR

CXCR4 is a GPCR involved in leukocyte trafficking. Small molecule antagonists of the receptor may treat inflammatory disease, cancer and HIV. Here we probe the binding of a tetrahydroisoquinoline-based antagonist (TIQ-10) to CXCR4 using saturation transfer double-difference (STDD) NMR. STDD spectra were acquired using extracts from Chinese Hamster Ovary cells expressing membrane-embedded CXCR4. The experiments demonstrate competitive binding between TIQ-10 and established antagonists and provide the TIQ-10 – CXCR4 binding epitope. Molecular modeling of TIQ-10 into the binding pocket provides a pose consistent with STDD-derived interactions. This study paves the way for future investigations of GPCR-ligand interactions in a biological milieu for use in chemical biology, biochemistry, structural biology, and rational drug design. [Display omitted] •Saturation transfer difference NMR for antagonists bound to the CXCR4 receptor in cellular membrane extracts is presented.•Competitive binding between two CXCR4 antagonists using STD NMR is established.•The STD-NMR binding epitope of a small molecule CXCR4 antagonist is identified.•A 3D model consistent with the experimentally-derived epitope is proposed.