Identification of a Potent, Selective Non-peptide CXCR2 Antagonist That Inhibits Interleukin-8-induced Neutrophil Migration

TLDR

IL‑8 and related ELR‑containing CXC chemokines are potent neutrophil chemoattractants that mediate inflammation, with IL‑8 signaling through both CXCR1 and CXCR2 while GROα, GROβ, GROγ and ENA‑78 signal exclusively via CXCR2. A high‑throughput screen identified a chemical lead that selectively inhibits CXCR2, which was then chemically optimized to produce a potent antagonist. The resulting compound, SB 225002, binds CXCR2 with an IC₅₀ of 22 nM, shows >150‑fold selectivity over CXCR1 and other G‑protein coupled receptors, potently blocks IL‑8– and GROα‑induced neutrophil chemotaxis in vitro, and selectively prevents IL‑8‑induced neutrophil margination in vivo, confirming CXCR2’s role in neutrophil recruitment and providing a useful tool for studying inflammatory diseases.

Abstract

Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)α, GROβ, GROγ, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROα, GROβ, GROγ, and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (<i>N</i>-(2-hydroxy-4-nitrophenyl)-<i>N</i>′-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of ¹²⁵I-IL-8 binding to CXCR2 with an IC₅₀ = 22 nm. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. <i>In vitro,</i> SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROα. <i>In vivo,</i> SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.