A CXCL8 receptor antagonist based on the structure of N-acetyl-proline-glycine-proline

A role for the collagen-derived tripeptide, N-acetyl proline-glycine-proline (NAc-PGP), in neutrophil recruitment in chronic airway inflammatory diseases, including COPD and cystic fibrosis, has recently been delineated. Due to structural similarity to an important motif for interleukin-8 (CXCL8) binding to its receptor, NAc-PGP binds to CXCR1/2 receptors, leading to neutrophil activation and chemotaxis. In an effort to develop novel CXCL8 antagonists, we describe the synthesis of four chiral isomers of NAc-PGP (NAc- l-Pro-Gly- l-Pro ( ll-NAc-PGP), NAc- l-Pro-Gly- d-Pro ( lD-NAc-PGP), NAc- d-Pro-Gly- l-Pro ( dl-NAc-PGP), and NAc- d-Pro-Gly- d-Pro ( dd-NAc-PGP)), characterize them by circular dichroism and NMR spectroscopy, compare their structures to the equivalent region of CXCL8, and test them as potential antagonists of ll-NAc-PGP and CXCL8. We find that ll-NAc-PGP superimposes onto the CXCR1/2 contacting E 29S 30G 31P 32 region of CXCL8 (0.59A rmsd for heavy atoms). In contrast, dd-NAc-PGP has an opposing orientation of key functional groups as compared to the G 31P 32 region of CXCL8. As a consequence, dd-NAc-PGP binds CXCR1/2, as demonstrated by competition with radiolabeled CXCL8 binding in a radioreceptor assay, yet acts as a receptor antagonist as evidenced by inhibition of CXCL8 and ll-NAc-PGP mediated neutrophil chemotaxis. The ability of dd-NAc-PGP to prevent the activation of CXC receptors indicates that dd-NAc-PGP may serve as a lead compound for the development of CXCR1/2 inhibitors. In addition, this study further proves that using a different technical approach, namely preincubation of NAc-PGP instead of simultaneous addition of NAc-PGP with radiolabeled CXCL8, the direct binding of NAc-PGP to the CXCL8 receptor is evident.