Anomeric modification of nona-sulfated tetrahyaluronan via dynamic ligation yields potent ligands of interleukin-8 and CXCR2 receptor, inhibiting their signaling

Complex carbohydrates are ubiquitous in biological systems and are involved in numerous diseases. Still only few carbohydrate-based drugs are known. Insufficient specificity, limited druglikeness and tedious synthetic access are major challenges of carbohydrate structures. In this study we are presenting a strategy for the fast modification of glycans at their anomeric position using a dynamic ligation screening assay enabling high-throughput fragment variation. A fluorescence polarization (FP) assay was developed for the protein target interleukin 8 (IL-8), a proinflammatory chemokine regulating the chemotaxis of leucocytes, especially neutrophils, into inflamed tissue. In this study, nona-sulfated tetrahyaluronan with an electrophilic aldehyde functionality was prepared as a primary fragment binding to IL-8 and a library of 1152 amine fragments was screened for the best fragment-glycoside combinations. Two hits with nanomolar KI values were identified in the FP assay and binding was confirmed by isothermal titration calorimetry (ITC). The best hit, 9s-HA4-aniline 13, was validated functionally by surface plasmon resonance (SPR) and inhibited complex formation of IL-8 with its receptor CXCR2. Inhibition of IL-8 by 9s-HA4-aniline blocked the activation of human polymorphonuclear leucocytes (PMN) by IL-8 but not by tumor necrosis factor (TNF) as a control cytokine and resulted in the down-regulation of CXCR2 and of L-selectin. Surprisingly, SPR and cell experiments showed that 9s-HA4-aniline was also binding CXCR2 receptors. The biological activity of 9s-HA4-aniline against interleukin-8 and CXCR2 receptors was rationalized by molecular docking and by molecular dynamics simulation of the protein-inhibitor complexes substantiating the possibility of glycoside drugs to target the IL-8 / CXCR-2 signaling pathway.