A Combinatorial Approach to Biophysically Characterise Chemokine-Glycan Binding Affinities for Drug Development

Abstract

Chemokine binding to glycosaminoglycans (GAGs) is recognised to be an important step in inflammation and other pathological disorders like tumor growth and metastasis. Although different ways and strategies to interfere with these interactions are being pursued, no major breakthrough in the development of glycan-targeting drugs has been reported so far. We have engineered CXCL8 towards a dominant-negative form of this chemokine (dnCXCL8) which was shown to be highly active in various inflammatory animal models due to its inability to bind/activate the cognate CXCL8 GPC receptors on neutrophils in combination with its significantly increased GAG-binding affinity [1]. For the development of GAG-targeting chemokine-based biopharmaceuticals, we have established a repertoire of methods which allow the quantification of protein-GAG interactions. Isothermal fluorescence titration (IFT), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and a novel ELISA-like competition assay (ELICO) have been used to determine K d and IC 50 values for CXCL8 and dnCXCL8 interacting with heparin and heparan sulfate (HS), the proto-typical members of the GAG family. Although the different methods gave different absolute affinities for the four protein-ligand pairs, the relative increase in GAG-binding affinity of dnCXCL8 compared to the wild type chemokine was found by all methods. In combination, these biophysical methods allow to discriminate between unspecific and specific protein-GAG interactions.

A C‐terminal CXCL8 peptide based on chemokine–glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation

“…Then, binding of each synthesized peptide was studied, to evaluate affinity for heparin. Heparin‐CXCL8 SPR confirmed binding as shown in Fig. .…”

“…To assess the GAG‐binding ability of synthesized C‐terminal peptides, SPR binding studies were performed. We first evaluated the binding of CXCL8 to a heparin‐coated chip following established protocols . Then, binding of each synthesized peptide was studied, to evaluate affinity for heparin.…”

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74–103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice

“…Bound CXCL8 or CXCL11 was incubated with biotinylated polyclonal rabbit anti-human CXCL8 or CXCL11 (PeproTech), diluted in blocking buffer, followed by detection with horseradish peroxidase-labeled streptavidin. Bound biotinylated CCL2 was directly detected using streptavidin-HRP (40). Subsequently, the plates were washed, the peroxidase activity was quantified by adding a horseradish peroxidase substrate solution, and conversion of 3,3Ј,5,5Ј-tetramethylbenzidine supplemented with 0.004% (v/v) H 2 O 2 was measured at 450 nm using a spectrophotometer.…”

The effect of the decoy molecule PA401 on CXCL8 levels in bronchoalveolar lavage fluid of patients with cystic fibrosis

“…Kd values for PA401 and CXCL8 binding to immobilized HS were determined by SPR measurements at 25 °C on a BiacoreX100 system (GE Healthcare, Uppsala, Sweden) as recently described (Gerlza et al, 2014 mM NaHCO 3 , 3 mM NaN 3 , pH 9.8)). The plate was covered and incubated at 4 °C overnight.…”