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The Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a body-own fragment of albumin and specific antagonist of the CXC-motif-chemokine receptor 4 (CXCR4). CXCR4 signaling is induced by its sole chemokine ligand CXCL12 and is involved in a plethora of functions including cell homing, differentiation, survival and angiogenesis. Consequently, dysregulation of CXCR4 is involved in a variety of disorders, such as cancer or inflammatory diseases, making CXCR4 an attractive drug target. EPI-X4 and derivatives with increased CXCR4 binding affinities represent promising leads as CXCR4 antagonists and have shown therapeutic activity in mouse models of inflammatory diseases. However, it is currently unclear how EPI-X4 and its derivatives interact with CXCR4. Here, by combining biomolecular simulations with experimental mutagenesis and activity studies we investigated the binding behavior of EPI-X4 to CXCR4 at the molecular level. Our work allowed us to show that the EPI-X4 peptide interacts primarily in the minor pocket of CXCR4 through its N-terminal residues. The biomolecular interactions highlighted by the computational studies are in good agreement with the experimental mutagenesis data. Moreover, we found that the N-terminal seven amino-acids of EPI-X4 (a 16-mer) and its improved derivatives (12-mers) are sufficient for CXCR4 binding, which led to the development of shorter leads with optimized CXCR4 antagonizing properties. Collectively, we here established how EPI-X4 binds to its receptor and used this knowledge for rational drug design. The new peptide variants developed by us are more potent in terms of inhibiting CXCR4-downstream signaling and cancer cell migration, without toxic effects.

Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cells’ (mEC) function and rapid recovery from catecholamine-dependency and septic shock. We here tested a single device, which consists of polystyrene-divinylbenzene core particles of 450 μm diameter with a high affinity for hydrophobic compounds. The current study aimed at the proof of concept that endothelial-specific damage mediators are adsorbed and can be recovered from hemoadsorption devices. Because of excellent clinical experience, we tested protein fractions released from a hemoadsorber in a novel endothelial bioassay. Video-based, long-term imaging of mEC proliferation and cell death were evaluated and combined with apoptosis and ATP measurements. Out of a total of 39 fractions recovered from column fractionation, we identified 3 fractions that caused i) inhibition of mEC proliferation, ii) increased cell death and iii) induction of apoptosis in mEC. When adding these 3 fractions to mEC, their ATP contents were reduced. These fractions contained proteins of approximately 15 kDa, and high amounts of nucleic acid, which was at least in part oxidized. The efficacy for endothelial cell damage prevention by hemoadsorption can be addressed by a novel endothelial bioassay and long-term video observation procedures. Protein fractionation of the hemoadsorption devices used is feasible to study and define endothelial damage ligands on a molecular level. The results suggest a significant effect by circulating nucleic acids – bound to an as yet undefined protein, which may constitute a major danger-associated molecular pattern (DAMP) in the exacerbation of inflammation when patients experience septic shock. Hemoadsorption devices may thus limit endothelial damage, through the binding of nucleic acid-bearing aggregates and thus contribute to improved endothelial barrier function.