An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation

Abstract

BackgroundAberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes including chronic inflammatory diseases. Thus, the chemokine receptor CXCR4 is a promising target for the therapy of inflammatory disorders, such as atopic dermatitis or allergic asthma. A natural fragment of serum albumin, named EPI-X4, has previous been identified as endogenous peptide antagonist and inverse agonist of CXCR4. The endogenous CXCR4 antagonist provides a promising basis for the development of improved analogues for the therapy of inflammatory diseases.ObjectiveTo increase the anti-CXCR4 activity of EPI-X4 and to evaluate the therapeutic potential of optimized analogs in mouse models of atopic dermatitis and asthma.MethodsMolecular docking analysis of the interaction of EPI-X4 with CXCR4 was performed to define critical interaction motifs and to rationally design analogs with increased activity. EPI-X4 derivatives were synthesized and CXCR4 binding and antagonizing activity determined in assays for antibody competition, inhibition of CXCR4-mediated HIV-1 infection, CXCL12-dependent Ca2+ mobilization, ERK and AKT phosphorylation and cell migration. Toxicity of peptides was evaluated in zebrafish embryos. The therapeutic efficacy of the lead peptide EPI-X4 JM#21 was determined in mouse models of atopic dermatitis and asthma.ResultsDocking analysis identified key interaction motifs of EPI-X4/CXCR4. Rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4 and resulted in the generation of the lead analog JM#21, which bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. JM#21 did not exert toxic effects in zebrafish embryos and efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Moreover, EPI-X4 and its improved derivative suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model.ConclusionThe rationally designed EPI-X4 derivative JM#21 is a potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.Graphical Abstract

Targeting of CXCR4 by the naturally occurring CXCR4 antagonist EPI-X 4 in Waldenström’s Macroglobulinemia

“…To investigate whether rational design can further enhance the anti-CXCR4 and therefore anti-WM activities of EPI-X4, we took advantage of two EPI-X4 derivatives, termed WSC02 and JM#21. These peptides were generated from EPI-X4 by structure-activity-relationship (SAR) studies and subsequent synthetic modulations inserting mutations in the lysine residues [23] and were also able to efficiently block the epitope 12G5 at low concentrations (Suppl. Fig.…”

“…Thus, generation of optimized derivatives of EPI-X4 will be crucial for opening the door to clinical application. We performed systematic, quantitative structure activity relationship (QSAR) studies to further improve the anti-CXCR4 activity of EPI-X4 and have solved NMR structures of EPI-X4, as well as first (WSC02) and second (JM#21) generation derivatives, and have used this information for computational modeling to elucidate their exact binding mode with CXCR4, and to predict peptides with further improved activity [23]. In iterative processes, we have now synthesized and tested more than 150 EPI-X4 derivatives (data not shown), enabling the identification of peptides with activities in the low nanomolar range.…”

Biomolecular models of EPI-X4 binding to CXCR4 allow the rational optimization of peptides with therapeutic potential

“…16 A further optimized 12-mer version, EPI-X4 JM#21, harbors three additional amino acid substitutions (V2L, K6R, V9L), which led to further increase in receptor affinity (Harms et al preprint). 17 In addition, EPI-X4 JM#21 showed increased efficiency for inhibition of CXCL12mediated receptor signaling (> 100-fold compared to EPI-X4, 10-fold compared to WSC02) and cancer cell migration (~ 1,500-fold compared to EPI-X4, 30-fold compared to WSC02).…”

“…Also, optimized EPI-X4 derivatives showed very promising therapeutic effects in mouse models for stem cell mobilization, allergic airway eosinophilia and atopic dermatitis. 16,17 Computational modeling has also been carried out to determine the interaction sites of different CXCR4 receptor ligands with the binding pockets of CXCR4. 18,19 Several molecular dynamics (MD) studies are reported, primarily (i) to characterize the structure and function of CXCR4 and agonist binding, 20-22 (ii) to study the interactions between CXCR4 and small molecule/peptide antagonists, [23][24][25] and (iii) to study the dimerization of CXCR4 [26][27][28] .…”

“…Next, we investigated the interactions of the optimized EPI-X4 derivatives WSC02 and JM#21, that showed potent therapeutic effects in mouse models of atopic dermatitis and asthma 16,17 . Both, WSC02 and JM#21, are C-terminally truncated analogues of EPI-X4 harboring 12 amino acids each.…”