EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

Zika virus (ZIKV) causes congenital neurologic birth defects, notably microcephaly, and has been associated with other serious complications in adults. The virus has been detected in human breast milk and possible transmissions via breastfeeding have been reported. Breast milk is rich in nutrients and bio-active substances that might directly affect viral infectivity. Thus, here, we analyzed the effect of human breast milk on ZIKV infection. We observed that fresh human breast milk had no effect on ZIKV, but found that upon storage, milk effectively suppressed infection. The antiviral activity is present in the fat-containing cream fraction of milk and results in the destruction of the structural integrity of viral particles, thereby abrogating infectivity. The release of the factor is time dependent but varies with donors and incubation temperatures. The viral titer of milk that was spiked with ZIKV decreased considerably upon storage at 37 °C for 8 h, was lost entirely after 2 days of 4 °C storage, but was not affected at −20 °C. This suggests that cold storage of milk inactivates ZIKV and that the antiviral factor in milk may also be generated upon breastfeeding and limit this transmission route of ZIKV.

C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC₅₀ values) and affinity constants (K_i values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 h. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody-competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.

CXCR4 expression and downstream signaling have been identified as key factors in malignant hematopoiesis. Thus, up to 40% of all patients with Waldenström’s macroglobulinemia (WM) carry an activating mutation of CXCR4 that leads to a more aggressive clinical course and inferior outcome upon treatment with the Bruton’s tyrosine kinase inhibitor ibrutinib. Nevertheless, little is known about physiological mechanisms counteracting CXCR4 signaling in hematopoietic neoplasms. Recently, the endogenous human peptide EPI-X4 was identified as a natural CXCR4 antagonist that effectively blocks CXCL12-mediated receptor internalization and suppresses the migration and invasion of cancer cells towards a CXCL12 gradient. Here, we demonstrate that EPI-X4 efficiently binds to CXCR4 of WM cells and decreases their migration towards CXCL12. The CXCR4 inhibitory activity of EPI-X4 is accompanied by reduced expression of genes involved in MAPK signaling and energy metabolism. Notably, the anti-WM activity of EPI-X4 could be further augmented by the rational design of EPI-X4 derivatives showing higher binding affinity to CXCR4. In summary, these data demonstrate that a naturally occurring anti-CXCR4 peptide is able to interfere with WM cell behaviour, and that optimized derivatives of EPI-X4 may represent a promising approach in suppressing growth promoting CXCR4 signaling in WM.

Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin–peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.

Semen is an important vector for sexual HIV-1 transmission. Although CXCR4-tropic (X4) HIV-1 may be present in semen, almost exclusively CCR5-tropic (R5) HIV-1 causes systemic infection after sexual intercourse. To identify factors that may limit sexual X4-HIV-1 transmission, we generated a seminal fluid–derived compound library and screened it for antiviral agents. We identified four adjacent fractions that blocked X4-HIV-1 but not R5-HIV-1 and found that they all contained spermine and spermidine, abundant polyamines in semen. We showed that spermine, which is present in semen at concentrations up to 14 mM, binds CXCR4 and selectively inhibits cell-free and cell-associated X4-HIV-1 infection of cell lines and primary target cells at micromolar concentrations. Our findings suggest that seminal spermine restricts sexual X4-HIV-1 transmission.