Discovery and Characterization of an Endogenous CXCR4 Antagonist

Abstract

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s)

“…It is necessary to develop more ubiquitous and effective HIV-1 inhibitor. Up to now, several natural small molecular peptides in human blood and tissues have been identified as HIV-1 entry inhibitors (Munch et al, 2007;Zirafi et al, 2015). However, it is tough to purify and characterize the endogenous antiviral factors circulating in the human body.…”

“…The group of Frank Kirchhoff identified a natural efficient inhibitor of the HIV-1 gp41 fusion peptide named VIRIP through a systematic screening of peptide library generated from human hemofiltrate (Munch et al, 2007). Through screening of hemofiltrate-derived peptide library, this group also discovered another endogenous effective and specific CXCR4 antagonist, a 16 amino acid long fragment of serum albumin, the most abundant protein in human plasma, as an inhibitor of CXCR4-tropic HIV-1 (Zirafi et al, 2015). Moreover, it has been reported that Palmitic acid (PA), which was isolated from Sargassum fusiforme, inhibited HIV entry through interference in gp120-CD4 interaction (Lee et al, 2009;Paskaleva et al, 2010).…”

New agents in HSC mobilization

“…Furthermore, EPI-X4 has a very short plasma half-life of ~17 min, which may limit its utilization as a therapeutic. However, the identification of several synthetic derivatives, specifically the dimeric derivative designated "WSC02" ((IVRWSKKVPCVS)×2) has been shown to possess superior plasma stability compared to EPI-X4 peptide and exhibited enhanced suppression of HSC migration towards SDF-1 [55]. These promising preliminary studies featuring EPI-X4 and its more stable synthetic derivatives warrant further investigation in their therapeutic potential [59].…”

“…Intriguingly, while all the prerequisites for the generation of EPI-X4 are ubiquitously available throughout the body, its presence and endogenous regulatory role, if any, on native BM HSC remains to be determined. Nevertheless, EPI-X4 has been demonstrated to reduce basal CXCR4 signalling activity and, therefore, also behaves as an inverse agonist, suggesting endogenous CXCR4 signalling may not be restricted to SDF-1 [55]. Of note, G-CSF induces HSC mobilization through dynamic changes in the BM microenvironment mediated in part by secretion of the proteolytic enzymes cathepsin K or cathepsin G, which are produced by osteoclasts and neutrophils, respectively [51,57,58].…”

“…In 2015, a naturally occurring 16-mer amino acid peptide fragment derived from human serum albumin termed "EPI-X4" (LVRYTKKVPQVST-PTL) was discovered and revealed to be a potent CXCR4 antagonist and rapid mobilizer of murine HSPC [55,56]. EPI-X4 is produced from the abundantly present serum albumin precursor through enzymatic digestion by the aspartyl proteases Cathepsin D and E [55]. Intriguingly, while all the prerequisites for the generation of EPI-X4 are ubiquitously available throughout the body, its presence and endogenous regulatory role, if any, on native BM HSC remains to be determined.…”