Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells

Abstract

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4-and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

“…Human ␤-defensin-1 binds to CCR6 and is chemotactic for immature dendritic cells; tyrosyl-tRNA synthetase is processed into a secreted agonist for CXCR1, histidyl-tRNA synthetase activates CCR5, and seryl-tRNA synthetase induces the migration of CCR3-transfected cells (62)(63)(64). ␤-Defensin-3, ubiquitin, peptucin analogs, as well as a complex between HMGB1 and CXCL12 have been reported as CXCR4 agonists or antagonists (65)(66)(67)(68). The structural basis underlying the engagement of chemokine receptors by these atypical chemokines is poorly understood and is likely to differ for each chemokine-like function because the structure of each protein does not share similarity with others (25).…”

Two distinct CXCR4 antagonists mobilize progenitor cells in mice by different mechanisms

“…34 Thus, for example, circadian variations in levels of CXCL12 in the BM have been shown to correlate with retention and egress of HPCs, 35 and sustained overexpression of CXCL12 using an adenovirus and other drugs that are CXCR4 agonists have been shown to mobilize HPCs into the blood. [36][37][38] CXCL12 is produced locally in damaged tissues and is reported to be critical for the mobilization and recruitment of BM stem/progenitor cells to the site of injury. [39][40][41][42][43][44][45] Taken together, these studies indicate that the relative level of CXCL12 in the blood and BM is one factor that regulates progenitor cell retention/mobilization under homeostasis and in disease.…”

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

“…Allosteric agonists and antagonists that bind at alternative sites to the orthosteric site (13) can provide certain advantages such as a greater GPCR subtype specificity and G protein-selective pathway engagement (14). Libraries of pepducins can be readily targeted to a GPCR of interest based on the sequences of the intracellular loops, including orphan GPCRs, and tested for allosteric activity by high throughput screening (15,16). This approach has proven useful in delineating the functional significance of GPCRs in cancer (17)(18)(19)(20) and cardiovascular (6,(21)(22)(23)(24) and inflammatory diseases (25)(26)(27)(28), and now pepducins have just entered human clinical trials (29); however, their mechanism of activation is largely unknown.…”