Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.BRET | lipid-anchored peptide | beta-arrestin | protein-protein interaction | cell signaling

Sesquiterpene lactones are of considerable interest due to their potent bioactivities, including cancer cell cytotoxicity and antineoplastic efficacy in in vivo studies. Among these compounds, artesunate, dimethylaminoparthenolide, and L12ADT peptide prodrug, a derivative of thapsigargin, are being evaluated in current cancer clinical or preclinical trials. Based on the structures of several antitumor sesquiterpene lactones, a number of analogues showing greater potency have been either isolated as natural products or partially synthesized, and some potential anticancer agents that have emerged from this group of lead compounds have been investigated extensively. The present review focuses on artemisinin, parthenolide, thapsigargin, and their naturally occurring or synthetic analogues showing potential anticancer activity. This provides an overview of the advances in the development of these types of sesquiterpene lactones as potential anticancer agents, including their structural characterization, synthesis and synthetic modification, and antitumor potential, with the mechanism of action and structure-activity relationships also discussed. It is hoped that this will be helpful in stimulating the further interest in developing sesquiterpene lactones and their derivatives as new anticancer agents.

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.

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