Cross-talk between the CXCL12 chemokine receptors CXCR4 and ACKR3: contribution of oligomers populations to receptors signaling and function – OCHRE

Directional cell migration (i.e. chemotaxis) orchestrated by chemokines (CKs) and their receptors (CKRs) regulates organ development and plays key roles in adult physiology at the interface between tissue homeostasis and immune surveillance. Some CKs/CKrs have pivotal and non-redundant homeostatic functions, far beyond cell migration, such as CXCL12 and its two receptors; the G protein coupled receptor CXCR4 and ACKR3/CXCR7, which belongs to the atypical subgroup of CKRs that do not signal through G proteins and act as CKs’ scavenger. A large body of work has been devoted to the CXCL12/CXCR4 pair since the discovery of its role in HIV pathogenesis and later on, its implication as well as that of ACKR3, in a wide spectrum of diseases including immune/infectious diseases and cancers making CXCL12/CXCR4-ACKR3 prime targets for therapeutic intervention.
An important issue is the reported potential of ACKR3 to regulate CXCR4-expressing cells chemotaxis via shaping CXCL12 extracellular gradients. Nevertheless, our preliminary results clearly indicate that CXCL12 binds and activates CXCR4 long before it binds on ACKR3, suggesting that the scavenging function of ACKR3 toward CXCL12 is probably not the only mechanism explaining the cross regulation between both receptors. In this proposal, we push forward the hypothesis that the existence of CXCR4/ACKR3 oligomers should also be considered. However, data supporting their existence in native conditions are still lacking and, accordingly, building novel tools and models to tackle this issue is a major and ambitious goal for the field.
Therefore, our multidisciplinary consortium that combines a unique set of complementary expertise in physiology, pharmacology, structural biology, medicinal chemistry and computational modeling of GPCRs aims at (i) characterizing the pharmacological/structural properties of CXCR4/ACKR3 oligomers, (ii) investigating their existence in native tissues and (iii) their role in physiology that will rely largely on (iv) the development of selective ligands for such complexes. Based on original in-house setup assays, our preliminary data strongly support the concept that CXCR4/ACKR3 oligomers represent unique binding/signaling entities, the original features of which will be further deepen. To investigate the existence of oligomers in native tissues, we have developed time-resolved FRET experiments based on the use of fluorescent nanobodies targeting CXCR4 or ACKR3. We have thus pioneered in demonstrating the existence of CXCR4 oligomers in cells endogenously expressing CXCR4, an original finding that we aim to extend to cells expressing both receptors (human & murine) and to native tissues from different mouse strains and models of human skin (3D epithelial cells cultures). We will put an emphasis in the elucidation of CXCR4/ACKR3 oligomers structure, which is critical to understand receptors crosstalk and to design the selective ligands required for assessing CXCR4/ACKR3 oligomers physiological roles. More specifically, we will analyze oligomers by the innovative Cryo-EM analysis at near-atomic resolution based on multifunctional 3D-DNA origami systems as supports to control the spatial positioning and density of CXCR4/CXCR7 receptors. Finally, two complementary strategies will be implemented to develop CXCR4/ACKR3 oligomers selective ligands: one is based on the development of hetero-bivalent ligands resulting from the tethering of ligands selective for CXCR4/ACKR3 and the other on structure-virtual screening to design small compounds able to stabilize/disrupt CXCR4/ACKR3 dimer interface.
We anticipate to generate a comprehensive map of the structural and biological features of CXCR4/ACKR3 oligomers that will benefit to drug discovery programs and also to offer insights into technical development thus providing a strong guide for other CKRs oligomers characterization.