Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

Abstract

AbstractCXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in homeostatic and pathological states, and also participates in organogenesis, HIV-1 infection and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, so far only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. CXCL12 activation of CXCR4 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation towards chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues on TMV and TMVI of CXCR4. Using this structural motif as a template, we performedin silicomolecular modeling followed byin vitroscreening of a small compound library to search for allosteric antagonists of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small compound that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients bothin vitroandin vivowithout altering ligand binding or receptor internalization.Significance StatementCXCR4 is a ubiquitous chemokine receptor that regulates leukocyte trafficking and arrest in homeostatic and pathological states. Yet, the only commercial CXCR4 antagonist approved for clinical use is plerixafor (AMD3100), a small compound that blocks the ligand-binding site. Unfortunately, its clinical application is limited by poor pharmacokinetics and adverse effects associated with long-term administration. Here, we performedin silicoanalyses of a small aromatic compound library followed byin vitroscreening to identify allosteric CXCR4 antagonists that abrogate the ability of cells to sense chemoattractant gradients without altering other ligand-mediated functions such as blockade of cAMP production or receptor internalization. The selected compounds also actedin vivo,as demonstrated by reduced tumorigenesis and metastasis in a zebrafish tumor model. Our study describes a new approach to selectively alter some GPCR functions without the need for abolishing all receptor functionality.