When Light Meets Chemokine Receptors: Photocontrolling ACKR3 and CXCR4 Function
Photopharmacology integrates photochemical principles with pharmacology to achieve light-dependent regulation of biological targets using photoswitchable molecules. Light-driven control enables temporal and spatial accuracy, offering new possibilities to study dynamic signaling processes while limiting undesired systemic effects.G protein-coupled receptors (GPCRs) represent the largest group of proteins targeted by therapeutic drugs and play central roles in numerous physiological functions. Within GPCRs, the chemokine receptors CXCR4 and ACKR3 are critically involved in immune cell trafficking, tissue development, inflammation, and cancer progression. However, conventional pharmacological tools targeting these receptors act globally and lack the ability to reversibly modulate receptor activity in space and time. This PhD thesis reports the development and pharmacological characterization of azobenzene-based photoswitchable ligands designed to enable optical control of CXCR4 and ACKR3. To support ligand discovery and evaluation, chemokine receptor binding assays based on NanoBRET technology were established and expanded into a multiplexed format. Using these platforms, the first photoswitchable CXCR4 antagonist was identified, alongside the first photoswitchable agonist and inverse agonist for ACKR3, allowing reversible and bidirectional modulation of receptor activity with light. The compounds were characterized using multiple GPCR-relevant assays, including binding studies, β-arrestin2 recruitment assays, FRET-based biosensors, and fluorescence imaging of ligand binding. In addition, assay formats suitable for in situ photoswitching were assessed and adapted. Overall, this work demonstrates that chemokine receptor activity can be modulated with high spatiotemporal precision using photoswitchable ligands. The tools and methodologies developed in this thesis expand the photopharmacological toolkit for GPCR research and provide new opportunities to investigate CXCR4 and ACKR3 signaling dynamics, with potential relevance for future light-controlled therapeutic strategies.
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