Highly Selective Drug-Derived Fluorescent Probes for the Cannabinoid Receptor Type 1 (CB1R)

The cannabinoid receptor type 1 (CB1R) is one of the central elements of the endocannabinoid system regulating a variety of signaling cascades. Extensive efforts on CB1R have validated its essential roles in physiology such as appetite regulation, pain perception, memory formation, and thermoregulation. Yet, there is a surprising lack of clear understanding of its cellular signaling, distribution, and expression dynamics. CB1R visualization in real-time is therefore crucial for addressing these open questions in cannabinoid research. Using various highly selective drug-like CB1R ligands with a defined pharmacological profile, we investigated their potential for constructing CB1R fluorescent probes by a reverse design-approach. A modular design concept with a diethyl glycine-based building block as centerpiece allowed the straightforward modular synthesis of novel probe candidates. Supported by computational docking studies, this systematic approach led to the identification of novel pyrrole-based CB1R fluorescent probes. The probes demonstrated CB1R selectivity in radioligand binding profiling and inverse agonist activity in a cAMP assay. Application in time-resolved fluorescence resonance target-engagement studies and CB1R live cell imaging exemplify the great versatility of the tailored pyrrole-based fluorescent probes. These validated fluorescent probes aim to deepen the understanding of mechanistic aspects of CB1R localization, trafficking, and activation essential for the function and role of this receptor in pathological conditions.