Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Azoacetylenes for the Synthesis of Arylazotriazole Photoswitches.

We report a modular approach toward novel arylazotriazole photoswitches and their photophysical characterization. Addition of lithiated TIPS-acetylene to aryldiazonium tetrafluoroborate salts gives a wide range of azoacetylenes, constituting an underexplored class of stable intermediates. In situ desilylation transiently leads to terminal arylazoacetylenes that undergo copper-catalyzed cycloadditions (CuAAC) with a diverse collection of organoazides. These include complex molecules derived from natural products or drugs, such as colchicine, taxol, tamiflu, and arachidonic acid. The arylazotriazoles display near-quantitative photoisomerization and long thermal Z-half-lives. Using the method, we introduce for the first time the design and synthesis of a diacetylene platform. It permits implementation of consecutive and diversity-oriented approaches linking two different conjugants to independently addressable acetylenes within a common photoswitchable azotriazole. This is showcased in the synthesis of several photoswitchable conjugates, with potential applications as photoPROTACs and biotin conjugates.

Optical Control of Cannabinoid Receptor 2-Mediated Ca2+ Release Enabled by Synthesis of Photoswitchable Probes.

Cannabinoid receptor 2 (CB2) is a promising target for the treatment of neuroinflammation and other diseases. However, a lack of understanding of its complex signaling in cells and tissues complicates the therapeutic exploitation of CB2 as a drug target. We show for the first time that benchmark CB2 agonist HU308 increases cytosolic Ca2+ levels in AtT-20(CB2) cells via CB2 and phospholipase C. The synthesis of photoswitchable derivatives of HU308 from the common building block 3-OTf-HU308 enables optical control over this pathway with spatiotemporal precision, as demonstrated in a real-time Ca2+ fluorescence assay. Our findings reveal a novel messenger pathway by which HU308 and its derivatives affect cellular excitability, and they demonstrate the utility of chemical photoswitches to control and monitor CB2 signaling in real-time.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs.

Off-tissue effects are persistent issues of modern inhibition-based therapies. By merging the strategies of photopharmacology and small-molecule degraders, we introduce a novel concept for persistent spatiotemporal control of induced protein degradation that potentially prevents off-tissue toxicity. Building on the successful principle of bifunctional all-small-molecule Proteolysis Targeting Chimeras (PROTACs), we designed photoswitchable PROTACs (photoPROTACs) by including ortho-F4-azobenzene linkers between both warhead ligands. This highly bistable yet photoswitchable structural component leads to reversible control over the topological distance between both ligands. The azo-cis-isomer is observed to be inactive because the distance defined by the linker is prohibitively short to permit complex formation between the protein binding partners. By contrast, the azo-trans-isomer is active since it can engage both protein partners to form the necessary and productive ternary complex. Importantly, due to the bistable nature of the ortho-F4-azobenzene moiety employed, the photostationary state of the photoPROTAC is persistent, with no need for continuous irradiation. This technique offers reversible on/off switching of protein degradation that is compatible with an intracellular environment and, therefore, could be useful in experimental exploration of biological signaling pathways-such as those crucial for oncogenic signal transduction. Additionally, this strategy may be suitable for therapeutic intervention to address a variety of diseases. By enabling reversible activation and deactivation of protein degradation, photoPROTACs offer advantages over conventional photocaging strategies that irreversibly release active agents.