Visible‐Light Photoswitchable Benzimidazole Azo‐Arenes as β‐Arrestin2‐Biased Selective Cannabinoid 2 Receptor Agonists

Abstract

The cannabinoid 2 receptor (CB2R) has high therapeutic potential for multiple pathogenic processes, such as neuroinflammation. Pathway‐selective ligands are needed to overcome the lack of clinical success and to elucidate correlations between pathways and their respective therapeutic effects. Herein, we report the design and synthesis of a photoswitchable scaffold based on the privileged structure of benzimidazole and its application as a functionally selective CB2R “efficacy‐switch”. Benzimidazole azo‐arenes offer huge potential for the broad extension of photopharmacology to a wide range of optically addressable biological targets. We used this scaffold to develop compound 10 d, a “trans‐on” agonist, which serves as a molecular probe to study the β‐arrestin2 (βarr2) pathway at CB2R. βΑrr2 bias was observed in CB2R internalization and βarr2 recruitment, while no activation occurred when looking at Gα16 or mini‐Gαi. Overall, compound 10 d is the first light‐dependent functionally selective agonist to investigate the complex mechanisms of CB2R‐βarr2 dependent endocytosis.

Bridging the Binding Sites 2.0: Photoswitchable Dualsteric Ligands for the Cannabinoid 2 Receptor

“…We have previously used this assay to characterize other (photoswitchable) CB ligands. 13,18,19 Reference compound CP55,940 and compound 2 are both full agonists in this assay with EC 50 = 57.2 and 27.9 nM, respectively. Initial screening was done at 10 μM, as this technique in our laboratory has lower sensitivity for substances yielding results in the micromolar range.…”

“…When designing photoswitchable ligands by introducing/incorporating a photoswitchable moiety into a lead structure, the affinity is often decreased to some extent. However, a certain affinity loss is tolerated if the obtained tool compound enables reversible photocontrol over its targeted receptor. , The potential dualsteric photoswitchable compound 17 - para displays “ cis -on” behavior in calcium mobilization (see Figure ), which is consistent in both assays and therefore makes it the most interesting derivative for obtaining optical control over CB 2 R activation.…”

“…We previously used this approach to develop a photoswitchable dualsteric M1 receptor ligand . CB 2 R agonist 2 was also previously used by us to obtain a CB 2 R-selective “affinity switch” as well as a photoswitchable biased CB 2 R agonist. , Following the same strategy as for designing non-photoswitchable dualsteric CB 2 R ligands, two series of compounds were synthesized. In the first approach, we used the photoswitchable moiety as a linker between the orthosteric and allosteric parts of the molecule, thereby also elongating the structure.…”

“…To further evaluate the pharmacological profile of compounds 17 - para and to make sure not to overlook potentially biased ligands, the photoswitchable derivatives of B3 were again measured in a calcium mobilization assay using Chinese hamster ovary (CHO-K1) cells overexpressing h CB 2 R and Gα 16 . We have previously used this assay to characterize other (photoswitchable) CB ligands. ,, Reference compound CP55,940 and compound 2 are both full agonists in this assay with EC 50 = 57.2 and 27.9 nM, respectively. Initial screening was done at 10 μM, as this technique in our laboratory has lower sensitivity for substances yielding results in the micromolar range.…”

“…Figure ) by connecting PAM 1 to the orthosteric CB 2 R agonist 2 , first published by AstraZeneca in 2008 . Several orthosteric photoswitchable tool compounds have recently been reported for CB receptors by us and others. − The photopharmacological toolbox for studying CB 2 R activation so far contains a “ cis -on” affinity switch, a “ trans -on” β-arrestin2 (βarr2) biased efficacy switch, and a “ cis -on” molecular probe to control intracellular calcium flux. ,, To further develop our dualsteric scaffold and enlarge this toolbox, we applied a photopharmacological approach to obtain photoswitchable potential dualsteric CB 2 R ligands. To the authors’ knowledge, photoswitchable dualsteric ligands have only been developed for muscarinic acetylcholine receptors so far, − where compound “PAI” was effective in vivo using two-photon excitation .…”