Photoswitchable Antagonists for a Precise Spatiotemporal Control of β₂-Adrenoceptors

Abstract

β2-Adrenoceptors (β2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1–3, a series of photoswitchable azobenzene β2-AR antagonists that can be reversibly controlled with light. These new photochromic ligands are designed following the azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety present in many β2-AR antagonists. Using a fluorescence resonance energy transfer (FRET) biosensor-based assay, a variety of photopharmacological properties are identified. Two of the light-regulated molecules show potent β2-AR antagonism and enable a reversible and dynamic control of cellular receptor activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being more active in the dark and Photoazolol-2 demonstrating higher antagonism upon illumination. In addition, we provide a molecular rationale for the interaction of the different photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for the precise control of β2-AR by means of light.

A photoswitchable ligand targeting β1-adrenoceptor enables light-control of the cardiac rhythm

“…We have recently employed this strategy to obtain nanomolar antagonists for β 2 -AR. [28] Therefore, these structural determinants were conserved in the proposed molecules. To confer selectivity for β 1 -AR in front of β 2 -AR, we explored an interesting feature found in many β 1 -AR selective antagonists, where the oxyaminoalcohol substructure is repeatedly positioned in para -with respect to the other substituents of the phenyl ring (Figure 1).…”

“…In the activity tests we also included Photoazolol-3 ( PZL-3 ), a recently reported adrenergic photochromic ligand with a p -acetamido substituent. [28] This ligand, which also displays the p-AB scaffold proposed to selectively target β 1 -AR (Figure 1), was found to have negligible inhibitory potency against β 2 -AR. Nevertheless, the pharmacological activity of its two photoisomers was never assessed against β 1 -AR, thus raising interest for its potential as a β 1 -AR selective photoswitchable ligand.…”

“…Nevertheless, the pharmacological activity of its two photoisomers was never assessed against β1-AR, thus raising interest for its potential as a β1-AR selective photoswitchable ligand. [28] β1-AR photopharmacological properties of p-ABs were evaluated in HEK293 iSNAP β1AR H188 cells, which express β1-AR upon induction with doxycycline. A double stable cell line was generated with an EPAC S H188 CFP-YFP FRET biosensor, [35] which allowed to continuously monitor intracellular cAMP levels.…”

“…These results were in good agreement with the pharmacological properties reported for PZL-3 towards β2-AR. [28] Therefore, the three p-ABs displayed β1/β2 selectivity ratios ranging from 93.8 to 161.8 (Table 2), at the level of β1-AR selective ligands atenolol and metoprolol, which are approved drugs considered cardioselective (Figure 1). [31] On the other hand, the cis isomers of the three compounds showed moderate modulation of the two adrenoceptor subtypes and displayed lower β1/β2 selectivity ratios, ranging from 2.3 to 8.6 (Table 2).…”

“…Pharmacological data of pAzo- On the other hand, the functional β2-AR activity of the two pAzos was assessed using HEK293 H188 M1 cells, following a protocol recently reported in the literature. [28] Briefly, inhibitory dose-response curves of the ligands were obtained with a constant concentration of cimaterol (3 nM), both in the dark and upon illumination at 380 nm. Both pAzo-1 and pAzo-2 displayed low inhibitory potency against β2-AR (IC50 values within the µM range), and no significant light-induced changes in their pharmacological behavior (Figure S4 and Table S1).…”