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Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A 2A receptor (hA 2A R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA 2A R ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA 2A R. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA 2A R by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA 2A R, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB 2 R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB 2 R-selective bifunctional probe that covalently captures CB 2 R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB 2 R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Cardiotoxicity is a side effect that plagues modern drug design and is very often due to the off-target blockade of the human ether-à-go-go related gene (hERG) potassium channel. To better understand the structural determinants of this blockade, we designed and synthesized a series of 40 derivatives of clofilium, a class III antiarrhythmic agent. These were evaluated in radioligand binding and patch-clamp assays to establish structure-affinity relationships (SAR) for this potassium channel. Efforts were especially focused on studying the influence of the structural rigidity and the nature of the linkers composing the clofilium scaffold. It was shown that introducing triple bonds and oxygen atoms in the n-butyl linker of the molecule greatly reduced affinity without significantly modifying the pKa of the essential basic nitrogen. These findings could prove useful in the first stages of drug discovery as a systematic way of reducing the risk of hERG K(+) channel blockade-induced cardiotoxicity.