Development and use of Clickable and Irreversible Probes for Detection of G Protein‐Coupled Receptors

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One of the major challenges faced in G protein-coupled receptor (GPCR) drug discovery is a need for specific tools for target engagement and to study non-canonical measures of receptor behavior that have gone unappreciated in traditional pipelines, such as receptor internalization and localization. Antibody-based approaches are commonly employed to localize proteins of interest in different tissues or subcellular compartments, however, GPCR targeting antibodies notoriously lack specificity. Chemical probes such as ‘preassembled’ fluorescent ligands have been developed for a number of GPCRs; however, introducing fluorescent moieties may interfere with receptor affinity or selectivity. Separating a fluorescent ligand into two parts with a complementary ‘clickable’ handle installed on each, offers an alternative and advantageous ‘in situ labeling’ solution to a conventional single large molecular weight fluorescent ligand. This strategy creates a bifunctional ligand that requires a two-step labeling approach to detect GPCRs. Step 1 involves irreversible binding to the target GPCR (either chemoreactive or photoreactive). Step 2 employs a biorthogonal reaction (e.g. click chemistry) to attach a reporter molecule (e.g. fluorophore). We have developed bifunctional ligands based on either orthosteric or allosteric pharmacophores informed by pre-existing structure-activity relationships for a number of GPCRs. Lead bifunctional probes had a similar pharmacological profiles to parent molecules and were assessed for selectivity against related GPCRs. We show successful attachment of fluorophores by click chemistry. Bifunctional probes offer a set of new tools to aid structure elucidation as well as studying GPCR localization in recombinant and native systems.