Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators

G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively “tuning” GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand–GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu₅) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu₅ NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu₅ via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu₅-bound probe <i>via</i> tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu₅ and negative cooperativity with glutamate, interacted with the “common allosteric-binding site,” displayed slow binding kinetics, and could irreversibly label mGlu₅ following UV exposure. We depleted the number of functional mGlu₅ receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes <i>via</i> click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu₅ allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-<i>threo</i>-Methylphenidate

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and the inhibitor pharmacophore. To expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact nontropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-<i>threo</i>-methylphenidate. (±)-<i>threo</i>-4-Azido-3-iodomethylphenidate [(±)-<b>6</b>; <i>K</i>_<i>i</i> = 4.0 ± 0.8 nM] displayed high affinity for hDAT. Moreover, a radioiodinated analogue of (±)-<b>6</b> demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure–function studies

Bupropion Binds to Two Sites in the <i>Torpedo</i> Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analogue [¹²⁵I]-SADU-3-72

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(<i>N</i>-<i>tert</i>-butylamino)-3′-[¹²⁵I]-iodo-4′-azidopropiophenone (SADU-3-72). Based on inhibition of [¹²⁵I]­SADU-3-72 binding, SADU-3-72 binds with high affinity (IC₅₀ = 0.8 μM) to the <i>Torpedo</i> nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC₅₀ = 1.2 μM) than in the resting state. Photolabeling of <i>Torpedo</i> nAChRs with [¹²⁵I]­SADU-3-72 followed by limited <i>in-gel</i> digestion of nAChR subunits with endoproteinase Glu-C established the presence of [¹²⁵I]­SADU-3-72 photoincorporation within nAChR subunit fragments containing M1–M2–M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1–M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu²⁶⁵, βLeu²⁵⁷) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr²¹³ in αM1. These results establish the presence of two distinct bupropion binding sites within the <i>Torpedo</i> nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket