Steroid‑based Tide Quencher 1 probes enable real‑time mapping of novel non‑canonical cholesterol sites on the M ₁ muscarinic receptor

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C‑terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length (γ-aminobutyric acid vs. L-glutamic acid) and C-3/C‑5 stereochemistry (3α/3β/5α/5β). Fluorescence-quenching assays with CFP‑tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub‑micromolar EC₅₀ values. The most potent N‑terminal probe (3α5α‑PRG‑Glu‑TQ1 (5)) achieved 300 nM potency, while the best C‑terminal probe (3α5β‑PRG‑Glu‑TQ1 (3)) reached 1 μM potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N‑site; K57, Y62, W150 at the C‑site) mediating binding, a prediction confirmed by alanine‑scan mutagenesis that markedly reduced quenching at the N‑terminus and only modestly affected the C‑terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence‑quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high‑throughput compatibility, and the ability to dissect lipid‑GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure‑guided drug discovery targeting allosteric cholesterol sites.