Abstract

The steroid SC17599 (17α-acetoxy-6-dimethylaminomethyl-21-fluoro-3-ethoxypregna-3,5-dien-20-one) has μ-opioid actions in vivo. The ability of SC17599 to interact with opioid receptors has been studied using radioligand and [³⁵S]guanosine-5′-O-(3-thio)triphosphate (GTPγS) binding assays. SC17599 bound to μ-opioid receptors in SH-SY5Y neuroblastoma cells and to recombinant receptors expressed in rat C6 glioma cells and Chinese hamster ovary cells with good affinity and with greater than 100-fold selectivity for μ- over both δ- and κ-opioid receptors. Binding was much reduced when aspartate 147 in the wild-type μ-opioid receptor was replaced with asparagine. The affinity of SC17599 for the μ-opioid receptor was decreased in the presence of sodium ions, indicating agonist activity. SC17599 stimulated the binding of [³⁵S]GTPγS in a naloxone-reversible manner with good potency and maximal effect equivalent to that of the μ-opioid agonists fentanyl and [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin. In rat brain membranes, SC17599-mediated stimulation of [³⁵S]GTPγS binding was reversed by the antagonist naltrexone. SC17599 lacks an aromatic ring andpara-hydroxyl substituent considered critical in the pharmacophore for μ-opioids. The structural relationship between SC17599 and more traditional opioid ligands was investigated through genetic algorithm-based modeling techniques for pharmacophore generation (GASP) and ligand-receptor docking (GOLD). The relatively planar and electron-rich A ring of the steroid compensated for the lack of aromaticity. Modeling of ligand-receptor docking showed that both morphine and SC17599 occupy the same binding pocket within the transmembrane helix bundle of the μ-opioid receptor and that the relationship between their binding modes largely mimicked the pharmacophore alignment.