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Synthesis and Activity of Dafachronic Acid Ligands for the C. elegans DAF-12 Nuclear Hormone Receptor

Division of Endocrinology and Metabolism, Department of Internal Medicine (K.K.S, R.J.A.), and the Department of Pharmacology and the Howard Hughes Medical Institute (Z.W., D.L.M., C.L.C., D.J.M.), University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Richard J. Auchus, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: richard.auchus{at}UTSouthwestern.edu; or David J. Mangelsdorf, Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050. E-mail: davo.mango{at}UTSouthwestern.edu.

The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-⁷-dafachronic acid isomer as the most potent compound, but the (25S)-⁴-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-⁷-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC₅₀ values of 23 and 33 nM, respectively, as are (25S)- and (25R)-⁴-dafachronic acids, with EC₅₀ values of 23 and 66 nM, respectively. The (25S)- and (25R)-⁵-isomers were much less potent, with EC₅₀ values approaching 1000 nM, and saturated 5- and 5β-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-⁷-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-⁷-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25.

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