title: Synthesis and activity of dafachronic acid ligands for the C. elegans DAF-12 nuclear hormone receptor.
abstract: 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)-Delta(7)-dafachronic acid isomer as the most potent compound, but the (25S)-Delta(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)-Delta(7)-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC(50) values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Delta(4)-dafachronic acids, with EC(50) values of 23 and 66 nm, respectively. The (25S)- and (25R)-Delta(5)-isomers were much less potent, with EC(50) values approaching 1000 nm, and saturated 5alpha- and 5beta-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)-Delta(7)-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Delta(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.
authors.affiliation: Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Kamalesh K
authors.initials: KK
authors.lastname: Sharma
authors.forename: Zhu
authors.initials: Z
authors.lastname: Wang
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Motola
authors.forename: Carolyn L
authors.initials: CL
authors.lastname: Cummins
authors.forename: David J
authors.initials: DJ
authors.lastname: Mangelsdorf
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002460:Cell Line; D002782:Cholestenes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D008024:Ligands; D015394:Molecular Structure; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015534:Trans-Activators / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D002782:Cholestenes; C410905:DAF-12 protein, C elegans; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; C533800:dafachronic acid