title: Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2).
abstract: Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Felipe
authors.initials: F
authors.lastname: de Jesus Cortez
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Suzawa
authors.affiliation: Chemistry and Chemical Biology Graduate Program, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Sam
authors.initials: S
authors.lastname: Irvy
authors.affiliation: Pharmaceutical Sciences and Pharmacogenomics Graduate Program, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: John M
authors.initials: JM
authors.lastname: Bruning
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Sablin
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fletterick
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Holly A
authors.initials: HA
authors.lastname: Ingraham
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, United States of America.|Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, United States of America.
authors.forename: Pamela M
authors.identifier: http://orcid.org/0000-0002-2308-1048
authors.initials: PM
authors.lastname: England
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D056945:Hep G2 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D062105:Molecular Docking Simulation; D015335:Molecular Probes / Q000737:chemistry*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*
chemical_list: D004220:Disulfides; D008024:Ligands; D015335:Molecular Probes; C082631:NR5A2 protein, human; D018160:Receptors, Cytoplasmic and Nuclear