ABSTRACT
Steroidogenic factor-1 (SF-1) is a phospholipid-sensing nuclear receptor expressed in the adrenal glands, gonads, and hypothalamus which controls steroidogenesis and metabolism. There is significant therapeutic interest in SF-1 because of its oncogenic properties in adrenocortical cancer. Synthetic modulators are attractive for targeting SF-1 for clinical and laboratory purposes due to the poor pharmaceutical properties of its native phospholipid ligands. While small molecule agonists targeting SF-1 have been synthesized, no crystal structures have been reported of SF-1 in complexes with synthetic compounds. This has prevented the establishment of structure-activity relationships that would enable better characterization of ligand-mediated activation and improvement in current chemical scaffolds. Here, we compare the effects of small molecules in SF-1 and its close homolog, liver receptor homolog-1 (LRH-1), and identify several molecules that specifically activate LRH-1. We also report the first crystal structure of SF-1 in complex with a synthetic agonist that displays low nanomolar affinity and potency for SF-1. We use this structure to explore the mechanistic basis for small molecule agonism of SF-1, especially compared to LRH-1, and uncover unique signaling pathways that drive LRH-1 specificity. Molecular dynamics simulations reveal differences in protein dynamics at the pocket mouth as well as ligand-mediated allosteric communication from this region to the coactivator binding interface. Our studies, therefore, shed important insight into the allostery driving SF-1 activity and show potential for modulation of LRH-1 over SF-1.
Subject(s)
Models, Molecular , Molecular Dynamics Simulation , Receptors, Cytoplasmic and Nuclear , Small Molecule Libraries , Steroidogenic Factor 1 , Ligands , Phospholipids/chemistry , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Small Molecule Libraries/chemistry , Steroidogenic Factor 1/agonists , Steroidogenic Factor 1/chemistry , Humans , Crystallography, X-RayABSTRACT
Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.
Subject(s)
Cushing Syndrome/veterinary , Dog Diseases/drug therapy , Steroidogenic Factor 1/agonists , Adrenal Gland Neoplasms/metabolism , Adrenal Glands/metabolism , Animals , Cell Line, Tumor , Cell Survival , DNA , Dogs , Female , Hydrocortisone/metabolism , Male , Quinolones/pharmacology , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.
Subject(s)
Receptors, Cytoplasmic and Nuclear/agonists , Small Molecule Libraries/pharmacology , Steroidogenic Factor 1/agonists , Amino Acid Sequence , Animals , Crystallography, X-Ray , HEK293 Cells , Humans , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Homology, Amino Acid , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Steroidogenic Factor 1/chemistry , Steroidogenic Factor 1/metabolism , Transcriptional Activation/drug effectsABSTRACT
CONTEXT: Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). OBJECTIVE: Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). MAIN OUTCOME MEASURES: Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and reverse transcription quantitative PCR were used. RESULTS: SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. CONCLUSION: SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.
Subject(s)
Adrenal Cortex Neoplasms/drug therapy , Adrenocortical Carcinoma/drug therapy , Cell Proliferation/drug effects , Phenols/therapeutic use , Steroidogenic Factor 1/agonists , Adrenal Cortex Neoplasms/pathology , Adrenocortical Carcinoma/pathology , Drug Evaluation, Preclinical , Drug Inverse Agonism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Phenols/pharmacology , Quinolones/pharmacology , Steroids/metabolism , Tumor Cells, CulturedABSTRACT
Steroidogenic factor SF-1, a constitutively active nuclear hormone receptor, is essential to the development of adrenal and gonadal glands and acts as a shaping factor of sexual determination and differentiation. Its effects are exerted primarily through the control of the synthesis of steroid hormones. The functional cell-based assay Receptor Selection and Amplification Technology (R-SAT) was used to identify potent and selective SF-1 inverse agonists through the screening of a chemical library of drug-like small-molecule entities. Among them, 4-(heptyloxy)phenol (AC-45594), a prototype inverse agonist lead, was used to show that SF-1 constitutive activity can be pharmacologically modulated by a synthetic ligand. In a physiological system of endocrine function, the expression of several reported SF-1 target genes, including SF-1 itself, was inhibited by treatment with AC-45594 and analogs. Thus, pharmacological modulation of SF-1 is critical to its function as an endocrine master regulator and has potentially important consequences to diseases in which SF-1 activity is critical.