STRUCTURE-FUNCTION STUDIES OF THE NR5A NUCLEAR RECEPTORS Open Access
D'Agostino, Emma (Fall 2020)
Abstract
Nuclear receptors (NRs) comprise 48 ligand-regulated transcription factors. NRs have unique, high-affinity ligands, making them excellent pharmacological targets. Many NRs respond to lipids or lipid metabolites and have proven challenging to target due to the promiscuity and metabolic lability of lipids. This work focuses on two phospholipid (PL)-regulated NRs which together form the human NR5A subfamily: steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). The NR5A receptors regulate development, metabolism, and steroidogenesis, and are putative therapeutic targets for obesity, diabetes, inflammatory bowel diseases, and several cancers. Small molecule screens for NR5As have yielded few results. Recombinantly expressed NR5As co-purify with bacterial PLs that are difficult to displace in compound screens; an easily accessible direct binding assay has not been previously described, complicating efforts to validate and improve lead compounds; and the largely hydrophobic binding pocket provides few residues to anchor a scaffold.
Here, we describe a novel fluorescence polarization competition assay which directly detects ligand binding and quantifies binding affinities. We demonstrate the development of agonists which combine elements of earlier LRH-1 agonists with PLs to drive improvements in efficacy, leading to the first LRH-1 agonist with in vivo efficacy in a murine model of colitis. We solve the first synthetic agonist-bound crystal structure of SF-1, giving insight into the mechanism of action of this receptor for future small molecule development. Finally, we show that SF-1, while previously reported to function exclusively as a monomer, can also dimerize in vitro and in cells and that the oligomerization state is ligand responsive. Together, these studies significantly progress our ability to study the NR5A receptors and our understanding of their ligand-mediated mechanisms of action.
Table of Contents
Table of Contents
Abbreviations
CHAPTER 1: THE NR5A NUCLEAR RECEPTORS
Introduction
NR5A Structure
Expression
Biological Roles/ Function
Putative Ligands
NR5As in Disease
Synthetic Modulation of NR5A Receptors
Questions Addressed in This Work
Figures and Tables
Table 1.1. Nuclear receptor family therapeutic landscape.
Figure 1.1. NR5A structure.
Figure 1.2. NR5A Mammalian Phospholipid Ligands.
Figure 1.3. NR5A synthetic modulators.
References
CHAPTER 2: DEVELOPMENT OF A VERSATILE AND SENSITIVE DIRECT LIGAND BINDING ASSAY FOR HUMAN NR5A NUCLEAR RECEPTORS1a
1This chapter adapted from the previously published work D’Agostino EH, Cornelison JL, Mays SG, Flynn AR, Patel A, Jui NT, Ortlund EA. Development of a Robust Direct Binding Assay for Phospholipid-Sensing Human NR5A Nuclear Receptors. ACS Med Chem Lett. 2019 Nov 21; 11(3):365-370.
Abstract
Introduction
Results
Probe Design
Assay Development
High-Affinity Probe Increases Sensitivity For Detecting Mammalian Phospholipid Binding
Affinity Correlates with Biological Activity and Receptor Stability for Synthetic Agonists
FP Competition Assay Accurately Quantifies Binding of Synthetic Modulators
Discussion
Methods
Protein Expression and Purification.
Generation of apo LRH-1.
Fluorescence Polarization.
Differential Scanning Fluorimetry.
Figure 2.1. Structure-guided design of NR5A probe.
Scheme 2.1. Chemical synthesis of 6N-FAM (6)a
Figure 2.2. Validation of fluorescence polarization.
Figure 2.3. FP assay detects lipid binding.
Figure 2.5. FP measurements for synthetic ligands.
Supplementary Figure S2.1. Summary Ki table.
Supplementary Figure 2.2. SF-1 binding to R1 compounds.
Supplementary Figure S2.3. LRH-1 binding to R1 compounds.
Supplementary Figure S2.4. Representative thermal shift curves.
Supplementary Figure S2.5. Detailed Chemical Syntheses
References
CHAPTER 3: TAPPING INTO A PHOSPHOLIPID-LRH-1 SIGNALING AXIS YIELDS A POWERFUL ANTI-INFLAMMATORY SIGNALING AGENT WITH IN VIVO ACTIVITY AGAINST COLITIS1a
Abstract
Introduction
Results
Structure-guided design of PL-mimics.
Modifications improve binding affinity and activity.
Structural basis for improved binding and activity.
Effects on LRH-1 conformation and coregulator recruitment.
10CA reduces expression of lipogenic genes in the liver.
Efficacy of 10CA in organoid and murine models of colitis.
Discussion
Methods
Chemical synthesis.
Cell culture.
Reporter gene assays.
Calculation of Relative Efficacy (RE).
Protein purification
Generation of apo LRH-1.
Mutagenesis.
Ligand binding assay.
Hydrogen-deuterium exchange mass spectrometry (HDX-MS).
In vitro NR-coregulator recruitment by MARCoNI
Crystallography.
Structure Determination.
Molecular dynamics simulations.
Agonist treatment and RNA extraction in Huh7 cells.
Animals.
Viral overexpression and drug treatment.
NanoString Gene Expression Analysis
Humanized LRH-1 Mouse Intestinal Enteroid Culture.
RNA Isolation and RT-qPCR.
Figures
Figure 3.1. Phospholipid mimetic design, binding affinity, and activity.
Figure 3.2. Phospholipid mimetics make PL-like interactions with LRH-1 ligand-binding pocket.
Figure 3.3. Longer-tailed PL mimetics stabilize the AFS in HDX-MS.
Figure 3.4. Coregulator recruitment profiling by MARCoNI.
Figure 3.5. 10CA activates LRH-1 in hepatocytes and in the liver.
Figure 3.6. Efficacy of 10CA in organoid and in vivo models of colitis.
Table S3.1. Summary of linker lengths and key biological parameters for LRH-1 agonists.
Figure S3.1. Fluorescence polarization competition: phosphorylcholines.
Figure S3.2. Fluorescence polarization competition: carboxylic acids.
Figure S3.3. Fluorescence polarization competition: diols.
Figure S3.4. Luciferase reporter assays with diols.
Table S3.2: X-ray data collection and refinement statistics.
Figure S3.5. 6HP cores of 10CA and 9ChoP adopt the same position as RJW100 6HP core.
Figure S3.6. Fluorescence polarization: pocket mouth mutants.
Figure S3.7. Fold reduction in Ki for Y516A versus K520A mutation versus WT Ki.
Figure S3.8. Peptide coverage and deuterium uptake in HDX-MS experiments.
Figure S3.9. Protein purification and MARCoNI.
Table S3.3. Modified colitis disease activity score.
Figure S3.10. 10CA treatment does not activate mouse LRH-1.
Figure S3.11. 10CA does not alter gene expression in enteroids lacking LRH-1.
References
CHAPTER 4: CRYSTAL STRUCTURE OF STEROIDOGENIC-FACTOR 1 IN COMPLEX WITH HIGH-AFFINITY SYNTHETIC AGONIST
Abstract
Introduction
Results
Agonist design.
6N-10CA is a potent, stabilizing SF-1 agonist.
Analysis of SF-1-6N-10CA crystal structure.
Discussion
Methods
Chemical synthesis.
Purification – Wildtype SF-1.
Purification – CysLite SF-1.
Crystallization.
Structure Determination.
Fluorescence Polarization.
Luciferase.
Differential Scanning Fluorimetry.
Figures
Figure 4.1. 6N-10CA is the highest affinity, most potent SF-1 agonist to date.
Table 4.1: X-ray data collection and refinement statistics.
Figure 4.2. 6N-10CA and DPPE co-occupy chain B, but not chain A.
Figure 4.3. DPPE and 6N-10CA ligand binding pocket-interactions.
Supplemental Figure 4.1. Omission of 6N-10CA or DPPE confirms ligand co-occupancy.
References
CHAPTER 5: DIMERIZATION OF STEROIDOGENIC-FACTOR 1
Abstract
Intro
Results
Split luciferase assay reveals SF-1 homodimer.
The SF-1 LBD dimerizes in a ligand-dependent manner.
Full-length SF-1 dimerizes in vitro.
Ligand disrupts SF-1-SF-1 interaction in cells.
Discussion
Methods
Split luciferase screen.
Purification – CysLite SF-1.
Protein Purification: Full-Length.
Fluorescence Polarization.
Analytical Ultracentrifugation.
Figure 5.1 Split luciferase screen.
Figure 5.2 SF-1 LBD dimerizes in a ligand-driven manner.
Figure 5.3 SF-1-FL purification.
Figure 5.4. FL-SF-1 dimerizes in vitro on DNA.
Figure 5.5. Ligand disrupts dimerization in cells.
References
CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS
Conclusions
LRH-1
SF-1
Future Directions
LRH-1 Small Molecule Development
SF-1 Small Molecule Studies
SF-1 Dimer
Figures
References
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