STRUCTURE-FUNCTION STUDIES OF THE NR5A NUCLEAR RECEPTORS 公开

D'Agostino, Emma (Fall 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/6h440t67j?locale=zh
Published

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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