LRH-1 agonist development: tapping the therapeutic potential of an orphan nuclear hormone receptor Open Access
Mays, Suzanne (Fall 2018)
Abstract
Liver Receptor Homolog-1 (LRH-1) is an orphan nuclear hormone receptor that controls bile acid, cholesterol, glucose, and one-carbon homeostasis. Until recently, LRH-1 was thought to be insensitive to ligands and intractable as a therapeutic target. Discoveries that LRH-1 is activated by certain phosphatidylcholines (PCs) and is regulated by availability of dietary PCs has given rise to the intriguing hypothesis that LRH-1 senses PCs as a measure of nutrient availability in order to direct an appropriate metabolic response. The discovery that LRH-1 is ligand-regulated also opens the door for pharmaceutical targeting of this receptor. In this work, I address challenges with development of LRH-1 synthetic modulators using an approach that integrates structural biology with biophysical, biochemical, and cellular studies. This work provides the first detailed investigation into mechanisms used by synthetic molecules to switch LRH-1 into the active state. Insights gained from mechanistic studies led to the development of two classes of new agonists. The first class exploits a polar region within the hydrophobic binding pocket, producing a new lead molecule that is 100 times more potent than the previous best agonist. The second class was inspired by knowledge of PC-driven activation of LRH-1, yielding highly active agonists designed to act as PC mimetics. Together, these studies have provided powerful tools to probe LRH-1 biology that also have potential as therapeutic agents for metabolic diseases.
Table of Contents
List of Figures. 7
List of Tables. 9
List of Abbreviations. 10
Chapter 1. Structure and Regulation of the Orphan Nuclear Hormone Receptor, LRH-1. 15
1.1 Nuclear receptors: hormone-regulated transcription factors. 15
Table 1.1. Human Nuclear Receptors and Cognate Ligands. Table has been adapted from (Gronemeyer et al., 2004) 17
1.2 Liver Receptor Homolog-1. 19
1.3 Regulation of LRH-1 by ligands. 22
1.4 Metabolic processes controlled by LRH-1. 24
1.4.1 Bile acid biosynthesis. 24
1.4.2 Reverse cholesterol transport. 25
1.4.3 De Novo Lipogenesis. 25
1.4.4 Endoplasmic-reticulum (ER) stress resolution. 26
1.4.5 Glucose homeostasis. 26
1.4.6 Methyl metabolism. 26
1.5. Potential role as an integrator of metabolism via phosphatidylcholine sensing. 27
Chapter 2: Targeting LRH-1 with Agonists in Metabolic Diseases. 30
2.1 LRH-1 as a therapeutic target for metabolic diseases. 30
2.2 Preclinical studies involving LRH-1. 31
2.3 Therapeutic potential beyond NAFLD and diabetes. 31
2.4 Previous strategies for discovery of LRH-1 modulators. 33
2.4.1 Antagonists. 33
2.4.2 Agonists. 35
2.5 Need for mechanistic studies to guide the design of LRH-1 modulators. 38
Chapter 3. Crystal Structures of LRH-1 with Synthetic Agonists Reveal a Novel Mechanism of Receptor Activation 40
3.1 Abstract. 40
3.2 Introduction. 41
3.3 Results. 44
3.3.1. The Crystal Structure of RJW100 Bound to LRH-1. 44
3.3.2 RJW100 Selectively Destabilizes Components of the AFS Relative to DLPC.. 50
3.3.3 Dramatic Repositioning of RJW100 Compared to a Closely Related Synthetic Agonist. 52
3.3.4 The crystal structure of LRH-1 bound to a RJW100 diastereomer. 52
3.3.5 Discovery of a Novel LRH-1 Interaction Mediated by the RJW100 Hydroxyl Group. 55
3.3.6 Differences in π-π-Stacking with Residue H390 among LRH-1 Agonists. 57
3.3.7 Role of T352 and H390 in LRH-1 Activation by Synthetic Agonists. 60
3.3.8 Capacity for productive π-π-stacking with residue H390 influences agonist positioning. 63
3.4 Discussion. 67
3.5 Materials and Methods. 69
3.5.1 Materials and reagents. 69
3.5.2 Protein expression and purification. 69
3.5.3 Crystallization. 70
3.5.4 Structure determination. 70
3.5.5 Structure analysis. 71
3.5.6 Mutagenesis. 71
3.5.7 Cell culture. 71
3.5.8 Reporter gene assays. 71
3.5.9 Hydrogen-deuterium exchange mass spectrometry. 71
3.5.10 Differential scanning fluorimetry (DSF)—... 72
3.5.11 Model construction for molecular dynamics simulations. 73
3.5.12 Molecular dynamics simulations. 73
3.6 Acknowledgements. 74
Chapter 4. Structure and Dynamics of the Liver Receptor Homolog 1–PGC1α Complex. 75
4.1 Abstract. 75
4.2 Introduction. 76
4.3 Results. 78
4.3.1. Coregulator binding affinities for full-length LRH-1. 78
4.3.2. Crystal structure of LRH-1 with PGC1α. 81
4.3.3. PGC1α strengthens the coactivator charge clamp and communication within the LRH-1 AFS 85
4.3.4. Differential effects of PGC1α and Tif2 on LRH-1 allosteric communication. 88
4.3.5. Ligand participation in allosteric signaling within the LRH-1-Tif2 complex. 92
4.4 Discussion. 92
4.5 Materials and Methods. 95
4.5.1 Materials and Reagents. 95
4.5.2 Protein purification. 96
4.5.3 Fluorescence polarization coregulator binding assays. 96
4.5.4 Crystallization. 97
4.5.5 Structure determination. 97
4.5.6 Hydrogen-deuterium exchange (HDX) mass spectrometry. 98
4.6.7 Model construction for molecular dynamics simulations. 99
4.6.8 Molecular dynamics simulations. 99
4.6 Acknowledgments. 100
Chapter 5. Discovery of the First Low Nanomolar LRH-1 Agonist Through Structure-Guided Design 101
5.1 Abstract 101
5.2 Introduction. 102
5.3 Results. 105
5.3.1 Locking the agonist in place with polar interactions. 105
5.3.2. Limitations of the sulfamate agonist. 110
5.3.3 Discovery of a low nanomolar LRH-1 agonist by enhancing polar interactions. 112
5.3.4 Activation of LRH-1 in vivo. 115
5.3.6 Compound 33 stabilizes the AFS, strengthens allosteric signaling, and promotes coactivator recruitment. 122
5.4 Discussion. 125
5.5 Materials and Methods. 127
5.5.1 Chemical Synthesis of Cpd33. 127
5.5.2 Biology: materials and reagents. 130
5.5.3 Protein purification. 130
5.5.4 Differential scanning fluorimetry (DSF). 130
5.5.5 Crystallography. 131
5.5.6 Structure Determination. 131
5.5.7 Tissue culture. 131
5.5.8 Reporter gene assays. 131
5.5.9 Calculation of Relative Efficacy (RE). 132
5.5.10 Mutagenesis. 132
5.5.11 Model Construction for Molecular Dynamics Simulations. 133
5.5.12 Molecular Dynamics Simulations. 133
5.5.13 Coregulator Recruitment Assays. 134
5.5.14 Animal Studies. 135
5.5.15 Gene expression analysis. 135
5.5.16 Hydrogen-deuterium exchange mass spectrometry. 136
5.6 Acknowledgements. 139
Chapter 6. Development of Synthetic Phospholipid Mimetics as LRH-1 Agonists. 140
6.1 Abstract 140
6.2 Introduction. 141
6.3 Results. 144
6.3.1 Synthesis and evaluation of the “full” phospholipid hybrid. 144
6.3.2 Scaffold simplification improves both synthetic accessibility and agonist activity. 149
6.4 Conclusions. 152
6.5 Materials and Methods. 156
6.5.1 Chemical synthesis. 156
6.5.2 Reporter gene assays. 159
6.6 Acknowledgements. 159
Chapter 7. Discussion and Future Directions. 161
7.1 Discussion. 162
7.1.1. Tuning potency through modifications at the RJW100 hydroxyl site. 163
7.1.2 R4-modified compounds: the “PL mimics”. 166
7.1.3. Possibility for hybrid agonists with dually modified R1 and R4 groups?. 167
7.2. Applications for this diverse compound library. 172
7.2.1 High affinity ligand enables the development of a competitive binding assay. 172
7.2.2 Tools to elucidate the ligand-dependent transcriptional program of LRH- 172
7.2.3 Computational modeling of ligand-driven LRH-1 activation. 173
7.3. Future directions for evaluating agonists as therapeutic agents. 174
7.3.1 LRH-1 Selectivity. 174
7.3.2 Stereospecific effects. 175
7.3.3 Pharmacokinetics and Toxicity. 176
7.3.4 Efficacy in Disease Models. 176
7.4 Concluding Remarks. 178
References. 179
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