Development and characterization of synthetic modulators of nuclear receptor LRH-1: Driving divergent signaling using a common molecular scaffold Restricted; Files Only
Cato, Michael (Spring 2023)
Abstract
Liver receptor homolog-1 (LRH-1) is a nuclear receptor that controls a wide variety of physiological processes. LRH-1 is well characterized in the liver and intestines, where it regulates bile acid biosynthesis, lipid and glucose homeostasis, intestinal cell renewal, and local steroidogenesis. These roles have made LRH-1 an attractive target for the treatment of disease states characterized by liver and intestine dysfunction. While phospholipids activate LRH-1, these molecules are poor tools for targeting this receptor because of their metabolic instability, insolubility, and poor potency. We have previously used structure-guided techniques to design two lead synthetic LRH-1 agonists. One molecule (“6N”) binds deep within the pocket, while the other (“10CA”) targets residues at the mouth of the pocket contacted by activating phospholipids. In this work, we explore how these two molecules differ from one another mechanistically and provide evidence for the utility of a molecule that combines these designs into one (“6N-10CA”). Studies indicate that 6N-10CA displays enhanced binding and efficacy and that contacting the pocket mouth drives divergent signaling, depending on the cellular context. Additional studies explore different pocket mouth-contacting groups and cross-reactivity with other nuclear receptors, with a strong focus on the close homolog of LRH-1, steroidogenic factor-1 (SF-1). By modifying groups at the pocket mouth, we enhance LRH-1 efficacy, while specific moieties contacting residues deep within the pocket promote LRH-1 selectivity. While LRH-1 activity is desired in the context of many metabolic diseases, it has been implicated in several cancers, including breast, pancreatic, and gastric cancer. Therefore, we explored whether lead agonists could be used as scaffolds for developing small molecules that antagonize or degrade LRH-1. The lead antagonist from these studies (“ANT3”) decreases LRH-1 activity by disrupting allosteric signaling on the receptor, while the lead degrader (“PROTAC-2”) reduces target gene expression by decreasing LRH-1 protein levels. Altogether, these studies demonstrate the successful strategies employed to enhance LRH-1 agonism or inhibit its activity and provide exciting new molecular tools that can be used to target this receptor in clinical and laboratory settings.
Table of Contents
CHAPTER 1: INTRODUCTION
1.1 Overview of the nuclear receptor superfamily: 2
1.2 Nuclear receptor structure: 4
1.3 Nuclear receptor activation: 6
1.4 LRH-1 and the NR5A subfamily: 9
1.5 LRH-1 as a developmental factor: 9
1.6 LRH-1 as a master metabolic regulator: 10
1.7 Additional roles of LRH-1: 13
1.8 LRH-1 as a cancer-promoting factor: 14
1.9 LRH-1 as a therapeutic target: 16
1.10 Modulation of LRH-1 activity by coregulators and post-translational modifications: 16
1.11 Modulation of LRH-1 activity by phospholipids: 19
1.12 History of LRH-1 synthetic agonist development: 27
1.13 History of LRH-1 synthetic antagonist development: 30
1.14 Remaining questions and challenges for LRH-1 synthetic agonists: 32
1.15 Remaining questions and challenges for LRH-1 synthetic inhibitors: 33
1.16 Summary of past and current work: 37
CHAPTER 2: EXPLORING INDIVIDUAL AND COMBINED EFFECTS OF LEAD SMALL MOLECULE AGONISTS OF NUCLEAR RECEPTOR LRH-1
2.1 Abstract: 39
2.2 Introduction: 39
2.3 Results: 44
2.3.1 Biochemical characterization: 44
2.3.2 Gene expression: 47
2.3.3 Coregulator binding and signaling: 50
2.3.4 Structural studies: 53
2.3.5 Compound-mediated dynamics: 56
2.4 Discussion and Conclusions: 63
2.5 Materials and methods: 67
2.5.1 Chemical synthesis: 67
2.5.2 Cell culture: 67
2.5.3 Protein expression and purification: 68
2.5.4 Ligand binding assays: 69
2.5.5 Reporter assays: 70
2.5.6 Protein stability: 71
2.5.7 RT-qPCR: 71
2.5.8 RNA-seq: 73
2.5.9 Peptide binding: 74
2.5.10 Crystallography and structure determination: 74
2.5.11 Molecular dynamics simulations: 75
2.5.12 HDX-MS: 78
2.5.13 DNA binding: 81
2.6 Supplemental information: 82
2.7 HPLC purity spectra: 96
2.8 Acknowledgements: 97
2.9 Author contributions: 97
2.10 Funding: 97
2.11 Conflicts of interest: 98
CHAPTER 3: EXPLORATION OF ALTERNATIVE PHOSPHOLIPID-MIMICKING GROUPS ON SMALL MOLECULE AGONISTS OF LRH-1
3.1 Abstract: 100
3.2 Significance: 100
3.3 Introduction: 101
3.4 Results: 105
3.5 Discussion: 119
3.6 Materials and methods: 121
3.6.1 Cell culture: 121
3.6.2 Data analysis and visualization: 122
3.6.3 Protein purification: 122
3.6.4 Fluorescence polarization competition assays: 123
3.6.5 Thermal stability assays: 124
3.6.6 Luciferase reporter assays: 124
3.6.7 RT-qPCR: 125
3.6.8 X-ray crystallography: 126
3.6.9 Model construction: 127
3.6.10 Molecular dynamics simulations: 128
3.6.11 Cross-reactivity studies: 130
3.7 Supplemental information: 132
3.8 Acknowledgements: 137
3.9 Author contributions: 137
3.10 Funding: 137
3.11 Conflicts of interest: 137
CHAPTER 4: COMPARISON OF SMALL MOLECULE AGONISM IN NR5A RECEPTORS LRH-1 AND SF-1
4.1 Abstract: 139
4.2 Introduction: 139
4.3 Results: 144
4.3.1 NR5A synthetic agonist design: 144
4.3.2 Biochemical characterization of small molecules: 147
4.3.3 Exploration of NR5A specificity: 149
4.3.4 Crystal structure of SF-1-6N-10CA: 153
4.3.5 Mutational analysis of residue contacts: 156
4.3.6 Molecular dynamics analysis of ligand contacts and allostery: 158
4.4 Discussion: 162
4.5 Materials and methods: 166
4.5.1 Cell Culture: 166
4.5.2 Protein purification of wildtype SF-1: 166
4.5.3 Protein purification of CysLite SF-1: 167
4.5.4 Differential scanning fluorimetry: 167
4.5.5 Fluorescence polarization: 167
4.5.6 Luciferase reporter: 168
4.5.7 Sequence alignment: 169
4.5.8 Crystallization: 169
4.5.9 Structure Determination: 170
4.5.10 Mutagenesis: 171
4.5.11 Molecular dynamics simulations: 171
4.6 Supplemental information: 175
4.7 Acknowledgements: 177
4.8 Author contributions: 177
4.9 Funding: 177
4.10 Conflicts of interest: 178
CHAPTER 5: CONVERSION OF LRH-1 SMALL MOLECULE AGONIST INTO ANTAGONISTS
5.1 Abstract: 180
5.2 Introduction: 180
5.3 Results: 185
5.3.1 Rationale and design of RJW100-derived antagonists: 185
5.3.2 Compound binding and effects on LRH-1 activity: 189
5.3.3 Small molecule-mediated LRH-1 target gene expression: 192
5.3.4 Combining ANT3 with pocket-mouth contacting groups: 195
5.3.5 Using accelerated MD simulations to predict compound orientation: 197
5.3.6 MD simulations reveal compound-driven effects on LRH-1 allostery: 200
5.3.7 Effects of ANT3 on breast cancer viability and proliferation: 204
5.4 Discussion: 207
5.5 Materials and methods: 209
5.5.1 Cell culture: 209
5.5.2 Data analysis and visualization: 210
5.5.3 Protein expression and purification: 210
5.5.4 Ligand binding: 211
5.5.5 Reporter assays: 211
5.5.6 Protein thermal stability: 212
5.5.7 Coregulator binding: 213
5.5.8 Gene expression analysis: 214
5.5.9 Accelerated molecular dynamics simulations: 216
5.5.10 Classical molecular dynamics simulations: 218
5.5.11 Apoptosis/viability assay on spheroids: 219
5.5.12 Live/dead assay on 2D monolayer: 220
5.6 Supplemental information: 221
5.7 Author contributions: 225
5.8 Funding: 225
5.9 Conflicts of interest: 225
CHAPTER 6: CONVERSION OF LRH-1 SMALL MOLECULE AGONIST INTO PROTAC DEGRADERS
6.1 Abstract: 227
6.2 Introduction: 228
6.3 Results: 232
6.4 Discussion: 242
6.5 Materials and methods: 243
6.5.1 Cell culture: 243
6.5.2 Protein expression and purification: 243
6.5.3 Ligand binding: 244
6.5.4 Gene expression analysis: 245
6.5.5 RNA-seq: 246
6.5.6 Western blotting: 247
6.5.7 Cell proliferation assays: 248
6.6 Supplemental information: 249
6.7 Acknowledgments: 251
6.8 Author contributions: 251
6.9 Funding: 251
6.10 Conflicts of interest: 252
CHAPTER 7: DISCUSSION AND FUTURE DIRECTIONS
7.1 Introduction: 254
7.2 Effects of deep pocket and pocket mouth contacts: 255
7.3 Modification of pocket mouth-contacting groups: 256
7.4 Cross-reactivity of small molecules with homolog SF-1: 257
7.5 Development of LRH-1 inhibitors using an agonist scaffold: 259
7.6 Modulation of LRH-1 activity through the AF-B: 260
7.7 Untangling mechanisms of ligand-mediated LRH-1 transcriptional output: 263
7.8 Combining designs: 266
7.9 Concluding remarks: 268
APPENDIX: MODIFICATION OF BRIDGEHEAD SUBSTITUENT ON RJW100 SCAFFOLD
8.1 Abstract: 271
8.2 Introduction: 271
8.3. Rationale and chemistry: 275
8.4 Biochemical characterization of compounds without bridgehead styrene: 278
8.5 Search for alternative bridgehead groups: 280
8.6 Biochemical characterization of aniline compounds: 286
8.7 Structure of LRH-1 LBD bound to compound 15: 290
8.8 Discussion: 292
8.9 Materials and methods: 292
8.9.1 Cell culture: 292
8.9.2 Protein expression: 293
8.9.3 Ligand binding assays: 293
8.9.4 Reporter Assays: 294
8.9.5 MARCoNI Assay: 295
8.9.6 Crystallography and structure determination: 296
8.10 Supplemental information: 298
8.11 Acknowledgements: 302
8.12 Funding: 302
8.13 Conflicts of interest: 302
REFERENCES: 303
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File download under embargo until 22 May 2025 | 2023-04-01 11:52:11 -0400 | File download under embargo until 22 May 2025 |
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