Design and Synthesis of New Liver Receptor Homolog-1 Modulators and Probes Restricted; Files Only

Cornelison, Jeffery (Fall 2020)

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The nuclear receptor known as Liver Receptor Homolog-1 (LRH-1) plays an integral role in many bodily processes with repercussions for human health, processes such as inflammation, cell differentiation, and glucose tolerance. The ability to target LRH-1 specifically and potently could lead to advances in the development of pharmaceuticals to treat disease. Diseases such as diabetes, non-alchoholic fatty liver disease, inflammatory bowel disease, and breast and pancreatic cancers could all have potential treatments through LRH-1 modulation.

           Historically, LRH-1 modulation by small molecules has been quite difficult because of the large and highly hydrophobic pocket in which LRH-1 binds its ligands. This results in very few polar residues available to anchor a scaffold in place and leads to unpredictable binding poses in the pocket. Here we describe the rational design of new LRH-1 ligands based on a previously reported weakly binding lead. Optimization of key interactions deep in the pocket and expansion of the framework into new areas enabled the development of new compounds able to activate LRH-1 (through agonism) much more potently and powerfully than before.

           Using the knowledge gained by these studies towards agonism, a novel probe was developed to study LRH-1 binding in a way that was previously impossible. The probe allowed for the development of a new assay to quantify direct binding to LRH-1. With the insight granted by this new assay, a series of antagonists were developed that could turn down LRH-1 activity. While the vast majority of reported LRH-1 modulators act as agonists to increase LRH-1 activity, the new compounds described here are able to use similar binding modes to oppositely alter LRH-1 activity, with the potential to ameliorate phenotypes associated with cancer.

Table of Contents

Chapter 1: Introduction to Liver Receptor Homolog-1 (LRH-1) 1

1.1 Nuclear Receptors (NRs) and Gene Regulation. 2

1.2 Gene Targets of LRH-1 and Downstream Effects. 3

1.3 Modulators of LRH-1. 5

1.3.1 Natural Modulators. 5

1.3.2 Synthetic Modulators. 6

1.3.3 LRH-1 Co-Crystal Structures with Modulators. 8

Chapter 2: Improved Agonists by Enhanced Deep-Pocket Interactions. 11

2.1 Introduction. 12

2.2 Results and Discussion. 13

2.2.1 Locking the Agonist in Place with Polar Interactions. 13

2.2.2 Discovery of the First Low Nanomolar LRH-1 Agonist. 16

2.2.3 DPP Contacts Drive LRH-1 Activation by 6N. 18

2.2.4 Compound 6N Stabilizes the AFS, Strengthens Allosteric Signaling, and Promotes Coactivator Recruitment. 21

2.2.5 Compound 6N Promotes Expression of Intestinal Epithelial Steroidogenic Genes in Humanized LRH-1 Mouse Enteroids. 24

2.3 Conclusions. 26

2.4 Supporting Information. 29

2.4.1 Biology Supplementary Materials and Methods. 29

2.4.2 Chemistry Supplementary Materials and Methods. 41

Chapter 3: LRH-1 Direct Binding Assay Enabled by New Chemical Probe. 97

3.1 Introduction. 98

3.2 Results and Discussion. 100

3.2.1 Probe Design. 100

3.2.2 Assay Development 101

3.2.3 High-Affinity Probe Increases Sensitivity for Detecting Mammalian Phospholipid Binding. 102

3.2.4 Affinity Correlates with Biological Activity and Receptor Stability for Synthetic Agonists. 104

3.2.5 FP Competition Assay Accurately Quantifies Binding of Synthetic Modulators. 105

3.3 Conclusions. 106

3.4 Supporting Information. 108

Chapter 4: Combining Agonist Leads Yields a Highly Potent and Efficacious Hybrid Compound. 127

4.1 Introduction. 128

4.2 Results and Discussion. 129

4.3 Conclusions. 132

4.4 Supporting Information. 134

Chapter 5: Redesigned Synthetic Route for Alternative Agonists. 141

5.1 Introduction. 142

5.2 Results and Discussion. 143

5.3 Conclusions. 151

5.4 Supporting Information. 152

Chapter 6: Agonist Scaffolds Repurposed for Antagonism.. 191

6.1 Introduction. 192

6.2 Results and Discussion. 195

6.3 Conclusions. 201

6.4 Supporting Information. 203

Bibliography. 237

Chapter 1. 237

Chapter 2. 238

Chapter 3. 241

Chapter 4. 244

Chapter 5. 244

Chapter 6. 246

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