Part 1. Discovery of a Fluorinated Enigmol Analog with Enhanced Pharmacokinetic and Anti-Tumor Properties. Part 2. Preferential Activation of Metabotropic Glutamate Receptor 3 over Metabotropic Glutamate Receptor 2 公开

Miller, Eric James (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/rn301161g?locale=zh
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Abstract

Part 1: Sphingolipids are a structurally diverse class of biomolecules that perform various functions within the cell. Via acute regulation of intracellular concentrations of sphingosine and sphingosine-1-phosphate, which signal for cell growth arrest and cell proliferation respectively, the sphingolipid metabolic pathway balances cell growth and cell death signaling, rendering modulation of this pathway a reasonable strategy for cancer treatment. Our lab hypothesized that a sphingosine mimic incapable of phosphorylation by sphingosine kinase would slow tumor progression. Such a compound, Enigmol, was developed in our lab without the hydroxyl group that is phosphorylated by sphingosine kinase. It was subsequently hypothesized that fluorinated analogs with higher lipophilicity would demonstrate enhanced efficacy against prostate cancer due to increased tumor uptake. Accordingly, three fluorinated Enigmol analogs were synthesized. Pharmacokinetic studies demonstrated that while plasma drug concentrations correlated with fluorine content, tissue concentrations did not. Although the most lipophilic analog CF3-Enigmol achieved higher tissue concentrations than Enigmol, less lipophilic CF2-Enigmol accumulated at even higher concentrations. Furthermore, while CF3-Enigmol was less efficacious than Enigmol in mouse xenograft models of prostate cancer, CF2-Enigmol demonstrated improved tumor growth inhibition.

Part 2: The metabotropic glutamate receptors (mGluRs) are class C GPCRs that are classified into groups I - III based on sequence homology, signal transduction, and pharmacology. Several group II mGluR agonists have demonstrated preclinical neuroprotective activity, highlighting the potential of these receptors (mGluR2 and mGluR3) as drug targets for treating neurodegeneration. However, studies using mGluR2-/- and mGluR3-/- knockout mice revealed that the neuroprotective actions of selective group II mGluR agonists are entirely mediated through mGluR3. Thus, the project goal was to achieve selective mGluR3 activation. Investigation began with synthesis of a series of N-acetyl-L-aspartyl glutamate (NAAG) analogs. During assay implementation, it was unexpectedly discovered that chloride affects activation of mGluR3 but not mGluR2. Although NAAG and NAAG analogs proved to be inactive at mGluR3, computational molecular modeling, pharmacology, and site-directed mutagenesis elucidated that chloride preferentially activates mGluR3 over mGluR2. Furthermore, these studies support a proposed mechanism of chloride discrimination between mGluR3 and mGluR2.

Table of Contents

Part 1. Discovery of a Fluorinated Enigmol Analog with Enhanced Pharmacokinetic and Anti-Tumor Properties

1.1. Introduction

1.1.a. Roles of Sphingolipid Biochemistry and Cancer………………………………………1

1.1.b. Enigmol, a 1-Deoxy-Sphingoid Base with Anti-Cancer Activity……………………….4

1.1.c. Design, Synthesis, and in vitro Cytotoxicity of Enigmol Analogs………………………7

1.2. Results and Discussion

1.2.a. Design and Synthesis of CF2-Enigmol………………………………………………11

1.2.b. Cytotoxicity against Human Prostate Cancer Cells in vitro……………………………22

1.2.c. 1st Scale Up Preparation of CF2-Enigmol……………………………………………26

1.2.d. Pharmacokinetics of Fluorinated Enigmol Analogs…………………………………31

1.2.e. 2nd Scale Up Preparation of CF2-Enigmol……………………………………………36

1.2.f. Synthesis and in vitro Cytotoxicity of CF2-Enigmol Metabolites………………………52

1.2.g. Anti-Tumor Activity of Fluorinated Enigmol Analogs in vivo………………………..54

1.3. Conclusions…………………………………………………………………………………..58

1.4. Experimental Methods

1.4.a. Synthetic Procedures………………………………………………………………..59

1.4.b. cLogP Calculations………………………………………………………………….87

1.4.c. Human Prostate Cancer Viability Assays…………………………………………….88

1.4.d. Rat Plasma Pharmacokinetic Assays (i.v.)……………………………………………88

1.4.e. Rat Plasma Pharmacokinetic Assays (p.o.)…………………………………………...89

1.4.f. Rat Tissue Distribution Assays (p.o.)………………………………………………...90

1.4.g. Mouse Xenograft Studies…………………………………………………………....91

1.4.h. Bioanalytical LC-MS/MS Assays……………………………………………………92

Part 2. Preferential Activation of Metabotropic Glutamate Receptor 3 over Metabotropic Glutamate Receptor 2

2.1. Introduction

2.1.a. Seven-Transmembrane Receptors and G Protein Coupling………………………….94

2.1.b. Metabotropic Glutamate Receptors…………………………………………………98

2.1.c. Subtype-Selective mGluR3 Activation and Neuroprotection……………………….104

2.2. Results and Discussion

2.2.a. Design of NAAG Analogs…………………………………………………………109

2.2.b. Synthesis of NAAG Analogs………………………………………………………115

2.2.c. GloSensor Assay Measures Group II and III mGluR Activity………………………119

2.2.d. mGluR3 Activity of NAAG and Analogs………………………………………….129

2.2.e. Group II mGluR Antagonist-Mediated Inverse Agonism………………………….130

2.2.f. Pharmacological Characterization of Chloride……………………………………...134

2.2.g. Implications for Selective mGluR3 Drug Design…………………………………..146

2.3. Conclusions…………………………………………………………………………………148

2.4. Experimental Methods

2.4.a. Synthetic Procedures………………………………………………………………149

2.4.b. Molecular Modeling……………………………………………………………….159

2.4.c. GloSensor Assays………………………………………………………………….162

2.4.d. LANCE cAMP Assays…………………………………………………………….165

Part 3. Overall Conclusions

3.1. Crossover between Sphingolipids and mGluRs…………………………………………..166

3.2. References…………………………………………………………………………………..171

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