Biological and Chemical Analysis of Small Molecule Activators of Anti-Inflammatory and Antioxidant Nrf2-Keap1 Signaling

BIOLOGICAL AND CHEMICAL ANALYSIS OF SMALL MOLECULE ACTIVATORS OF ANTI-INFLAMMATORY AND ANTIOXIDANT NRF2-KEAP1 SIGNALING

by TONIBELLE NICOLAY GATBONTON-SCHWAGER

Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy

Dissertation Advisors: Dr. John J. Letterio and Dr. Gregory P. Tochtrop

Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY

May 2014

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of Tonibelle Nicolay Gatbonton-Schwager candidate for the Ph.D. degree*.

(signed) Monica M. Montano, Ph.D. (chair of the committee)

Gregory P. Tochtrop, Ph.D. John J. Letterio, M.D. John J. Mieyal, Ph.D. Marvin T. Nieman, Ph.D. Sanjay Gupta, Ph.D.

(date) December 20, 2013

* We also certify that written approval has been obtained for any proprietary material contained therein.

Dedication

To the loved ones that have inspired me to go down this path – lola Primitiva P. Nicolay, uncle Ismael P. Nicolay, and ninang Lilia P. Nicolay. The short time we had will inspire me for a lifetime.

To my mama (Alma) and papa (Alvaro) for their never-ending sacrifice. For my brother and sister for always reminding me who I am. For my auntie Angie and uncle Rick Capati for their role as my second parents. For the Schwager clan for their enthusiasm for my project.

To those who believed in me more than I did, the critics that challenged me, and the dreamers who have inspired me.

To my husband Dan Schwager, for his extraordinary patience, support and unconditional love that allowed me to keep on following my dreams. Last but not least, to my ‘anti-inflammatory’ and ‘anti-stress’ natural product Arabidopsis Thaliana…I hope your work can also be your passion.

This body of work would not have been possible without your love and support.

Table of Contents

Table of Contents.………………………………………………………………….………i

List of Tables.………………………………………………………………….…….……v

List of Figures.………………………………………………………………….……..….vi

Acknowledgements.……………………………..…………………………….………….ix

List of Abbreviations.…………………………………………………………….………xi

Abstract …………………………………………………………………………………...1

Chapter 1: Background and Significance

1.1 Inflammatory Response

1.1.1 Overview of the Acute Inflammatory Response………………………………3

1.1.2 Inflammation and Oxidative Stress Leading to Chronic Inflammation……….5

1.1.3 Chronic Inflammation as a Cause of Disease…………………………………6

1.2 Regulation of Inflammation Through NF-κB Signaling

1.2.1 NF-κB Signaling Pathway…………………………………………………….7

1.2.2 Activation of NF-κB Signaling………………………………………………..8

1.2.3 Crosstalk Between NF-κB and Nrf2……………………………….………….9

1.3 Anti-inflammatory and Antioxidant Nrf2-Keap1 Signaling

1.3.1 Historical Overview of the Discovery of the Nrf2-Keap1 Signaling Pathway. 9

1.3.2 Components of Nrf2-Keap1 Signaling

NF-E2 related factor-2 (Nrf2).……………………...…….……………….…11

Kelch-like ECH-associated protein1 (Keap1)……………………………….12

Antioxidant response element (ARE)/Electrophile response element (EpRE)13

1.3.3 Nrf2 Regulation

i Mechanistic models of Nrf2 activation………………………………………13

Nrf2 regulation independent of Keap1………………………………………14

Positive and negative regulators of the Nrf2 pathway……………………….15

1.3.4 Cytoprotective Roles of Nrf2 Target Genes…………………………………17

1.3.5 Importance of the Nrf2-Keap1 Pathway in Disease…………………………18

1.3.6 Regulation of Anti-inflammatory Response Through Nrf2………………….20

1.4 Small Molecule Activators of Nrf2-Keap1

1.4.1 Historical Perspective………………………………………………………..22

1.4.2 Exogenous Phytochemical Activators of Nrf2

Phytochemicals that readily react with Keap1-cysteines………………..…..23

Phytochemicals requiring biotransformation in order to react with Keap1-

cysteines……………………………………………………………………...24

Triterpenoids…………………………………………………….…………...25

1.4.3 Endogenous Electrophile Activators of Nrf2……………………...…………26

1.4.4 Other Classes of Nrf2 Activators……………………...……………………..27

1.5 Strategy to Investigate Small Molecule Activators of Nrf2

1.5.1 Defining the Determinants of Nrf2 Activation by Triterpenoids……………28

1.5.2 Diversity Oriented Synthesis as an Alternative Approach for the Discovery of

Effective Nrf2 Inducers………………………………………………………29

1.5.3 Bryonolic Acid and Lanosterol as Lead Material for DOS……………….…30

1.5.4 Identification of a Negative Feedback Loop of Biological Oxidant Formation

Regulated by 4-HNE…………………………………………………………30

Chapter 1 Figures……………...……………………………………………………..32

Chapter 2. Bryonolic Acid: A Large-Scale Isolation and Evaluation of Heme Oxygenase

1 Expression in Activated Macrophages.

2.1 Abstract…………………………………………………………………………….38

2.2 Introduction………………………………………………………………………...39

2.3 Results and Discussion……………………………..……………………………...39

2.4 Experimental Section……………………………..……………………………...... 43

Chapter 2 Figures……………………………………………………………...…….…49

Chapter 3: Bryonolic Acid Transcriptional Control of Anti-inflammatory and

Antioxidant Genes in Macrophages in Vitro and in Vivo.

3.1 Abstract………………………………………………………………………….…54

3.2 Introduction………………………………………………………………………...55

3.3 Results and Discussion…………………..…………………..…………………….57

3.4 Experimental Section…………………..…………………..………………………68

Chapter 3 Figures…….…...... ………………………………………………………….73

Chapter 4: Identification of Small Molecule Nrf2 Activators Through Diversity Oriented

Synthesis

4.1 Abstract…………………..…………………..…………………………………….84

4.2 Introduction…………………..…………………..………………………………...85

4.3 Results and Discussion…………………..…………………..………………….....88

4.4 Experimental Section…………………..…………………..…………………...... 94

Chapter 4 Figures and Tables…………...…….………………...... 98

Chapter 5: Identification of a Negative Feedback Loop in Biological Oxidant Formation

Regulated by 4-Hydroxy-2-(E)-Nonenal

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5.1 Abstract…………………..…………………..…………………………………..109

5.2 Introduction…………………..…………………..………………………………109

5.3 Results and Discussion…………………..…………………..…………………..111

5.4 Experimental Section…………………….……………….……………………...117

Chapter 5 Figures…….……...…………….…………………………………..……...130

Chapter 6: Summary, Conclusions and Future Directions……………………………..139

6.1 Summary and Future Directions - Chapter 2. Bryonolic Acid: A Large-Scale

Isolation and Evaluation of Heme Oxygenase 1 Expression in Activated

Macrophages……………………………….……………………………………..141

6.2 Summary and Future Directions - Chapter 3. Bryonolic Acid Transcriptional

Control of Anti-inflammatory and Antioxidant Genes in Macrophages in Vitro and

in Vivo……………………...………………..……………………………………142

6.3 Summary and Future Directions - Chapter 4. Identification of Small Molecule Nrf2

Activators Through Diversity-Oriented Synthesis…………………………...... …145

Chapter 6 Figures……………...……………..…………………………………….....150

Appendix

1 A1.1. H-NMR (400 MHz, pyridine-d5) of bryonolic acid………………………...155

13 A1.2. C-NMR (100 MHz, pyridine-d5) of bryonolic acid, 40-180 ppm……...... 156

13 A1.3. C NMR (100 MHz, pyridine-d5) of bryonolic acid, 15-55ppm……………157

Bibliography……………………………………………………………………………158

List of Tables

Table 4.1. Anti-inflammatory activity of skeletally diverse triterpenoids……………..102

Table 4.2. Cell viability measurement of cells treated with derivatives……………….103

Table 4.3. Anti-inflammatory activity of functionalized derivatives……………….…105

Table 4.4. Cell viability measurement of cells treated with functionalized derivatives.106

List of Figures

Figure 1.1. Activation of NF-κB signaling pathway by LPS………………...…………32

Figure 1.2. Activation of Nrf2-Keap1 signaling..……………………………………….33

Figure 1.3. Thiol-reactive, α, β-unsaturated carbonyl that acts as a Michael acceptor

crucial for Nrf2 activation...…………………………………………………..….34

Figure 1.4. Phytochemicals with thiol-reactive motif.………………………...……..….35

Figure 1.5. Oxidation of tBHQ to thiol-reactive tBQ.…………………………..……....36

Figure 1.6. Cyclization of 2,3 oxido-squalene to form triterpenoids……………………37

Figure 2.1. Structure and numbering of bryonolic acid…………………….………...…49

Figure 2.2. iNOS, COX-2 and HO-1 immunoblot analysis of induced RAW 264.7 cells treated with bryonolic acid.……………………….………………………………..…….50

Figure 2.3. HPLC traces of extracts……………………………………………………..51

Figure 2.4. Bryonolic acid production in Cucurbita pepo L. roots.……………………..52

Figure 2.5. Maximum and total bryonolic acid production in roots.……………………53

Figure 3.1. Bryonolic acid and oleanolic acid structures.……………………………….73

Figure 3.2. NO levels and iNOS expression in LPS-activated RAW 264.7 cells treated with bryonolic acid………………………………………………………………………74

Figure 3.3. HO-1 expression in RAW 264.7 cells treated with bryonolic acid…………75

Figure 3.4. qRT-PCR analysis of Nrf2 target genes in RAW 264.7 cells treated with bryonolic acid.……………………………………………………………………………76

Figure 3.5. NO suppression and HO-1 induction activity comparison of bryonolic acid and oleanolic acid…………………….………………………………………………….77

Figure 3.6. Comparative analyses of induction of HO-1 expression by triterpenoids.….78

Figure 3.7. Immunoblot analysis of cytoplasmic and nuclear Nrf2 in bryonolic acid and oleanolic acid treated RAW 264.7 cells………………………………………………….79

Figure 3.8. Comparative qRT-PCR analysis of Nrf2 target genes in RAW 264.7 cells treated with bryonolic acid compared with oleanolic acid. …………………...………...80

Figure 3.9. Selective regulation of HO-1 by bryonolic acid ……………………..……..81

Figure 3.10. Immunoblot analysis of HO-1 induction in vitro and in vivo by bryonolic acid. ………………….…………………………………………………...……………...82

Figure 3.11. Positive and negative controls of bryonolic acid treated cells……...……..83

Figure 4.1. Reagents and conditions for DOS of lanosterol derivatives…………...……98

Figure 4.2. Reagents and conditions for DOS of bryonolic acid derivatives………….100

Figure 4.3. Reagents and conditions for A-ring functionalization..……………………104

Figure 4.4. Immunoblot and qRT-PCR analyses of iNOS in LPS-activated cells treated with triterpenoid derivatives……………………………………...…………………….107

Figure 4.5. Activation of Nrf2 by linear derivatives 14f and 26f……………………...108

Figure 5.1. Nitrite levels in 4-HNE treated LPS-activated RAW 264.7 cells……...…130

Figure 5.2. Viability of LPS-activated RAW 264.7 cells treated with 4-HNE…...……131

Figure 5.3. Synthesis and activity of C5–C12 4-hydroxy-2-(E)-alkenal derivatives….132

Figure 5.4. Viability of LPS-activated RAW 264.7 cells treated with C5–C12 4-hydroxy-

2-(E)-alkenal derivatives…………………..…………..…………..…………..………..133

Figure 5.5. iNOS enzyme activity and expression analysis of LPS-activated RAW

264.7 cells treated with 4-HNE. …………………..…………..…………..……………134

Figure 5.6. Immunoblot analysis of Nrf2 protein levels in the cytoplasm and nucleus and qRT-PCR analysis of Nrf2 target genes in RAW 264.7 cells treated with 4-HNE…….135

vii

Figure 5.7. Nitrite levels and cell viability of LPS-activated primary macrophage cells treated with 4-HNE …………………………...……..…………..……………….……136

Figure 5.8. Conjugated 4-HNE-GSH measurement………………………………...…137

Figure 5.9. A model for 4-HNE control of LPS-induced nitric oxide production...….138

Figure 6.1. Clinical score of EAE mouse model treated with bryonolic acid…..….….150

Figure 6.2. Immunoblot analysis of iNOS and HO-1 in triterpenoid-treated cells....….151

Figure 6.3. Immunoblot analysis of iNOS, COX-2, and HO-1 in triterpenoid-treated cells……………………………………………………………………………………..152

Figure 6.4. Differential regulation of iNOS, COX-2, and HO-1 protein by lanosterol derivatives …………………………………………………………………...…………153

Figure 6.5. In vivo treatment of mouse model of inflammation with 14f…….…….….154

viii

Acknowledgements

I would like to first acknowledge past mentors who have helped and encouraged me along this scientific path. First and foremost, I would like to thank my high school teacher Bill Reiswig for setting me up with my very first lab experience using a microscope under the guidance of Dr. Minoo Ahdieh at Immunex Corporation. To Dr.

Lee Hartwell at Fred Hutchison Cancer Research (FHCRC), I would like to express my gratitude for giving me a chance to work in a lab with world-class scientists. To my most influential mentor Dr. Antonio Bedalov at FHCRC, thank you for your inspiring enthusiasm and your infectious passion for science. Before meeting you, I never understood how someone other than a brewer could be so excited about a plate of yeast cells. Thank you for pushing me beyond my limits and believing in my potential.

I would like to thank Drs. John Letterio and Greg Tochtrop for their tremendous support and encouragement throughout my graduate career and for giving me the opportunity to work independently. Thank you for giving me the freedom to explore and allowing the lab to be my playground. I also would like to thank my committee members

Dr. Monica Montano, Dr. Sanjay Gupta, and Dr. John Mieyal, and former committee member Dr. Toni Berdis for their constructive advice and support. I would also like to acknowledge the mentorship and honest advise provided by Dr. Toni Berdis. I would like to thank the members of the Cancer Center who allowed me to use their equipment, which was integral to my research: Dr. David Danielpour, Dr. Clark Distelhorst, and Dr.

Mark Jackson. I would like to thank Cami Thompson, Ivona Golczak, Diane Dowd, and

(superwoman) Vida Tripodo for all of your support navigating and evading the perils of graduate school funding and administration.

Thanks to members of the Letterio lab, especially Eric Lam who has been my partner in crime. Sharing similar graduate school ups and downs helped the down days to be more tolerable. To members of the Tochtrop lab including Emily Barker, Dr. Yong

Han, Dr. Vasily Ignatenko, and Dr. Sushaban Sadhukhan: I hope that all future collaborations are as energizing and fruitful as our work together. I would like to especially acknowledge Emily Barker for her encouragement and support for navigating motherhood during my graduate career. I would also like to thank our Letterio lab mom and former lab manager, Janet Robinson for her care and resourcefulness. The Letterio lab is not the same without you.

I would also like to acknowledge the funding support from NIH fellowship,

F31CA134211 and the Reuter Foundation.

Finally, I would like to thank family and friends who have kept me sane throughout this process.

List of Abbreviations

4-HNE 4-hydroxynonenal

8-nitro-cGMP 8-nitro-guanosine 3′,5′-cyclic monophosphate

AHR aryl hydrocarbon receptor aPKC atypical protein kinase C

ARNT AHR nuclear translocator

AIMS antioxidant inflammation modulators

AP-1 activator protein-1

ARE antioxidant response element

ATO arsenic trioxide

BA 3β-hydroxy-D:C-friedoolean-8-en-29-oic acid, bryonolic acid

BCL-3 B-cell lymphoma 3

BHT butylated hydroxytoluene

BMDM bone marrow derived macrophages

BTB Broad complex, Tramtrack, and Bric-a-Brac

C. pepo L. Cucurbita pepo L

CAT catalase

CBP CREB (cAMP Responsive Element Binding protein) binding

protein

CDDO 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid

CHD6 chromo-ATPase/helicase DNA binding protein

CK2 protein casein kinase 2

CLRs C-type lectin receptors

CNC cap ‘n’ collar

CO carbon monoxide

COPD chronic obstructive pulmonary disease

COX-2 cyclooxygenase-2 cPKC classical protein kinase C

Cul3 Cullin

DAMPS damage-associated molecular patterns

DOS diversity oriented synthesis

EAE experimental autoimmune encephalomyelitis

EGCG epigallocatechin gallate eNOS endothelial eNOS

EpRE electrophile response element

GCLC glutamate-cysteine ligase catalytic subunit

GR glutathione reductase

GSH glutathione

GST(s) glutathione S-transferase(s)

HO-1 heme oxygenase-1

HIF-1α hypoxia-inducible factor-1α iNOS inducible nitric oxide synthase

IκB inhibitor of κB

IKK IκB (inhibitor of κB) kinase ip intraperitoneal

IRF interferon regulatory factor IRF

xii

IVR intervening region

Keap1 Kelch-like ECH-associated protein1

LPO lipid peroxidation

LPS lipopolysaccharide

MCP-1 monocyte chemoattractant protein-1

MEFs mouse embryonic fibroblasts

NAC N-acetylcysteine

Neh Nrf2-ECH homology

NF-κB nuclear factor of kappa light polypeptide gene enhancer in B-cells

NFAT nuclear factor of activated T cells nNOS neuronal nitric oxide synthase nPKC novel protein kinase C

Nrf2 nuclear factor erythroid 2 p45-related factor

NO nitric oxide

– NO2 nitrite

NLRs NOD-like receptors

NQO1 NAD(P)H dehydrogenase, quinone 1

NQOs NAD(P)H:quinone oxidoreductases

– O2 superoxide

OA oleanolic acid

OA-NO2 nitro-oleic acid

ONOO– peroxynitrite

PAMPs Pathogen-associated molecular patterns

xiii

PGE2 prostaglandin E2

PKC Protein kinase C

PRRs pattern recognition receptors

Prx1 peroxiredoxin 1

PUFAs polyunsaturated fatty acids

RAW RAW 264.7, leukemic mouse macrophage cell line

RIP receptor-interacting protein

RLRs retinoic acid-inducible gene (RIG)-I-like receptors

ROS reactive oxygen species

ROS/RNS reactive oxygen/nitrogen species sMAF small masculoaponeurotic fibrosarcoma

SOTs synthetic oleanane triterpenoids

STAT3 signal transducer and activator of transcription 3 tBHQ tert-butylhydroquinone

TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

TLRs toll-like receptors

TLR-4 toll-like receptor 4

TNFR tumor necrosis factor receptor

TRAF TNF receptor associated factors

UA ursolic acid

VCAM-1 vascular adhesion molecule-1

XRE xenobiotic-responsive element

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Biological and Chemical Analysis of Small Molecule Activators of

Anti-inflammatory and Antioxidant Nrf2-Keap1 Signaling

Abstract

TONIBELLE NICOLAY GATBONTON-SCHWAGER

The immune system provides a cellular defense mechanism against harmful agents in the form of inflammatory and antioxidant responses mediated by the Nrf2-

Keap1 signaling pathway. Activators of Nrf2-Keap1 signaling include endogenous molecules such as reactive oxygen species (ROS) and electrophiles, and exogenous molecules including xenobiotics, phytochemicals, and potent synthetic phytochemical derivatives. This body of work furthers the current understanding of the biology and chemistry of small molecule activators of Nrf2 with a focus on exogenously derived natural triterpenoids and related semi-synthetic triterpenoids, and endogenously derived lipid peroxidation product 4-hydroxynonenal (4-HNE). Synthetic oleanane triterpenoids

(SOTs) are potent antioxidant inflammation modulators (AIMs) that target Nrf2 and demonstrate preventive and therapeutic effects against a variety of inflammation- mediated chronic diseases. The therapeutic potential of SOTs and the pleiotropic biological profile of natural triterpenoids led us to examine whether the steroid skeletal structure is a requirement for the activation of the Nrf2-Keap1 signaling pathway. We hypothesized that rearranging the triterpenoid skeletal structure would yield novel and effective Nrf2-Keap1 modulators. To test our hypothesis, we developed a unique synthetic approach utilizing Diversity Oriented Synthesis (DOS) techniques to rearrange

1 the carbocyclic triterpenoid skeletal structure of naturally occurring triterpenoids bryonolic acid and lanosterol. In addition to our triterpenoid studies, we examined whether 4-HNE, an endogenous activator of Nrf2, exhibits auto-regulatory features.

During oxidative stress, polyunsaturated fatty acids (PUFAs) are oxidized to form 4-HNE, which has been implicated in the pathology of a variety of diseases. Since 4-HNE production is initiated by ROS, we hypothesized that activation of Nrf2 by 4-HNE suppresses biological oxidant formation, leading to the inhibition of 4-HNE production.

The triterpenoid studies demonstrated that rearrangement of the triterpenoid skeleton altered potency and that the steroid skeletal structure is not a requirement for Nrf2 activation. We also validated the DOS approach as a method for identifying effective

Nrf2 modulators. Understanding structural requirements for activation of Nrf2 will facilitate the identification of novel Nrf2 activators. Additionally, the discovery that 4-

HNE regulates its own formation via a negative feedback loop has implications that could lead to new approaches for controlling production and limit the deleterious effects of this molecule.

2 Chapter 1. Background and Significance

1.1 Inflammatory Response

1.1.1 Overview of the Acute Inflammatory Response

Circa 30 BC–38 AD Roman encyclopedist Celsus first described inflammation defined by classic symptoms: redness, swelling, heat, and pain. The Greek physician

Galenus later added loss of tissue function as the fifth symptom of inflammation. We now define inflammation as the body’s immediate response to infections, toxins, and tissue injury, resulting in the activation and recruitment of immune cells to eliminate the stimulus, repair damaged tissue, and return the damaged area to normal homeostasis.1, 2

The acute inflammatory response is a short, rapid, and highly regulated response that is triggered by endogenous and exogenous stimuli such as infections (bacterial, viral, fungal, and parasitic), foreign species (toxins and xenobiotics), trauma (from physical and chemical agents), tissue necrosis (including myocardial infarct and physical or chemical injury), and immune reactions (against environmental factors or “self”). Pathogen- associated molecular patterns (PAMPs) and damage-associated molecular patterns

(DAMPs) are derived from exogenous and endogenous stimuli and trigger the inflammatory response by binding to pattern recognition receptors (PRRs) expressed on the surface of immune cells such as macrophages, dendritic cells, and neutrophils.3

Activation of PRRs such as toll-like receptors (TLRs), NOD-like receptors (NLRs),4 C- type lectin receptors (CLRs) and retinoic acid-inducible gene (RIG)-I-like receptors

(RLRs) results in signaling cascades that activate transcription factors that mediate inflammation, production and release of inflammatory signaling molecules that further activate the inflammatory process, and physiological changes such as vasodilation and

3 extravasation of fluid for recruitment of additional immune cells at the site of damage.

Macrophages and other phagocytic cells are the first line of defense at the site of infection or injury, eliminating cellular debris and pathogens through phagocytosis.

Cytokines and chemokines are important inflammatory signaling molecules produced and secreted by many cell types and are responsible for recruitment, migration, and stimulation of immune cells, which further drives the inflammatory response.

Macrophages secrete inflammatory cytokines such as IL-1 and TNFs, stimulating a variety of immune cells, which include neutrophils, eosinophils, basophils, and lymphocytes. The functions of these activated immune cells are to target bacteria, fungi, and larger parasites, release histamine to increase capillary permeability for leukocyte mobilization, release antibodies, and target both virus-infected cells and tumor cells.

Important inflammatory signaling molecules also include vasoactive amines such as histamine and serotonin, which mediate vasodilation and increase vascular permeability for immune cell recruitment. Transcriptional activation of inflammatory enzymes such as inducible cyclooxygenase-2 (COX-2) results in production of arachidonic acid metabolites such as prostaglandins and leukotrienes involved in vascular permeability, leukocyte chemotaxis, and platelet aggregation. Another key enzyme induced during inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO), a signaling molecule important for vasodilation and direct bacterial killing. Cytokines such as IFN-γ, IL-1, TNF-α, and IL-6 further activate signal transduction cascades that lead to activation of transcription factors including nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor-1α (HIF-

4 1α), activator protein-1 (AP-1), nuclear factor of activated T cells (NFAT), and Nrf2, which mediate the inflammatory process and cellular stress response.

During the resolution phase of inflammation, mediators switch from pro- inflammatory prostaglandins to anti-inflammatory lipoxins. Pro-resolving factors such as

5 6 prostaglandin E2 (PGE2), omega-3 fatty acids, IL-10, TGF-β, annexin A1, lipoxins, resolvins, protectins, and maresins (small lipid mediators derived from arachidonic acid and omega-3 fatty acids through lipoxygenase pathways)7-9 are important signaling molecules during the resolution phase of inflammation. This phase includes clearance of the inflammatory stimuli such as the pathogen or cellular debris from tissue injury, removal of the inflammatory cells, recruitment of anti-inflammatory repair macrophages, recruitment of regulatory T cells, and activation of anti-inflammatory nuclear receptors, followed by repair of tissue and normalization of vasculature to return the tissue to homeostasis.

1.1.2 Inflammation and Oxidative Stress Leading to Chronic Inflammation

Recruitment of inflammatory cells such as macrophages, mast cells, and leukocytes at the site of inflammation leads to an increased release and accumulation of reactive oxygen/nitrogen species (ROS/RNS) and is part of normal physiology during inflammation.10, 11 Oxidative stress occurs when oxidants such as ROS/RNS, free radicals, and reactive metabolites overwhelm the body’s protective mechanism for detoxification and elimination of these reactive species via the antioxidant and cytoprotective system regulated by the Nrf2-Keap1 signaling pathway. Failure to remove the inflammatory trigger and eliminate these oxidants leads to oxidative stress and

5 chronic inflammation. Aberrant production of ROS/RNS during inflammation can alter the structure and function of DNA and various proteins.12-18 Protein damage can occur when the unpaired electron in NO interacts with ribonucleotide reductase,19 inhibiting its function, and by inactivating enzymes involved in the oxidative stress response20 and

21 DNA repair. In addition, auto-oxidation of NO forms N2O3 species, which deaminate

– and cross-link DNA, causing damage. NO readily reacts with superoxide (O2 ) to form peroxynitrite (ONOO–). This persistent inflammation and oxidative stress results in a chronic inflammatory state, which is a prolonged deregulated process characterized by cellular infiltration of monocyte/macrophages, lymphocytes, plasma cells, and excessive reactive species. Persistent infections, inappropriate activation of the immune system

(immune-mediated inflammatory diseases), prolonged exposure to inflammatory agents, and oxidative stress are all causes of chronic inflammation and result in destruction of tissues. Many diseases have an underlying chronic inflammatory state; therefore a carefully regulated inflammatory response is crucial to properly defend against harmful insults and return the damaged area to homeostasis without causing further damage.22

1.1.3. Chronic Inflammation as a Cause of Disease

A variety of diseases have a pathophysiological inflammatory component, including autoimmune disorders, neurodegenerative and cardiovascular diseases such as rheumatoid arthritis, atherosclerosis, chronic obstructive pulmonary disease, asthma, multiple sclerosis, inflammatory bowel disease, obesity, and cancer. As early as 1867,

Rudolf Virchow proposed that diseases such as cancer are inflammatory-based as a result of his observation that the tumor microenvironment is infiltrated with inflammatory

6 macrophages. Recent data indicate that the surrounding stroma (associated matrix, immune cells, and inflammatory cells) interacts with cancer cells and fosters an environment for angiogenesis and cancer development.23, 24

1.2 Regulation of Inflammation Through NF-κB Signaling

1.2.1 NF-κB Signaling Pathway

Several transcription factors are activated during the inflammatory process.

However, one of the key transcription factors playing a central role during this response is NF-κB. There are three major components to the NF-κB pathway: (1) the IκB

(inhibitor of κB) kinase (IKK) complex, which is activated by many signaling pathways;

(2) the inhibitory IκB protein, which sequesters NF-κB in the cytoplasm; and (3) the transcription factor NF-κB. The NF-κB family consists of five members NF-κB1 (p50),

NF-κB2 (p52), RelA (p65), RelB, and c-Rel (Rel) with 15 possible homo- and heterodimerizations with differential DNA-binding and transactivating activities. Under basal conditions, inactive NF-κB is sequestered in the cytoplasm by IκB proteins, which include IκBα, IκBβ, IκBε, IκBζ, B-cell lymphoma 3 (BCL-3), IκBns, and precursor proteins NF-κB2 (p100) and NF-κB1 (p105). Activation of this pathway leads to phosphorylation of IκB by IKK and subsequent ubiquitin mediated proteosomal degradation of IκB. NF-κB is released from the cytoplasm, allowing it to translocate and accumulate in the nucleus and bind to DNA κB sites in promoter and enhancer regions of target genes (Figure 1.1, p. 32). The homo- and heterodimerization of NF-κB subunits contributes to the many flexible κB sequences that can be recognized by NF-κB, thus

7 resulting in a wide variety of target genes.25, 26 In addition to regulating inflammatory response enzymes, NF-κB regulates transcription of genes important for the development and maintenance of the immune system, skeletal system and epithelium, and also contributes to the control of cell survival, differentiation, and proliferation.27-29 Aberrant

NF-κB activity results in chronic inflammation or autoimmune diseases30 including obesity,31 malignancy,32 and metabolic diseases,31, 33 thus serving as an important therapeutic target.

1.2.2 Activation of NF-κB Signaling

NF-κB can be activated via either the canonical or non-canonical pathway. The canonical pathway is activated by ligand binding to cytokine receptors including tumor necrosis factor receptor (TNFR), IL-1 receptor (IL-1R), PRRs such as toll-like receptor 4

(TLR4), and antigen receptors. Binding of a ligand to one of these receptors results in the activation of IKKβ (IKK2), which forms a complex with other IKK members such as

IKKα (IKK1), and IKKγ (NEMO, regulator protein). The activated IKKβ complex phosphorylates IκB proteins (such as IκBα), leading to its degradation and release of an active NF-κB complex. The non-canonical/alternative pathway is activated by members of the TNF cytokine family which include CD40, BAFF, and lymphotoxin-β34 and is dependent on IKKα, but not IKKγ (NEMO). Activation of IKKα in this pathway leads to p100 phosphorylation and p52/RelB complex formation. Regardless of the activation pathway, signaling cascades converge at IKK, resulting in NF-κB activation. Although many signaling pathways activate NF-κB upstream of the IKK complex, intermediates including receptor-interacting protein (RIP) and TNF receptor associated factors (TRAF)

8 families of proteins are required to activate IKK and serve as a point of convergence for

NF-κB activation.

1.2.3 Crosstalk Between NF-κB and Nrf2

In addition to the activation of the NF-κB pathway by extracellular ligands, intracellular activators include responses to DNA damage, ROS, and recognition of intracellular pathogens mediated by NLR family of proteins. The NF-κB pathway can also be activated via crosstalk with other signaling pathways including p53, MAP kinase

(MAPK), interferon regulatory factor (IRF), and the Nrf2-Keap1 signaling pathway.35

Bioinformatics analysis of promoter regions of NF-κB and Nrf2 revealed an NF-κB binding site in the promoter region of Nrf2, suggesting regulation of NF-κB by Nrf2.36

Keap1 has been shown to directly bind to the IKK complex, IKKβ, resulting in IKKβ ubiquitination and degradation.37, 38 However, further studies are needed to fully elucidate the mechanistic underpinnings of Nrf2 and NF-κB cross-regulation.

1.3 Anti-inflammatory and Antioxidant Nrf2-Keap1 Signaling

1.3.1 Historical Overview of the Discovery of the Nrf2-Keap1 Signaling Pathway

The Nrf2-Keap1 signaling pathway modulates over 600 genes that encode anti- inflammatory and antioxidant enzymes important for cell survival.39 The key regulators of this pathway are the transcription factor Nrf2 and its transcriptional inhibitor Kelch- like ECH-associated protein1 (Keap1). Nrf2 is a basic leucine zipper transcription factor discovered in a screen for proteins that bind tandem NF-E2/AP1 DNA repeats in the regulatory region of the β-globin locus gene in humans.40 Studies on the β-globin locus

9 gene in chicken erythroid cells identified a small masculoaponeurotic fibrosarcoma

(sMaf) family of proteins that heterodimerize with Nrf2.47, 41, 42 The sMafs and Nrf2 heterodimer mediate the cellular response to Nrf2 inducers, electrophiles and oxidants in vivo.43 Previous work identified a cis-regulatory antioxidant response element (ARE) located in the promoter region of several detoxifying genes, which is required for expression of phase II detoxifying enzymes. Gene expression analysis of

NAD(P)H:quinone oxidoreductase 1 (NQO1), which detoxifies quinones to a less reactive hydroquinone, led to the identification of Nrf2 as a positive regulator of NQO1 expression through Nrf2-ARE binding.44 Additional detoxifying gene targets regulated by Nrf2 through ARE binding include glutamine-L-cysteine ligase (GLCL) which encodes a rate-limiting enzyme for de novo synthesis of glutathione,45 and heme- oxygenase 1 (HO-1) which encodes the HO-1 enzyme responsible for heme catabolism.46

Genes that are regulated by Nrf2 total 645 basal and 654 inducible direct targets.39

Characterization of the first Nrf2 knockout mice (Nrf2-/-)47 lead to studies demonstrating that Nrf2 is essential for protection from butylated hydroxytoluene (BHT) induced pulmonary injury.48 Although it is established that the binding of Nrf2 to ARE in promoter regions of cytoprotective genes is essential for the transcriptional induction of phase II enzymes, the mechanism of Nrf2 activation by inducers was unknown until the discovery of the transcriptional repressor of Nrf2, Keap1.49 Mass spectrometric analysis of Keap1 cysteine residue modification by dexamethasone was the first direct evidence demonstrating that sulfhydryl groups on Keap1 act as sensors that regulate the transcriptional activation of Nrf2-dependent cytoprotective genes.50 Additional studies in

Nrf2-/- mice further established the cytoprotective role of the Nrf2 signaling pathway as

10 shown by the increased susceptibility of Nrf2-/- mice to toxins,51-53 oxidative stress,54-56 inflammation,57, 58 and inflammation-mediated diseases such as cancer.59-61 The characterization of Nrf2 as essential for the induction of cytoprotective anti-inflammatory and antioxidant enzymes and identification of the mechanism of activation through the

Keap1 sensor led to increased interest in identifying potent Nrf2 inducers. As a result, potent Nrf2 inducers are now in clinical trials for the treatment of a variety of inflammatory-mediated diseases.

1.3.2 Components of Nrf2-Keap1 Signaling

NF-E2 related factor-2 (Nrf2)

Nrf2 belongs in the cap ‘n’ collar (CNC) family of factors, which include Nrf1,

Nrf2, Nrf3, p45 NF-E2, BACH1 and BACH2.62 Nrf1 and Nrf2 are ubiquitously expressed but have distinct roles. While Nrf1 is essential for embryonic development, this is not the case with Nrf2 as evidenced by the viability of Nrf2-/- mice with no developmental defects.47 Nrf3 is an endoplasmic reticulum-associated protein63, 64 that plays a role in smooth muscle cell differentiation.65 Nrf2 contains 589 amino acids and six highly conserved Neh (Nrf2-ECH homology) domains: Neh1 – Neh6. The Neh2 domain is located at the N-terminus followed by Neh4, Neh5, Neh6, Neh1, and Neh3 at the C-terminus. Neh2 contains the DLG and the ETGE motifs, which bind to the Kelch region of Keap1 with different binding affinities, serving as the negative regulatory domain of Nrf2. Lysine residues located between the DLG and ETGE motifs serve as the site for poly-ubiquitination and subsequent rapid degradation, resulting in a t1/2 < 20 min for Nrf2.66, 67 Neh4 and Neh5 are proline rich transactivation domains, which

11 cooperatively bind to co-activator CBP (CREB (cAMP Responsive Element Binding protein) Binding protein).68 Neh6 is a Keap1-independent negative regulator of Nrf2.69

Neh1 is the binding domain for small Maf and DNA. At the carboxy-terminus, Neh3 serves as a binding domain for transcriptional co-activator chromo-ATPase/helicase

DNA binding protein (CHD6), which promotes transcription of ARE-dependent genes.70

Kelch-like ECH-associated protein1 (Keap1)

Under basal conditions Nrf2 is sequestered in the cytoplasm by Keap1, which serves as an adaptor for the Cullin (Cul3)-based E3 ubiquitin ligase complex targeting

Nrf2 for ubiquitination and proteosomal degradation by the 26S proteosome.71, 72 Keap1 contains 624 amino acids and has 5 domains: N-terminal region, BTB, IVR, Kelch, and

C-terminal region. The Broad complex, Tramtrack, Bric-a-Brac (BTB) domain and the

Kelch domain are all protein-binding domains. BTB along with the N-terminal region of intervening region (IVR) facilitates Keap1 homodimerization and serves as a Cul3 binding domain. There are 25 and 27 cysteine residues in mice and humans, respectively, located throughout Keap1. However, these residues are not equally important for Nrf2 activation. The cysteine residues have different reactivity towards Nrf2 activators.

Mutation of Cys151 decreased Nrf2 activation by tBHQ, diethylmaleate and sulforaphane, while activation with CDDO-Im, mitroleic acid, and cadmium chloride remained

73 278 288 unaffected. Cys and Cys are important for basal repression for Keap1 as demonstrated by the inability of Keap1 with either cysteine mutation to repress Nrf2 under basal condition.74 Seven key cysteine residues in humans – Cys151, Cys257, Cys273,

Cys288, Cys297, Cys434, and Cys613 have been identified as important for the reactivity of

12 Keap1 to electrophiles, oxidants, and activators.50, 75, 76 These residues are the most reactive towards modification by electrophiles, oxidants, and activators, resulting in the release of Nrf2 from Keap1. Nrf2 translocates in the nucleus where it heterodimerizes with small Maf protein(s) and binds to ARE upstream of Nrf2-target genes, thereby resulting in their transcriptional activation (Figure 1.2, p. 33).43

Antioxidant response element (ARE)/Electrophile response element (EpRE)

An antioxidant response element (ARE), also referred to as an electrophile response element (EpRE), is a cis-regulatory element located upstream of Nrf2-target genes. AREs were first identified as a DNA regulatory sequence in the promoter region of glutathione S-transferase (GST) Ya subunit and in the NQO1 gene responsible for basal and inducible expression by aromatic and phenolic antioxidants.77-80 The original consensus ARE sequence was 5’-TGACnnnGC-3’ identified upstream of rat GST-Ya gene,81 but later expanded to 16-bp sequence from comparison analysis of ARE from rat, mouse, and human GST and NQO1 genes.82, 83 The ARE consensus sequence is not representable by a single sequence due to the variation among genes. However, the

A A C A typical sequence is 5’-T /CAnn / GTGA / GnnnGC / G-3’ where n is variable depending on the gene. The core ARE sequence also contains sequences for additional transcription factors to bind and regulate Nrf2.

1.3.3 Nrf2 Regulation

Mechanistic models of Nrf2 activation

Two mechanistic models for Nrf2 activation have been proposed: the ‘hinge and

13 latch’ model and the Keap1-Cul3 dissociation model. In the ‘hinge and latch’ model, activation of the Nrf2-Keap1 pathway results in the dissociation of the low affinity binding ‘latch’ DLG motif from Keap1 while the higher affinity ETGE motif or ‘hinge’ stays bound.84 As a consequence, newly synthesized Nrf2 accumulates and translocates to the nucleus, resulting in transcription of Nrf2-target genes. The premise for this model is that studies of Nrf2 activation with tert-butylhydroquinone (tBHQ) resulted in Nrf2- target gene activation, but the Nrf2-Keap1 complex remained undisrupted.85, 86 The second model for Nrf2 activation is the Keap1-Cul3 dissociation model wherein Keap1 and Cul3 are dissociated from each other, resulting in Nrf2 stabilization.87, 88 In this model, covalent modification of cysteine residues in the Cul3/BTB binding domain of

Keap1 disrupts the Keap1-Cul3 interaction, thereby dissociating the complex.89, 90

Nrf2 regulation independent of Keap1

At the transcription level, Nrf2 target genes can be regulated via binding of the transcription factor aryl hydrocarbon receptor (AHR) on the xenobiotic-responsive element (XRE) present in the promoter region in close proximity to ARE of many Nrf2- target genes including Nrf2 itself.78, 91 AHR ligands activate AHR, resulting in AHR and

AHR nuclear translocator (ARNT) transcription heterodimer formation. This complex transcriptionally activates many of the Nrf2-target genes and phase I enzymes that generate reactive intermediates that can activate the ARE-Nrf2-target genes.92 Potent

AHR inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can also activate

Nrf2.91, 93 Nrf2 activates its own expression as shown by increased Nrf2 protein and mRNA levels with Nrf2 activators.100

Positive and negative regulators of the Nrf2 pathway

There are many regulators of the Nrf2-Keap1 signaling pathway, but the primary regulator of Nrf2 is Keap1. Keap1 suppresses Nrf2 transcriptional activation by sequestering Nrf2 in the cytoplasm and targeting it for ubiquitination and degradation.

Positive regulators of Nrf2 include phytochemicals and derivatives, therapeutics, environmental agents and endogenous inducers that modify Keap1 cysteine residues.

Nrf2 is released from Keap1, allowing it to translocate into the nucleus and transcriptionally activate ARE-containing genes. Protein kinase C (PKC) is a serine/threonine kinase family grouped into three isoforms, classical (cPKC), atypical

(aPKC), and novel (nPKC), with each class requiring different cofactors for their different activities. PKCs play varying roles in controlling cell growth, differentiation, apoptosis, survival and carcinogenesis.94-100 In response to oxidative stress, aPKCs phosphorylate Ser-40 located in the Nrf2-Neh2 domain where the Kelch domain of

Keap1 binds, resulting in disruption of the Nrf2-Keap1 complex.101 Mutation of Nrf2

Ser-40 decreases Nrf2 nuclear translocation compared to wild-type, demonstrating that

Nrf2 phosphorylation is a regulatory mechanism for Nrf2 activation.102 Proteins such as p62, p21, and pGAM5 disrupt Nrf2-Keap1 interaction by binding to the Keap1 DC domain where Nrf2 binds. Cyclin dependent kinase (CDK) p21 bind to the ETGE and

DLG motifs on Nrf2 which disrupts Keap1 binding to Nrf2.103 Additionally, DJ-

1/PARK7 also disrupts Nrf2-Keap1 interaction, but the molecular mechanism of Nrf2 stabilization has not been identified. Several other kinases phosphorylate Nrf2 including

MAP kinases,104 PERK,105 and GSK3β,106-108 leading to increased Nrf2 nuclear

15 accumulation and activation. Transcriptional co-activators including RAC3/SRC,

CBP/p300, CARM1, PRMT1, and p/CAF positively regulate Nrf2 by enhancing transactivation activity. p300/CBP is an Nrf2 transcriptional co-activator that associates and acetylates Nrf2 at the Neh1 DNA binding domain.68, 109 Disrupting this interaction between Nrf2 and p300/CBP via mutation of the lysine residues disrupts the ability of

Nrf2 to bind DNA.109 In addition, p300/CBP also interacts with NF-κB and serves as a regulatory crosstalk mechanism between NF-κB and Nrf2. 110 Micro-RNAs such as miR-

200 bind to Keap1 mRNA promoting Nrf2 nuclear translocation. Nrf2-Neh2 domain phosphorylation by PKC, Nrf2-Neh4 and Neh5 phosphorylation by protein casein kinase

2 (CK2) disrupts Nrf2-Keap1 interaction and decreases Nrf2 nuclear translocation.111

Inhibition of CK2 reduces nuclear localization of Nrf2 and transcriptional activation of

Nrf2-target genes.111 Phosphorylation of Nrf2 by PERK, JNK1/2 and ERK1/2 also results in Nrf2 nuclear translocation, but the molecular mechanism by which these pathways regulate Nrf2-Keap1 signaling has not been elucidated.

In addition to Keap1 as a negative regulator of Nrf2, several mechanisms exist for the negative regulation of Nrf2. Tyrosine kinase Fyn phosphorylates Tyr-568 on Nrf2 resulting in Nrf2 nuclear export, ubiquitination and degradation. Mutation of Tyr-568 results in accumulation of nuclear Nrf2 and loss of interaction with the exporting protein

Crm1.112 In addition, phosphorylation of Nrf2 by p38 promotes Keap1 association and prevents nuclear translocation and transcriptional activation of Nrf2-target genes.113

Micro-RNA, miR-144 reduces Nrf2 protein levels,114 miR-28 facilitates mRNA and protein Nrf2 degradation,115 and miR-34 suppresses both Nrf2 expression and Nrf2 target gene Mgst1.116 ENC1 (ectoderm-neural cortex protein 1) regulates Nrf2 at a translational

16 level by decreasing Nrf2 protein levels.117 Scaffolding protein caveolin-1 competes with

Keap1 for Nrf2 binding, resulting in decreased expression of Nrf2 target genes.118, 119

Despite all of the evidence demonstrating different proteins, miRNA, and pathways that directly interact with Nrf2, it is unclear how these pathways are coordinate and impact regulation of the Nrf2 pathway.

1.3.4 Cytoprotective Roles of Nrf2 Target Genes

The Nrf2-Keap1 signaling pathway regulates 645 basal and 654 inducible genes.39

The genes that were initially identified to contain the ARE in the promoter region and which are inducible via Nrf2 activation are GST Ya subunit and the NQO1 gene.77-80

GST is a rate-limiting enzyme in the de novo synthesis of glutathione, which conjugates hydrophobic electrophiles and ROS. NQO1 encodes for detoxifying enzymes, including

NAD(P)H:quinone oxidoreductases (NQOs), which catalyze two-electron reduction and detoxification of quinones. Additional ARE-dependent Nrf2 regulated genes include

GCLC45 which is a catalytic subunit of glutathione synthethase, heme-oxygenase 1 (HO-

1) which catabolizes heme,46 several members of the GST family,87, 120, 121 and phase II enzymes which can catalyze detoxification reactions for the elimination of xenobiotics, redox homeostasis, and cell survival.122-127 Typically the most analyzed Nrf2 target genes include HO-1 and NQO1 due to their high expression during Nrf2 induction. Additional

Nrf2 target genes that are analyzed include catalase (CAT), GCLC, and glutathione reductase (GR) due to their important function in detoxifying harmful ROS/RNS.

17 1.3.5 Importance of the Nrf2-Keap1 Pathway in Disease

One of the most studied therapeutic roles of Nrf2-Keap1 activation is in cancer chemoprevention. Since the 1960’s it has been demonstrated that in addition to direct mechanism of action, an important property of anticancer agents is the ability to induce detoxifying enzymes to protect animal models from cancer inducing agents. It is now established that activation of Nrf2 offers protection against carcinogens and harmful agents128, 129 and that disruption of Nrf2 leads to increased susceptibility to the progression of inflammation leading to cancer formation.130 Chemopreventive activities have been demonstrated in several cancer models including colon, bladder, lung, stomach, breast, skin, and liver cancer where Nrf2 activation results in prevention or progression of cancer.131 Several Nrf2 activators have been investigated in clinical trials for cancer prevention, including glucosonylates and isothiocyanates for detoxification of lung cancer inducing toxins,132 protection of women with high risk for breast cancer,133 and curcumin for multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and Alzheimer’s disease.134-137 Although activation of Nrf2 is an established therapeutic means for amelioration and prevention of cancer, Nrf2 overexpression in some cancers leads to cancer progression. Mutations and polymorphisms in Nrf2 and Keap1 genes cause constitutive Nrf2 activation and induction of cytoprotective enzymes and drug efflux pumps resulting in protection of cancer cells and promote tumor growth.138 Patients with lung, head, and neck cancers with Keap1 or

Nrf2 mutations have poorer prognosis than patients without mutations.138 Nrf2 is constitutively active in breast, lung, head and neck, ovarian, and endometrial cancers and result in poor patient prognosis.139-145 Keap1 mutations appear with high frequency in

18 breast and gall bladder cancer,140, 146 lung cancer cell lines, and non-small-cell lung cancer.141 Given the alternative roles of Nrf2, suppression of Nrf2 activity inhibits tumor growth by improving the efficacy of cancer chemotherapeutics.147

Chronic Obstructive Pulmonary Disease (COPD) patients have decreased Nrf2 expression as well as decreased Nrf2-dependent gene expression in lungs.148 Nrf2 is ubiquitously expressed and found in epithelium and alveolar macrophages. Activation of

Nrf2 is a promising therapeutic approach in a variety of airway disorders, which include pulmonary fibrosis, asthma, cystic fibrosis, and acute lung injury/acute respiratory distress syndrome in which pathologies are associated with oxidative stress.149-152

The high lipid content and oxygen consumption in the brain makes it sensitive to oxidative stress as a result of mitochondrial dysfunction that produces ROS which has been implicated in the pathogenesis of chronic neurodegenerative diseases.153 Activation of Nrf2 in the brain has been demonstrated to be protective against oxidative stress and neurotoxicity and has been proposed to be an important target for Alzheimer’s and

Parkinson’s Diseases154-156 and could potentially be beneficial in other neurological disorders such as Huntington’s disease, Lou Gehrig’s disease, Down syndrome, and multiple sclerosis.157-159

Nrf2 activation has been shown to exhibit protective effects in the heart via suppression of oxidative stress which leads to cardiac hypertrophy and heart failure160 as well as therapeutic properties against cardiovascular associated diseases including atherosclerosis.161 Nrf2 has been shown to have protective roles in diabetic nephropathy and neuropathy against high glucose-induced oxidative damage123, 162-164 and thus serves

19 as a potential therapeutic target for treatment of diabetes and other metabolic disorders.165, 166

1.3.6 Regulation of Anti-inflammatory Response Through Nrf2

One of the most important anti-inflammatory effects of Nrf2 activation is mediated by the transcriptional activation of HO-1. HO-1 catalyzes the breakdown of inflammatory heme into carbon monoxide (CO), free iron, and biliverdin, which is further metabolized into bilirubin. Induction of HO-1 attenuates the inflammatory response in tissue injury induced oxidative stress and cytokinemia,167 an endotoxin shock model,168 as well as attenuated inflammation induced in different organs including airway mucus,169 vascular endothelium,170 colon,171 brain.172, 173 In addition, skin inflammation and contact hypersensitivity is reduced, and wound healing after epithelial injury in mice is accelerated when HO-1 is induced.174, 175 The heme metabolite CO has anti- inflammatory effects as demonstrated by decreasing ROS production, inhibition of NF-

κB in endothelial cells,176 reduced inflammatory response in the liver of thermally injured mice,176 ameliorated joint inflammation in collagen-induced arthritis mouse model177 and enhanced the host defense response to microbial sepsis in mice.178 Upregulation of HO-1 expression results in production of CO and biliverdin metabolites attenuated inflammatory responses such as neutrophil rolling, adhesion and migration in acute inflammation induced by carrageenan in adult BALB/c mice.179 Biliverdin is protective against polymicrobial sepsis,180 endotoxin-induced acute lung injury181 and colitis.182

Bilirubin, the reduced product of biliverdin, suppressed experimentally induced autoimmune encephalomyelitis and autoimmune hepatitis.183, 184 Bilirubin has

20 additionally been found to inhibit iNOS expression and NO overproduction, and protected mice and rats against endotoxic shock.185-187 Moreover, bilirubin attenuated vascular endothelial activation and dysfunction as well as inhibited vascular adhesion molecule-1 (VCAM-1)-mediated transendothelial leukocyte migration.188, 189 Although the basis for the anti-inflammatory action of heme metabolites has not been fully elucidated, there is clear evidence that HO-1 counteracts inflammation via conversion of heme to CO and biliverdin.

Inflammatory cytokines such as IL-1β, IL-6, IL-12p40 and TNF-α, are abundant in DSS-induced model of colitis in Nrf2-/- mice.190, 191 Activation of Nrf2 prevents overproduction of these pro-inflammatory cytokines.192, 193 Chemokines such as CXC and CC, which guide the migration of inflammatory cells to sites of infection or tissue damage, were overproduced in the lung of Nrf2-/- mice.194 Nrf2-dependent HO-1 expression was suggested to inhibit TNF-α-stimulated NF-κB activation and monocyte chemoattractant protein (MCP-1) secretion in human umbilical vein endothelial cells.195

These observations suggest that Nrf2 is an important regulator of NF-κB activation and chemokine overexpression in response to inflammatory stimuli.

Two of the principal enzymes induced during inflammation are COX-2 and iNOS which are responsible for the production of inflammatory mediators prostaglandins and

NO, respectively.196, 197 Nrf2-/- mice have been demonstrated to have increased iNOS and

COX-2 expression in inflammation models such as DSS-induced colitis.190, 191 It is documented that Nrf2 activators are able to inhibit iNOS expression through inhibition of

NF-κB. Activation of Nrf2 with chalcone derivatives inhibited TNF-α and IL-1β NF-κB- induced COX-2 expression,198 while carbazole analogue, LCY-2-CHO decreased IL-1β-

21 induced iNOS expression, and inhibited NF-κB activation.198 Dithiolethione inhibited

LPS-induced NF-κB nuclear translocation and hepatic iNOS and NO production.199

Additional Nrf2-activating agents, which inhibit NF-κB, include activators such as benzyl isothiocyanates,200 sulforaphane,201 1,4-dihydroquinone,202 EGCG,203 curcumin,203

203 204-206 15d-PGJ2, and triterpenoids. Taken together, it is evident that Nrf2 is a negative regulator of the expression of inflammatory enzymes such as iNOS and COX-2, and that activation of the Nrf2 pathway serves as an alternative approach to mediating inflammation regulated by the NF-κB pathway.

1.4 Small Molecule Activators of Nrf2-Keap1

1.4.1 Historical Perspective

Prior to the identification and characterization of the Nrf2-Keap1 signaling pathway, scientists had long studied natural and synthetic compounds that block or suppress carcinogenesis. As early as the 1960s structurally diverse compounds were demonstrated to enhance the activity of detoxifying or phase II xenobiotic-metabolizing enzymes, thereby protecting animals from chemical induced carcinogens.207 This class of anticancer agents includes compounds such as phenolic antioxidants, azo dyes, polycyclic aromatic hydrocarbons, flavonoids, coumarins, cinnamates, indoles, isothiocyanates, 1,2- dithiol-3-thiones, and thiocarbamates.208-211 It wasn’t until the 1980s that Paul Talalay and his colleagues discovered a common chemical feature for structurally diverse small molecules that induce detoxifying enzymes. These inducers can undergo a Michael reaction and it was predicted that there is a protein with reactive cysteine residues that acts as a sensor for small inducers of the detoxifying genes such as NQO1 and

22 glutathione S-transferases (GSTs).212 Following the identification of the Nrf2 transcriptional repressor, Keap1, several labs have demonstrated modification of Keap1 cysteine residues by inducers. This body of work laid the foundation for elucidating the mechanism of activation of detoxifying enzymes via the Nrf2-Keap1 signaling pathway.

It is now widely accepted that Keap1 cysteine residues functions as a sensor for electrophiles, oxidative stress, and Nrf2 inducers. Since the identification of Keap1, hundreds of structurally diverse Nrf2-Keap1 inducers derived from natural and synthetic sources have been identified. These Nrf2 inducers are classified based on their chemical structures: (1) Michael acceptors, (2) oxidizable diphenols and quinones, (3) sulfoxythiocarbamates, (4) dithiolethiones and diallyl sulfides, (5) vicinal dimercaptans,

(6) trivalent arsenicals, (7) selenium-based compounds, (8) polyenes, (9) hydroperoxides, and (10) heavy metals and metal complexes.213, 214

1.4.2 Exogenous Phytochemical Activators of Nrf2

Phytochemicals that readily react with Keap1-cysteines

Plants are a rich source of biologically active chemicals that can transcriptionally activate Nrf2. Although structurally diverse, these plant-derived ARE-inducers share a common thiol-reactive motif such as an α, β-unsaturated carbonyl that acts as a Michael acceptor crucial for Nrf2 activation (Figure 1.3, p. 34). Phytochemicals with this thiol- reactive motif include withanolides, chalcones, butenolides, oxidized terpenoids, curcuminoids, and electrophilic epithionitriles, which include isothiocyanates and organopolysulfides. Phytochemicals that act readily with Keap1-cysteines include curcuminoids, cinnamic acid esters, chalcones, and flavonoids. Curcumin, a principal

23 component of several Indian spices,137 exhibits a variety of therapeutic properties including anti-inflammatory, antioxidative, antiarthritic, antiischemic, and anticarcinogenic effects and has been evaluated in clinical trials for various diseases including multiple myeloma, pancreatic cancer, colon cancer, psoriasis, and Alzheimer’s disease.134, 215 Curcumin synthetic analogues such as salicylcurcuminoid and Bis[2- hydroxybenzylidene]acetone have displayed increased potency for activating detoxifying genes.216, 217 Open-chain flavonoids such as chalcones have a broad spectrum of activities including anti-proliferative, anti-inflammatory, and anti-infective properties.218,

219 Two important compounds from this class, isoliquiritigenin, found in the root of

Glycyrrhiza glabra220 and sofalcone, a synthetic derivative of sophoradin chalconoid found in herbs of Sophora tonkinensis, were shown to induce Nrf2-Keap1-ARE signaling in gastric epithelial cells.221 Flavonoids, which are found in high concentrations in fruits, vegetables, and teas have been reported to have ARE gene-induction properties. These include natural isoflavone sappanone A,222 and flavonoids-like benzofuran-containing substituted aurones223 and their corresponding indole derivatives, benzylidene-indolin-2- ones224 and synthetic flavonoids such as 4-bromo flavone225 and β-naphthoflavone.226

(Figure 1.4, p. 35)

Phytochemicals requiring biotransformation in order to react with Keap1-cysteines

Reactivity with thiolate groups is a critical process for activation of Nrf2.

However, a large number of phytochemicals that activate Nrf2 do not have this feature.

These include phytochemicals such as phenols, monosulfides, furans, and indoles that are required to be biotransformed, metabolically or chemically, in order to acquire reactivity

24 with the Keap1 thiol group. Phenolic compounds are some of the best-characterized Nrf2 inducers requiring electrophilic conversion to a thiol-reactive molecule in order to activate Nrf2. Phytochemicals in this category include quercetin, epigallocatechin gallate

(EGCG), caffeic acid, picceatanol, and kaempferol. Quercetin does not structurally have a reactive group that can interact with Keap1 thiols. However, it is readily oxidized in human blood plasma to a significantly more electrophilic quinone methide227 which is much more reactive toward thiolates. Conjugation products have been detected with glutathione (GSH), N-acetylcysteine (NAC),228, 229 and protein cysteine residues.230

Similarly, EGCG contains several aromatic 1,2-dihydroxy units, which can form quinones that react with thiolates in vitro and in vivo.231-233 Oxidation of tBHQ234 and

EGCG analogs,232 as well as a broad series of phenols235 such as 1,2- 1,4-, and 1,6- diphenols,236 to Michael acceptors have been shown to be prerequisites of the induction of Nrf2 (Figure 1.5, p. 36).

Triterpenoids

Triterpenoids are naturally occurring hydrocarbons, derived from cyclization of

2,3 oxido-squalene forming 5-6 membered rings (Figure 1.6, p. 37). Thousands of triterpenoids have been isolated and characterized to date that exhibit a broad range of biological activities including anti-inflammatory, anticancer, anti-ulcerogenic, antimicrobial, anti-plasmodial, anticariogenic, anti-viral, hepato- and cardio-protective, and analgesic activities.237 Some of the most studied triterpenoids are the oleanane and ursane triterpenoids which have interesting biological, pharmacological and medicinal activities including anti-carcinogenic activities.238, 239 In 1997, the Sporn and Gribble

25 group at Dartmouth Medical School began to explore the weak anti-inflammatory properties of these triterpenoids and utilized a medicinal chemistry approach of iteratively changing functional groups around the triterpenoid structure.240 They used an assay measuring NO output to assess iNOS suppression activity of their triterpenoid libraries which years later led to one of the most promising therapeutic and chemopreventive agents for cancer, the semi-synthetic triterpenoid 2-cyano-3,12- dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its derivatives.241, 242 Also known as

SOTs, CDDO analogs are 200,000-times more active than the natural parent compound,241, 242 oleanolic acid, and suppresses de novo synthesis of iNOS and COX-2 in activated macrophages.243 Subsequent studies identified additional biological activities including anti-inflammatory, anticancer and cytoprotective activities in a variety of tumor cells and animal models.53, 213, 241, 243-260 It wasn’t until Nrf2 and Keap1 were identified that SOTs were classified as potent AIMs that activate Nrf2 to mediate its therapeutic effects.

1.4.3 Endogenous Electrophile Activators of Nrf2

The Nrf2 transcription factor is a cytoprotective mechanism that protects the body from oxidative stress. Therefore, endogenous activators include products of oxidative stress such as ER stress, ROS/RNS such as NO, and lipid aldehydes. In addition 15 Δ-

PGJ2, nitro-fatty acids, shear stress, nitrated cGMP, toxic bile acids have been shown to be endogenous Nrf2 activators. Cyclopentanone prostaglandin 15-deoxy-Δ12,14- prostaglandin J2 (15Δ-PGJ2) is a known inflammatory mediator produced during the resolution phase of inflammation. Studies on the regulation of inflammation by Nrf2

26 have shown that 15Δ-PGJ2 forms Keap1 adducts, resulting in the induction of Nrf2 target genes HO-1 and peroxiredoxin 1 (Prx1), which exerts anti-inflammatory effects in carrageenan induced acute model of inflammation in mice.261 Nitro-fatty acids are electrophilic signaling mediators formed by nitration of fatty acids by NO and nitrite

(NO2)-dependent reactions during oxidative/nitrosative stress. Nitro-fatty acid nitro-oleic

262, 263 acid (OA-NO2) has been shown to modify several Keap1 cysteine residues.

Nucleic acid nitration product 8-nitro-guanosine 3′,5′-cyclic monophosphate (8-nitro- cGMP) plays an important role in nitric oxide signaling.264, 265 Proteomic analysis of

Keap1 revealed that 8-nitro-cGMP directly interacts with Keap1 via S-guanylation of

Cys-434 resulting in Nrf2 activation. Additional electrophilic endogenous products such as 4-HNE, acrolein, and nitric oxide have been shown to modify Keap1.266

1.4.4 Other Classes of Nrf2 Activators

Although arsenic containing compounds are considered environmental carcinogens, they also have therapeutic benefits, such as arsenic trioxide (ATO) for treatment of leukemia and psoriasis. ATO has been FDA approved for relapsed or refractory acute promyelocytic leukemia and has been shown to induce Nrf2 nuclear translocation and expression of NQO1 and HO-1 in human myeloma cell lines.267 Additional heavy metals that induce expression of Nrf2 genes include mercury, cadmium, and zinc.226 Although cadmium chloride is a carcinogen which induces oxidative stress, it is also found to induce ARE genes.268, 269 Organogold complex, an auranofin which is an antirheumetic drug has been shown to have anti-inflammatory effects through activation of Nrf2.270

27 1.5 Strategy to Investigate Small Molecule Activators of Nrf2

The purpose of these studies is to biologically and chemically analyze small molecule activators of anti-inflammatory and antioxidant Nrf2-Keap1 signaling. Within the last decade, Nrf2 has been demonstrated to be an important therapeutic target due to its cytoprotective effects. Significant research effort towards identifying and developing

Nrf2 activators has led to the progression of several of these molecules such as the semisynthetic triterpenoids, sulforaphane and dimethyl fumarate (BG-12) into clinical trials.

Understanding the structural requirements for activation of Nrf2 would facilitate the identification of novel Nrf2 activators entering the clinic. In addition to exogenously derived Nrf2 activators, byproducts of the inflammatory process and oxidative stress lead to activation of Nrf2-Keap1 signaling. However, excessive production of these byproducts leads to cellular stress. Determining how these markers of stress are regulated can lead to new approaches for controlling their production to limit the extent of their damage.

1.5.1 Defining the Determinants of Nrf2 Activation by Triterpenoids

Triterpenoids have recently been found to possess a wide range of biological activities, the most studied of which is their ability to activate Nrf2. A majority of our research effort has been spent determining whether the core skeletal triterpenoid structure plays an important role in their ability to activate Nrf2. Our approach rearranges the triterpenoid skeleton using DOS, followed by biological screening of anti-inflammatory activity via quantification of NO production. Carefully selected structurally diverse derivatives were further refined to increase potency by applying modifications

28 established to increase triterpenoid activity. Mechanistic analysis of biological activity determined the ability of derivatives to inhibit inflammation via suppression of NO and transcriptional activation of Nrf2 target genes.

1.5.2 Diversity Oriented Synthesis as an Alternative Approach for the Discovery of

Effective Nrf2 Inducers

Diversity Oriented Synthesis is a unique and efficient approach used to expand a collection of small molecules that modulate biological systems. This approach is different from what has been previously done in the discovery of synthetic oleanane triterpenoids (SOTs) where iterative changes around the triterpenoid skeleton result in changes in activity. Previous perturbation of oleanolic acid had been limited to iterative modulation of functional groups, defined as medicinal chemistry. The DOS approach aims to diversify the core carbocyclic skeleton of triterpenoids, which has not been explored previously. DOS was utilized on structural homologs of SOTs, lanosterol and bryonolic acid to determine the requirements for triterpenoid activity (Figure 1.6, p. 37).

Structure activity relationships were conducted by monitoring changes in expression of iNOS using a Griess assay as previously reported241 and via western-immunoblot techniques. Modifying the core structure through DOS will gain access to new chemical space, which will diversify the current collection of potential AIMs. Evaluating the activity of these new sets of molecules will lead to a better understanding of the molecular requirements for Nrf2 activation and activity. In addition, we also sought to validate DOS as a unique method for the identification of effective Nrf2 modulators.

29 1.5.3 Bryonolic Acid and Lanosterol as Lead Material for DOS

Bryonolic acid and lanosterol are naturally occurring triterpenoids that are structurally homologous to the parent compound of SOTs, oleanolic acid. These triterpenoids were chosen because of the rare unsaturation between the B/C-ring fusion essential for our DOS approach. In general, an unsaturated B/C ring fusion will undergo oxidative cleavage, followed by aldol addition/condensation to reform the carbon-carbon bond to afford skeletally diverse structures. Lanosterol is the parent of most mammalian steroids and is the major product from cyclization of 2,3-oxidosqualene (Figure 1.6, p.

37). It is also naturally abundant and commercially available. Bryonolic acid is strikingly similar in structure to oleanolic acid except that it contains an unsaturated B/C- ring fusion identical to lanosterol. Although not commercially available, bryonolic acid can be extracted and purified from zucchini sprouts (Curcubita pepo L.).271 Before DOS could be applied to bryonolic acid, we had to first develop a large-scale strategy for isolation of bryonolic acid from zucchini sprouts. In addition, due to the lack of biological and mechanistic analysis on bryonolic acid activity, we had to determine whether the compound is able to suppress the inflammatory response and activate Nrf2.

1.5.4 Identification of a Negative Feedback Loop of Biological Oxidant Formation

Regulated by 4-HNE

In addition to the triterpenoids, we extended our studies to an endogenously derived Nrf2-Keap1 modulator, 4-HNE. This project came into fruition when a fellow chemistry graduate student, Sushabhan Sadhukhan, and I began working collaboratively to study a molecule formed during oxidative stress. 4-HNE is a major lipid peroxidation

30 product and an established marker of oxidative stress associated with the pathology of various diseases. Given that 4-HNE is produced when there are high levels of ROS (e.g.

NO), we hypothesized that 4-HNE can activate the Nrf2-Keap1 pathway, activating anti- inflammatory and antioxidant genes, and thus controlling its own production by inhibiting nitric oxide production.

This dissertation begins with an overview of the role of inflammation and oxidative stress in the pathology of diseases with underlying chronic inflammation, followed by a comprehensive overview of the Nrf2-Keap1 signaling pathway, regulation of NF-κB mediated inflammation via activation of Nrf2, small molecule Nrf2 activators, and concluding with our strategy to analyze biological and chemical aspects of Nrf2 activators to identify effective modulators of this pathway. The following chapters

(Chapter 2 – 4) cover our work on triterpenoids, beginning with isolation of bryonolic acid as a starting material for our DOS strategy (Chapter 2: Bryonolic Acid: A Large-

Scale Isolation and Evaluation of Heme Oxygenase 1 Expression in Activated

Macrophages), followed by the activity characterization of bryonolic acid (Chapter 3:

Bryonolic acid transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo), and the application of DOS and biological analysis of bryonolic acid and lanosterol (Chapter 4: Identification of small molecule Nrf2 activators through Diversity-Oriented Synthesis). The work on 4-HNE is covered in Chapter 5

(Chapter 5: Identification of a Negative Feedback Loop in Biological Oxidant Formation

Regulated by 4-Hydroxy-2-(E)-Nonenal), followed by discussion of these works with suggestions for futures studies (Chapter 6: Discussion and Future Work).

31 Figure 1.1. Activation of NF-κB signaling pathway by LPS (1) leads to phosphorylation of IκB by IKK (2) and subsequent ubiquitin mediated proteosomal degradation of NF-κB inhibitor, IκB (3). NF-κB is released from the cytoplasm and translocate in the nucleus

(4) where it binds to DNA κB sites in promoter regions of target genes (5) inducing expression of inflammatory enzymes which include iNOS and COX-2 (6).

32 Figure 1.2. Activation of Nrf2-Keap1 signaling. Under basal conditions Nrf2 is sequestered in the cytoplasm by Keap1, which facilitates Nrf2 ubiquitination and proteosomal degradation (0). Modification of key cysteine residues by electrophiles, oxidative stress, and Nrf2 activators results in the release of Nrf2 from Keap1 (1). Nrf2 translocates in the nucleus (2) and heterodimerizes with small Maf protein(s) (3) which facilitates binding to ARE upstream of Nrf2-target genes and results in their transcriptional activation (4).

33 Figure 1.3. Thiol-reactive, α, β-unsaturated carbonyl that acts as a Michael acceptor crucial for Nrf2 activation.

34 Figure 1.4. Phytochemicals with thiol-reactive motif.

35 Figure 1.5. Oxidation of tert-butylhydroquinone (tBHQ) to thiol-reactive tert- butylquinone (tBQ).

36 Figure 1.6. Cyclization of 2,3 oxido-squalene forms triterpenoids which contain 30 carbons forming 5-6 membered rings.

37 Chapter 2. Bryonolic Acid: A Large-Scale Isolation and Evaluation of Heme

Oxygenase 1 Expression in Activated Macrophages

Barker, E. C*.; Gatbonton-Schwager*, T. N.; Han, Y.; Clay, J. E.;

Letterio, J. J.; Tochtrop, G. P.

Reprinted with permission from J. Nat. Prod. 2010, 73, (6), 1064-1068.

2.1 Abstract

Bryonolic acid (BA) is a triterpenoid found in the Cucurbitaceae family of plants.

Our interests in the immunomodulatory effects of this class of natural products led us to

discover that BA induces a marked increase in the expression of a phase 2 response

enzyme, heme oxygenase 1 (HO-1), in a dose-dependent manner. This phenotype has

translational implications in malarial disease progression, and consequently we developed

a large-scale isolation method for BA that will enable future in vitro and in vivo analyses.

We have determined ideal growth conditions and time scale for maximizing BA content

in the roots of Cucurbita pepo and analyzed BA production by HPLC. Large-scale

extraction yielded 1.34% BA based on dry weight, allowing for the isolation of BA on a

multigram scale.

38 *These authors contributed equally to this work.

2.2 Introduction

Bryonolic acid (3β-hydroxy-D:C-friedoolean-8-en-29-oic acid, BA) is a naturally occurring triterpenoid that has been identified in multiple species of the Cucurbitaceae family and in dispersed species of other reported plant families (Meliaceae,

Tetramelaceae, and Anisophylleaceae).272-275 It was first isolated in 1960 from roots of

Bryonia dioica Jacq. (Cucurbitaceae), with an initial structure reporting a carboxylic acid moiety at C-19 and unsaturation at C-12/C-13.272 A revised structure was subsequently published assigning unsaturation to the C-8/C-9 B−C ring fusion and the carboxylic acid moiety at C-20 (Figure 2.1, p. 49).276

Several biological activities have been reported for BA, including antiallergic properties in rodents277 and a panel of cytotoxic and antitumor activities in various cancer cell lines.278-280 Despite these reports, there has never been an investigation into the molecular underpinnings of these phenotypes. Our laboratory has central interests in how triterpenoid natural products signal through the phase 2 response, an activity that is best represented by the semisynthetic oleanane triterpenoids, which have been implicated in induction of the phase 2 response through the Nrf2:INrf2 (Keap1) signaling pathway.281,

282 In accordance with these interests, we investigated whether the molecular basis of

BA’s reported activities could be related to induction of expression through the phase 2 response.

2.3 Results and Discussion

The broad attention garnered by the oleanane triterpenoids is related to their potent anti-inflammatory activities, which are mechanistically linked to the inhibition of

39 expression of key inflammatory mediators, namely, inducible nitric oxide synthase

(iNOS) and cyclooxygenase-2 (COX-2).283, 284 When we investigated BA in this context, we were surprised that the expression profiles were markedly different (as compared to the oleanane triterpenoids), in that iNOS and COX-2 expression levels were only moderately perturbed (Figure 2.2A, p. 50). The most striking phenotype was a robust induction of heme oxygenase 1 (HO-1) levels. As seen in Figure 2.2B, p. 50, BA elicits robust HO-1 expression in RAW 264.7-treated cells in a dose-dependent manner after a

24 h treatment. HO-1 expression is induced by 3.3-fold and 14-fold compared to LPS control in the presence of 50 and 100 µM BA, respectively. In comparison to untreated cells, treatment with 50 and 100 µM BA increases HO-1 by 13-fold and 55-fold, respectively.

The implications of this observed phenotype have direct relevance to human disease. Plasmodium is a genus of parasites that cause malaria, resulting in more than

500 million infections and one million deaths per year.285 Recent studies have implicated

HO-1 expression as a key therapeutic target in treating malaria. This connection has been rationalized by the enzymatic activity of HO-1 (which converts heme to biliverdin),286-288 in conjunction with the clinical manifestations of malaria being linked to the hemolysis of red blood cells, and subsequent deposition of free heme to the vasculature.289, 290 In addition to this rationalized connection, in vivo studies comparing wild-type and Hmox−/−

(Hmox is the gene encoding HO-1) mice have demonstrated that HO-1 expression protects against the development of the cerebral form of malaria in Plasmodium-infected mice.286

40 To facilitate further studies of the in vitro and in vivo activity of BA, a protocol for robust isolation of BA is needed. To this end we have developed a reliable and scalable method for isolating gram quantities of BA from the roots of Cucurbita pepo L.

(C. pepo L.), which was chosen on the basis of literature precedence in addition to its ready commercial availability. Although BA has been isolated using alternate methods including callus cell culture,291, 292 these methods are most appropriate for analytical scale isolation and biosynthetic studies. The key to our approach is a scalable method for obtaining biomass that is rich in BA content. Our initial step in defining our strategy was to determine if BA production was dispersed throughout C. pepo L. or limited to specific plant tissues. HPLC traces demonstrate that BA production is confined to the fine hairy root structure of C. pepo L. (Figure 2.3, p. 51), which is consistent with previous BA isolation from the root (or radicle) portion of plants and seedlings in Cucurbitaceae.272, 278,

292-297

To address scalability in biomass accumulation, we compared two germination methods that are standard in the field: moist blotting paper and peat-based growth media.

In both cases, seeds and germinations of C. pepo L. were maintained in a medium that retained a moist environment, but did not contribute any level of nutrients to the germinations. For both approaches, roots were isolated every two days and evaluated for

BA content by HPLC. For germinations grown in a peat-based media, root isolation was continued for 40 days. The time-course and HPLC trace overlay show increasing production of BA from day 2 to day 16, peaking at 1.26 mg g-1 dry weight (Figure 2.4A and C, p. 52). BA content subsequently decreased from day 16 to day 40, suggesting that BA presence in the roots is most prevalent during the early germination stage.

41 Decreased BA content after day 16 may also be suggestive of further localization of BA to the fine hairs and extremities of the root system; these grew increasingly delicate with age and became more difficult to recover during the washing process.

In a parallel experiment, germinations were grown between moist blotting paper for 36 days. Germinations were not continued to day 40 as in the peat-based media method due to overgrowth and initial signs of morbidity by day 26. Under these growth conditions, BA content increased from day 2 to day 24 and plateaued thereafter, leveling at approximately 15 mg g-1 dry weight (Figure 2.4B, p. 52). Presumably, BA content changed little after germinations no longer maintained vitality. The increased BA content can be observed by HPLC peak height on select days between day 2 and day 24 (Figure

2.4D, p. 52) and reached a maximum of 16.1 mg g-1 dry weight.

Taken together, BA content in roots from moist blotting paper is roughly 10-fold that detected in roots from peat-based media. Maximum and total BA production per unit mass in roots from blotting paper germinations far outweighed the total amount produced in the peat-based growth media (Figure 2.5A, p. 53). On those days resulting in maximum BA content (day 16 for peat-based, day 24 for blotting paper) the magnitude of

BA production in germinations grown in blotting paper is apparent in the HPLC trace overlays for these respective days (Figure 2.5B, p. 53). In germinations grown in blotting paper, we do not observe a decrease in BA content following achievement of maximum content. This may be due to the capability of retaining 100% of root material when isolated from the blotting paper as opposed to the unavoidable root loss experienced when isolating root material from the peat-based germination media as discussed above.

42 To translate the above analytical scale observations to a large-scale isolation of

BA, we began scaling our blotting paper germinations of C. pepo L. On the basis of the data above, roots were collected from germinations grown between moist blotting paper for 18 days. At this time, germinations remained healthy and we anticipated that BA content was approaching its maximum (Figure 2.4B, p. 52). Lyophilized and powdered roots were combined in a Soxhlet extractor and subjected to a 3-day extraction using a solvent mixture of CHCl3 and MeOH to afford a BA-rich extract. Subsequent silica gel column purification and recrystallization resulted in the isolation of 200 mg of BA from

14.9 g of dry roots (1.34%). The process proved to be further scalable, as the same procedure resulted in isolation of 949 mg of BA from 80.5 g of roots (1.18%). Our results indicate that the method is scalable and multiple gram quantities of BA can be isolated proportionally from increased root masses.

Taken together, this is the first report to show that BA bioactivity is potentially due to induction of expression via the phase 2 response, as illustrated by the robust induction of HO-1 expression. In Chapter 3, we demonstrate that BA is also able to induce HO-1 expression in the absence of LPS (Figure 3.3C and D, p. 75). This phenotype is of clear translational significance, and given the clear importance of malaria, it is axiomatic that future studies of malaria will necessarily include BA. Because plants from the Cucurbitaceae family are grown throughout the world and could serve as an abundant source of the natural product in developing countries where malaria is widespread, BA is likely to become the target of many future biological studies. With the purification strategy reported here, the therapeutic value of this compound can now be carefully explored.

2.4 Experimental Section

General Experimental Procedures

Melting points were measured on an Electrothermal melting point apparatus

(Barnstead/Thermolyne) and are uncorrected. Optical rotation was determined using a

Perkin-Elmer 241 polarimeter. NMR spectra were recorded on a Varian AS400 spectrometer operating at 400/100 MHz (1H/13C). Chemical shifts are reported in ppm

1 13 using residual pyridine-d5 (δ 8.74 for H and 150.4 for C) as internal reference. HPLC separation was performed on an Agilent 1200 HPLC system and ZORBAX Eclipse

XDB-C18 column (5 µm particle size, 80 Å pore size, 4.6 mm i.d. × 250 mm) fitted with a guard column. HPLC operation and data analysis was performed using Agilent

ChemStation for LC 3D systems software, Rev. B.04.01.

Reagents and Chemicals

Cucurbita pepo L. seeds (Spineless Beauty hybrid) were purchased from Siegers

Seed Co. (Holland, MI). The peat-based medium, a mixture of peat, vermiculite, and perlite (Pro-Mix BX, Premier Horticulture), was purchased from a local distributor. All solvents were purchased from Fisher Scientific (Pittsburgh, PA). Pyridine-d5 was purchased from Cambridge Isotope Laboratories (Andover, MA). The leukemic mouse macrophage cells (RAW 264.7) were obtained as a gift from Dr. Michael Sporn from

Dartmouth College. The DMEM medium and PBS were obtained from ATCC

(Manassas, VA) and were supplemented with heat-inactivated fetal bovine serum (FBS) with low endotoxin (≤0.06 EU/mL) from Thermo Fisher Scientific (Waltham, MA). The

44 penicillin−streptomycin, RIPA buffer, Novex 4−20% Tris-glycine gel, and 0.2 µm PVDF membrane were from Invitrogen (Carlsbad, CA). The cells were induced with LPS from

Escherichia coli purchased from Sigma Aldrich (St. Louis, MO) and dissolved in PBS

(ATCC). The Complete Protease Inhibitor cocktail tablet was purchased from Roche

(Indianapolis, IN). The heme oxygenase 1 (HO-1) primary rabbit polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), while the secondary donkey anti- rabbit IgG (H+L)-HRP was from Southern Biotech (Birmingham, AL). The ECL Plus

Western Blot Detection Reagent was purchased from GE Biosciences (Piscataway, NJ).

Germination

Twenty planters (14 × 7 × 4 in3) were filled to 3.5 in. with moistened growing medium. Ninety-six seeds were planted under 1.5 in. of moistened growing medium.

Planters were watered from the bottom for 15 min. Planters were covered until seedlings began to break through the media (day 4). Following sprouting, planters were watered from the bottom for 20 min daily until plants were strong enough to be watered from the top (day 8). Plants were subsequently watered with 500 mL of water every other day for the 40-day growth duration.

Forty seeds were folded between a stack of moist blotting paper. Stacks were paired in a 9 × 12 in2 zip-seal bag, partially unsealed to allow for airflow. Twenty bags were placed in a dark incubator at 25.0 °C for 3 days. Upon germination, the bags were transported to a university greenhouse, where the temperature ranged from 20 to 29 °C over the course of the 40-day growth duration. The bags were rotated each day and kept moist as needed to maintain 100% humidity.

Germination Harvest Procedure

Roots from germinations grown in peat-based media were clipped from the stems and cleared of residual peat mixture in a water bath. Roots were blotted dry and frozen at

−80 °C. Roots from germinations grown in moist blotting paper were clipped from stems and frozen at −80 °C. Stems and leaves from 14-day-old germinations were also separated for verification of anatomical localization of BA.

Calibration for Bryonolic Acid Content

Eight standard BA solutions were made to give concentrations ranging from 25 to

400 µg/mL. A 40 µL amount of each solution was injected into the HPLC in triplicate.

A linear gradient elution was applied at a flow rate of 1.0 mL/min for 20 min from 85% to 100% MeCN in H2O. Both solvents contained 0.02% TFA (v/v). Elution was monitored at 205 nm. The calibration curve for BA content was constructed by plotting the average peak area as a function of the analyte concentration.

Analytical Extraction and HPLC Procedure

Roots, stems, and leaves were dried by lyophilization and ground to a fine powder.

The powder (200 mg) was extracted in 10 mL of MeOH at reflux for 3 h. The filtered extract was brought to a volume of 10 mL with fresh MeOH and analyzed by HPLC, using 40 µL injections repeated in triplicate. For root extracts and evaluation of BA content, a linear gradient elution was applied at a flow rate of 1.0 mL/min for 20 min from 85% to 100% MeCN in H2O. For root, stem, and leaf extracts and evaluation of

46 anatomical localization of BA, a linear gradient was applied at a flow rate of 1.0 mL/min for 60 min from 85% to 100% MeCN in H2O. Both solvents contained 0.02% TFA (v/v).

The system was run at ambient temperature. Elution was monitored at 205 nm. BA was quantified by referencing peak area to the linear calibration generated with standard BA solutions.

Preparative Extraction and Purification Approach

Powdered roots of C. pepo L. germinations (14.9 g lyophilized) were extracted for

3 days by Soxhlet using 700 mL of 2:1 CHCl3−MeOH heated to reflux with stirring. The extract was immobilized on silica gel (1:1) and evaporated to dryness. The crude extract was subjected to a short (2.5 in.) silica gel column (i.d. 1.5 in.), and the column was eluted with 80:20:1 hexanes−EtOAc−HOAc followed by 50:50:1 hexanes−EtOAc−HOAc and then 85:15:1 EtOAc−hexanes−HOAc to yield fractions containing BA and two impurities as observed by TLC. BA-containing fractions were combined, and repeated recrystallization in 40:1 CHCl3−THF resulted in the isolation of

200 mg of BA (1.34%). BA purification from 80.5 g of root extraction was executed similarly, except requiring 5 days for extraction and a larger chromatography column (i.d.

2.5 in).

Bryonolic acid: white powder (CHCl3−THF); mp 274−278 °C (discoloration prior to

25 1 melting began at 246−248 °C); [α]D +18 (c 15, pyridine); H NMR (pyridine-d5, 400

MHz) δ 1.02 (3H, s), 1.06 (3H, s), 1.08 (3H, s), 1.11 (3H, s), 1.23 (3H, s), 1.30 (3H, s),

1.44 (3H, s), 2.50 (1H, m), 2.60 (1H, d, J = 13.6) 2.78 (1H, d J = 15.6), 3.39 (1H, t, J =

13 8.0); C NMR (pyridine-d5, 100 MHz) δ 17.1 (CH3), 18.5 (CH3), 20.1 (CH2), 20.7(CH3),

47 21.6 (CH2), 22.9 (CH3), 26.0 (CH2), 28.5 (CH2), 29.1 (CH3), 29.2 (CH2), 31.0 (CH2), 31.2

(CH2), 31.7 (CH2), 31.8 (C), 32.0 (CH3), 33.9 (CH3), 35.6 (CH2), 36.0 (CH2), 38.0 (CH2),

38.2 (C), 38.3 (C), 39.9 (C), 41.1 (C), 42.7 (C), 45.7 (CH), 51.4 (CH), 78.5 (CH), 134.7

(C), 135.1 (C), 181.8 (C). NMR spectra are shown in the Supporting Information and are consistent with published spectroscopic data for BA.4, 25, 27, 28 Treatment of the isolated compound with diazomethane yields the expected methyl ester by comparison with reported 13C NMR data.29

Biological Data of Bryonolic Acid in RAW 264.7 Cells

RAW 264.7 cells were grown in DMEM medium with 10% FBS and 100 U/mL penicillin−100 µg/mL streptomycin. Cells were cultured in a 37 °C incubator with 5%

CO . The cells were plated at 2 × 106 cells/well in six-well plates and allowed to attach 2 for 2 h. Cells were then induced with 5 ng/mL LPS from E. coli and treated with BA dissolved in DMSO at increasing concentrations (5, 10, 50, 100 µM) for 24 h with a final concentration of 0.25% DMSO in the cells. Cells were lysed using RIPA lysing buffer with a Complete Protease Inhibitor tablet per 10 mL of lysing buffer. The lysates were loaded on a Novex 4−20% Tris-glycine gel, transferred into 0.2 µm PVDF membrane, and blocked with 5% milk for 1 h at room temperature. The membrane was probed for

HO-1 (1:1000 dilution) for 1 h at room temperature and with a secondary donkey anti- rabbit antibody (1:5000 dilution) under the same conditions as the primary antibody. For detection of the bands, the blot was incubated with ECL Plus Western Blot Detection

Reagent for 5 min at room temperature and quantified using a GE Healthcare Typhoon

9400 imager with the Image Quant software for band intensity calculation.

48 Figure 2.1. Structure and numbering of bryonolic acid.

49 Figure 2.2. Immunoblot analysis of induced RAW 264.7 cells treated with increasing concentrations of bryonolic acid for 24 h. (A) RAW 264.7 cells were activated with 5 ng/mL IFNγ and treated with increasing concentrations of bryonolic acid (BA) for 24 h.

Protein lysates were probed for HO-1, iNOS, COX-2 and β-actin. (B) RAW 264.7 cells were activated with 5 ng/mL LPS and treated with increasing concentrations of bryonolic acid (BA) for 24 h. Protein lysates were probed for HO-1 and β-actin. HO-1 protein levels were quantified via densitometry.

50 Figure 2.3. HPLC traces of extracts from the fine hairy root, stem root, and dicotyledon leaf body of 14-day-old germinations. Bryonolic acid is detected only in the root portion of Cucurbita pepo L.

51 Figure 2.4. Bryonolic acid production in Cucurbita pepo L. roots under two growth conditions: peat-based media (A) and moist blotting (B). Bryonolic acid content is observed to increase from day 2 to day 16 in peat-based media and from day 2 to day 24 in moist blotting paper, as observed by growing HPLC peak areas (C and D). At its maximum, BA content in roots from moist blotting paper is roughly 10-fold greater than that detected in roots from peat-based media, as is apparent in the respective scales.

52 Figure 2.5. Comparison of maximum and total BA production in roots from peat-based media versus roots from moist blotting paper (A). Comparison of HPLC peak area for maximum BA production under both conditions (B).

53 Chapter 3. Bryonolic Acid Transcriptional Control of Anti-inflammatory and

Antioxidant Genes in Macrophages in Vitro and in Vivo

Gatbonton-Schwager, T. N.; Letterio, J. J.; Tochtrop, G. P

Reprinted with permission from J. Nat. Prod. 2012, 75, (4), 591-598.

3.1 Abstract

Bryonolic acid (BA) is a naturally occurring triterpenoid with pleiotropic properties. This study characterizes the mechanisms mediating the anti-inflammatory and antioxidant activities of BA and validates the utility of BA as a tool to explore the relationships between triterpenoid structure and activity. BA diminishes the inflammatory mediator NO by suppressing the expression of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 macrophage cells.

In addition, BA robustly induces the antioxidant protein heme oxygenase-1 (HO-1) in vitro and in vivo in an Nrf2-dependent manner. Further analyses of Nrf2 target genes reveal selectivity for the timing and level of gene induction by BA in treated macrophages with distinct patterns for Nrf2-regulated antioxidant genes. Additionally, the distinct expression profile of BA on Nrf2 target genes relative to oleanolic acid suggests the importance of the triterpenoid scaffold in dictating the pleiotropic effects exerted by these molecules.

54 3.2 Introduction

Triterpenoids are one of the most functionally and structurally diverse classes of secondary metabolites ubiquitous in the plant kingdom. Triterpenoids are cyclized from oxidosqualene to form approximately 200 chemically diverse triterpene skeletons. More than 20,000 triterpenoids have been documented, with new structures continually being identified and studied for their biological activity. In addition to the impressive skeletal diversity of these molecules, they also possess a variety of biological activities including anti-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory, and tonic effects.298 One of the best-studied triterpenoids, oleanolic acid (OA) (Figure 3.1, p. 73), served as a platform for the discovery of the semisynthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO).283, 299 CDDO and its methyl ester derivative, CDDO-Me, are potent anti-inflammatory283, 300 and chemopreventive agents282, 301-303 that have advanced to phase III clinical trials for renal sparing effects in diabetic nephropathy.304, 305

We became interested in bryonolic acid (BA) (Figure 3.1, p. 73) in part because of its unique chemical attributes within the triterpenoid family (namely, the unsaturated

B–C ring fusion) and partly due to its interesting pleiotropic profile of biological activity.

The activities reported for BA include anti-allergic properties, inhibition of homologous passive cutaneous anaphylaxis in rats, delayed hypersensitivity in mice,276, 291 antitumor activity,278 and cytotoxicity toward various tumor cell lines.277, 296 Although reports have shown BA to be a promising natural anti-inflammatory agent, the mechanism of action mediating these effects has yet to be identified. We hypothesized that the previously observed BA phenotypes could be explained by the activation of the transcription factor

55 Nrf2.

The NF-E2-related factor 2 (Nrf2) was first isolated as a DNA-binding protein to tandem repeats in the β-globin locus region and had been targeted for prevention of chemical carcinogenesis even before its complete characterization.306 Nrf2 acts as an electrophilic and oxidative damage sensor and induces a battery of cytoprotective genes that detoxify reactive electrophiles and oxidants. Among the hundreds of genes regulated by Nrf2, the most studied include heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), catalase (CAT), glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione reductase (GR). Several classes of endogenous and exogenous ligands induce Nrf2, with triterpenoids being one of the most promising and clinically relevant examples. Upon induction, Nrf2 dissociates from Keap1, the principal cytoplasmic inhibitor of Nrf2 function.307 Nrf2 subsequently escapes ubiquitination and proteosomal degradation, translocates to the nucleus, and effectively upregulates the expression of cytoprotective and antioxidant genes.308

Here we provide the first demonstration of the molecular mechanisms contributing to the anti-inflammatory/anti-allergic effects of BA. Through in vitro analysis of BA activity in mouse macrophages and in vivo studies following systemic administration of BA in mice, we show potent suppression of iNOS expression and a robust induction of HO-1 by BA in an Nrf2-dependent manner. BA induces other antioxidant and cytoprotective genes and triggers a unique expression profile for Nrf2 target genes when compared to a structurally similar triterpenoid, OA. The observed differential regulation by BA of genes in this pathway is characterized by a rapid and more potent induction of HO-1 and NQO1, while other Nrf2 target genes such as CAT,

56 GCLC, and GR respond with a more gradual and modest increase in expression. This newly discovered ability of BA to regulate expression of inflammatory and antioxidant enzymes validates the utility of BA as a platform to explore the importance of the triterpenoid scaffold in defining the anti-inflammatory and chemopreventive properties of triterpenoids. In addition, BA may potentially uncover unique mechanism(s) in regulating the inflammatory and antioxidant pathway in comparison to the oleanane triterpenoids due to its different regulation of antioxidant genes. More specifically, these studies set the stage for an effort combining the application of synthetic chemistry and chemical biology screens that has the potential to yield diverse triterpenoid structures with selective therapeutic properties.

3.3 Results and Discussion

Bryonolic Acid Decreases NO Levels and iNOS Expression in LPS-Activated RAW

264.7 Cells

Initial studies of BA have shown in vivo anti-inflammatory properties in rats and mice by inhibiting the allergic response.276, 291, 309 However, the mechanisms mediating these effects have not been explored. In order to elucidate the anti-inflammatory effects of BA, we used an established in vitro model of LPS-activated RAW 264.7 leukemic mouse macrophage cells (RAW). Upon LPS activation of RAW cells, NO is produced, which spontaneously oxidizes to nitrite. In an initial experiment, LPS-activated RAW cells were treated with BA and nitrite levels were measured from cell culture supernatants. Treatment with BA reduced nitrite levels, demonstrating an IC50 value of

53.3 ± 3 µM after a 24 h treatment (Figure 3.2A, p. 74). RAW cells remained viable in

57 BA concentrations as high as 300 µM, but cytotoxicity was apparent at higher concentrations (gray shaded area), as measured by the MTT assay (Figure 3.2B, p. 74).

This decrease in viability may be attributed to higher DMSO exposure at this dose range

(1.5% at a concentration of 300 µM BA from the maximum soluble stock of 20 mM).

Due to the solubility of BA and possible toxicity from DMSO, the maximum concentration included in the calculated IC50 value and the subsequent cell culture experiments was 100 µM at 0.5% DMSO.

To determine the mechanism through which BA suppresses the production of NO following LPS exposure, we examined the expression profile of iNOS through immunoblot analysis in LPS-activated RAW cells. Treatment with 50 µM BA significantly reduced iNOS protein levels after a 24 h treatment compared to the 0.5%

DMSO control (Figure 3.2C, p. 74). At 100 µM BA, iNOS protein levels are no longer detectable. The iNOS mRNA levels in LPS-activated RAW cells were also reduced in the presence of 50 and 100 µM BA after a 24 h treatment (Figure 3.2D, p. 74). Thus, the decrease of NO production is mediated by the suppression of iNOS expression, as shown by the decrease in both protein and mRNA levels in the presence of BA. To further assess this effect, RAW cells were treated at various time points with 100 µM BA. LPS induced iNOS expression at 4 h, and BA suppressed iNOS levels at this early time point

(Figure 3.2E, p. 74).

58 Bryonolic Acid Induces Antioxidant Heme Oxygenase-1 Expression in RAW 264.7

Cells

Previous data have shown robust induction of HO-1 by other triterpenoids.280, 310

Therefore, we next determined whether BA might also induce HO-1. We observed a dose-dependent induction of HO-1 by BA, with HO-1 protein and mRNA levels induced at 50 µM and 100 µM after 24 h (Figure 3.3A and B, p. 75) in LPS-activated RAW cells.

Since LPS is known to induce HO-1, we next determined whether LPS is a requirement for HO-1 induction by BA. RAW cells were treated with BA in the absence of LPS and probed for HO-1. As seen in Figure 3.3C, p. 75, HO-1 was induced in the presence of

50 and 100 µM BA in the absence of LPS. HO-1 mRNA levels were induced in the presence of 10 µM BA with increasing expression at 100 µM BA (Figure 3.3D, p. 75).

To investigate the time course of HO-1 induction, RAW cells treated with 100 µM BA were harvested at different time points and probed for HO-1. BA induced HO-1 protein levels as early as 6 h, and expression peaked approximately 24 h after treatment. The induction extends beyond 48 h and is diminished by 72 h (Figure 3.3E, p. 75). This profile for HO-1 induction illustrates a long-term induction by BA amounting to a total of approximately 66 h. HO-1 mRNA levels were also induced at 4 h with a 4.1-fold change compared to control at time = 0 (Figure 3.4, p. 76). HO-1 mRNA levels continued to increase up to 20 h, at which time the peak induction demonstrated a 11.3-fold change in mRNA levels compared to control.

Bryonolic Acid Induces Nrf2 Target Genes

The induction of HO-1 by BA led us to question whether BA could also induce

59 other phase 2 genes. Previous data showed an inverse correlation between the expression of the inflammatory gene iNOS and phase 2 genes in triterpenoid-treated cells.280 Several phase 2 genes were probed including HO-1, NQO1, CAT, GR, and GCLC at various time points in BA-treated RAW cells (Figure 3.4, p. 76). Since Nrf2 also controls its own expression, we initially probed for Nrf2 mRNA levels. Nrf2 was induced 1.8-fold at 20 h in 100 µM BA-treated cells compared to control. Significant induction in expression for all of the genes occurred at the 20 h time point. NQO1 expression was significantly induced, demonstrating an 11.7-fold increase, similar in magnitude to the 11.3-fold increase in HO-1 mRNA levels. CAT was induced 1.7-fold at 16 h and almost doubled to 3.5-fold induction at 20 h. GCLC was induced 1.8-fold at 20 h and decreased to basal levels by 24 h. This pattern of induction is also similar for GR, which was induced 1.5- fold at 20 h and decreased to basal levels by 24 h.

Unique Dose–Response Profiles for Bryonolic Acid and Oleanolic Acid for NO

Suppression and HO-1 Induction

The numerous biological activities of triterpenoids led us to compare BA with the well-studied and structurally similar triterpenoid OA. We compared the ability of OA and BA to suppress NO production in RAW cells. Nitrite levels decreased 85% from control in OA-treated cells and 72% in BA-treated cells (Figure 3.5A, p. 77). Studies of

OA have led to the discovery of several potent synthetic triterpenoids, and selected OA derivatives are now in late phase clinical trials.311, 312 However, when we compared the ability of both naturally occurring triterpenoids to induce HO-1, we found that BA is considerably more effective than OA (Figure 3.5B, p. 77). At 50 µM, there is greater

60 HO-1 induction in BA-treated cells compared to OA-treated cells after 24 h. BA’s potency for inducing HO-1 is more apparent at the higher concentration of 100 µM. In addition, we compared the potency of HO-1 induction by BA with the structurally similar triterpenoids ursolic acid (UA), betulinic acid, boswellic acid, and glycyrrhetinic acid

(Figure 3.6, p. 78). We found that BA induces HO-1 more potently compared to ursolic acid and betulinic acid, but similarly to boswellic acid and glycyrrhetinic acid after 8 h of treatment. Interestingly, BA is more potent at inducing HO-1 mRNA levels at the earlier

4 h time point compared to all of these triterpenoids (2–6). We noted that unlike BA and

OA, UA, betulinic acid and glycyrrhetinic acid are both toxic to cells as early as 8 h after treatment.

Bryonolic Acid Induces Translocation of Nrf2 into the Nucleus

The induction of HO-1 is regulated by the transcription factor Nrf2. In the presence of an inducer, Nrf2 is released from its cytoplasmic inhibitor Keap1 and is translocated into the nucleus, where it then binds to genes containing antioxidant response element (ARE) sites and induces transcription of the antioxidant phase 2 enzymes. In order to determine the mechanism of HO-1 induction by BA, we examined whether BA is able to promote translocation of Nrf2 into the nucleus. We treated RAW cells with 50 and 100 µM BA at various time points and probed both cytoplasmic and nuclear fractions for Nrf2. We observed that exposure to BA decreased cytoplasmic Nrf2 in treated cells as early as 1 h (Figure 3.7, p. 79), and this decrease was more evident in the presence of 100 µM BA. This reduction in cytoplasmic Nrf2 was more evident in

BA-treated cells from 1 to 4 h after treatment. Nrf2 accumulates in the nucleus in both

61 BA- and OA-treated cells and remained nuclear throughout the time course, whereas less accumulation was observed in the control. These results show that BA is more potent at inducing Nrf2 nuclear translocation when compared to OA.

Differential Regulation of Nrf2 Target Genes by Bryonolic Acid

We next determined whether the observed enhanced BA-induced Nrf2 activation relative to OA correlates with distinct expression profiles for Nrf2 target genes.

Quantitative RT-PCR analyses revealed peak HO-1 induction in OA-treated cells beginning 2–4 h after treatment, while peak HO-1 induction in BA-treated cells occurred

16–20 h after treatment (Figure 3.8A, p. 80). HO-1 expression levels in OA-treated cells decreased to basal levels, while HO-1 expression levels in BA-treated cells remained elevated at later time points as compared with OA. In addition, another prototypical target of Nrf2, NQO1, was induced in a similar manner to HO-1 (Figure 3.8B, p. 80), while the expression profiles of CAT and GR were disparate in BA- and OA-treated cells.

Whereas OA-treated cells showed a peak induction followed by a steep decline in gene expression, BA-treated cells demonstrated only a gradual increase in expression of these genes over the entire time course (Figure 3.8C and E, p. 80). This differential regulation exerted by BA does not affect non-Nrf2 regulated genes, β-actin, or GAPDH (Figure 3.9, p. 81). Taken together, these data show that subtle structural differences between triterpenoids with a similar carbocyclic skeleton affect the ability of these small molecules to modulate expression of the Nrf2 target genes in a different manner.

62 Bryonolic Acid Activity in Primary Macrophage Is Dependent on the Nrf2-Keap1

Pathway

In order to test the requirement for Nrf2 in the BA induction of HO-1, we performed a series of experiments in primary peritoneal macrophages and probed for HO-

1. Consistent with the observed activity in the RAW cell line, BA induced HO-1 expression in a dose-dependent manner in primary peritoneal macrophages, and it did so with greater potency (induction observed at 10 µM BA in primary macrophages in comparison with 50 µM in the RAW cells; Figure 3.10A, p. 82). However, when we treated primary macrophages from Nrf2-deficient mice (Nrf2-/-), BA no longer induced

HO-1. Thus, the induction of HO-1 by BA is dependent upon the Nrf2-Keap1 pathway.

Bryonolic Acid Induces HO-1 in Vivo in an Nrf2-Dependent Manner

In order to determine whether this demonstrated capacity of BA to induce HO-1 can be exerted following systemic exposure to BA in vivo, wild-type mice were treated with 500 mg/kg BA by intraperitoneal (ip) injection and sacrificed 8 h after treatment.

Mouse livers were then harvested, as hepatocytes have been reported to exhibit highly inducible HO-1 expression.313 Mouse livers that had been homogenized and probed for

HO-1 after 8 h of BA treatment showed significant induction of HO-1, as determined by immunoblot analysis (Figure 3.10B, p. 82). The same experiment performed in Nrf2-/- mice similarly demonstrated that in the absence of an intact Nrf2-Keap1 pathway, BA was unable to induce HO-1 in vivo. Taken together, these data show that BA potently induces HO-1 in a manner dependent on the Nrf2-Keap1 pathway.

63 Our goal in this study was to understand the molecular mechanism of the anti- inflammatory activity of BA and to validate the use of BA as a platform for studies designed to explore the relationship of structure to the pleiotropic effects of triterpenoids.

This is the first definitive report demonstrating a molecular mechanism through which

BA exerts potent anti-inflammatory activity, by reducing NO levels via suppression of iNOS expression as shown in LPS-activated macrophages, in a dose- and time-dependent manner. We previously reported that BA induces HO-1 in vitro in LPS-activated RAW cells314 and herein report the induction of HO-1 expression in a dose-dependent and time- dependent manner independent of LPS. In addition, we show that BA induces HO-1 in an Nrf2-dependent manner in primary mouse macrophages and in hepatocytes in vivo following systemic administration of BA. It is possible that BA is metabolized in the liver prior to reaching additional organs. This could explain why we did not observe significant HO-1 induction in the spleen, lung, or brain. Administration of BA at higher concentrations or via a different route may induce HO-1 in organs other than the liver.

These data, together with the observed induction of antioxidant genes, provide a mechanism to explain how BA exerts the previously reported anti-allergic and anti- inflammatory properties observed in preclinical models in mice and rats.276, 291 More importantly, the observed effects of BA on the production of iNOS and HO-1 are consistent with the reported role of these molecules in allergy and inflammation as highlighted in several studies. For example, iNOS expression is significantly increased after an allergen challenge in a preclinical anaphylaxis mouse model315 and is highly expressed in several forms of dermatoses in humans.316 Furthermore, induction of HO-1 inhibits allergic inflammation in mice317 and in humans,318 and it is intriguing that the

64 anti-allergic properties of several molecules have been attributed to an effect on HO-1 expression or activity.319 Although the anti-inflammatory activity of HO-1 is established, further studies are required to determine how HO-1 contributes to the anti-inflammatory activity of BA .320-322

Surprisingly, BA exhibits a unique expression profile compared with OA, inducing the characteristic robust HO-1 expression while yielding a different expression profile of the Nrf2 target genes from the structurally similar OA. In comparison to other structurally similar triterpenoids ursolic acid, betulinic acid, boswellic acid, and glycyrrhetinic acid, BA induces HO-1 mRNA at an earlier time point (Figure 3.6, p. 78).

In the absence of Nrf2, BA failed to induce HO-1, suggesting that BA acts primarily through this pathway. This is further corroborated by an analysis of Nrf2 nuclear translocation in which we observed a marked decrease in cytoplasmic Nrf2 and an increase in nuclear accumulation of Nrf2 in BA-treated cells. The chemopreventive and chemotherapeutic effects of the oleanane-derived semisynthetic triterpenoids (CDDO and

CDDO derivatives) have been attributed to the potent induction of HO-1- and the Nrf2- dependent genes.161, 310, 313, 323-328 The synthetic effort leading to the discovery of CDDO originated from improvement upon the weak anti-inflammatory activity of OA. Since

BA is more potent in comparison with OA at inducing HO-1, combined with the extended gradual induction of other Nrf2-dependent genes, we can anticipate BA to be an excellent platform for the development of potent anti-inflammatory and chemopreventive agents.

While one might expect that targeting the Nrf2 pathway with any triterpenoid would result in similar upregulation of antioxidant and cytoprotective genes,313, 329, 330 our

65 studies indicate that the minimal structural differences between the BA and OA triterpenoids influence their capacity to modulate a similar set of target genes. There are multiple interpretations for the observed HO-1 phenotype and the unique expression profile of BA. One possibility is that BA is targeting a pathway different from Nrf2, which results in the difference of gene expression between BA and OA. However, the mechanistic studies in which we explored BA effects in primary mouse macrophages in vitro and in hepatocytes in vivo show that BA failed to induce HO-1 without an intact

Nrf2 pathway. These data support the conclusion that the observed BA phenotype is mediated through the Nrf2 pathway. A second interpretation is that the primary target of

BA is Nrf2, but there exists structural specificity that dictates the differential regulation of Nrf2 target genes. There is precedence for this interpretation, as several ligands including OA have been shown to bind to the farnesoid x receptor (FXR) and selectively modulate the expression of specific target genes important for bile acid regulation.331

Our results suggest that BA may be acting in a similar fashion while targeting the Nrf2 pathway. In addition, although Nrf2 is a key transcription factor for the induction of antioxidant genes, several studies have shown differences in the regulation of these genes.

Small Maf accessory proteins that heterodimerize with Nrf2 have been shown to play a role in the differential regulation of HO-1 and NQO1.332, 333 However, further studies are required to identify the cognate binding partner of BA and to determine the molecular underpinnings of the observed expression profile.

It has been suggested that inducers of the phase 2 response are also suppressors of inflammation. Triterpenoids have been shown to coordinately regulate markers of inflammation and phase 2 response and that inducer potency is a reliable predictor of

66 anti-inflammatory activity.280, 334 However, it is interesting to note that this is not the case for the observed BA phenotype, where HO-1 induction is more potent and suppression of iNOS is weaker in BA-treated cells compared with OA. Further mechanistic studies on BA will hopefully uncover novel regulation of both the phase 2 and inflammatory pathway by triterpenoids.

Translationally, the capacity of BA to induce HO-1 and other target genes could be leveraged through the use of BA as a platform for developing novel selective modulators of Nrf2-dependent gene expression. By fully exploring the triterpenoid scaffold, there is potential to unlock the full potential of triterpenoids as selective inflammatory regulators. Such an effort could lead to the design of synthetic triterpenoids that may modulate expression of specific genes in a selected disease context.

For example, the HO-1 phenotype observed in vivo has clear translational implications in the context of malaria, caused by infection of the Plasmodium genus of parasites. Many of the clinical manifestations of infection by Plasmodium are directly linked to the hemolysis of red blood cells and release of hemoglobin and the effects of hemoglobin degradation products. Hemoglobin released from red blood cells is oxidized by reactive oxygen species (ROS)335 during inflammation, resulting in free heme which is deleterious to the host. In addition, hemoglobin serves as an amino acid source for the parasite.336, 337

HO-1 plays an important role in modulating the inflammatory response by breaking down the deleterious heme and produces anti-inflammatory byproducts, effectively protecting the host from developing cerebral and noncerebral forms of malaria.286, 338 The importance of this mechanism is supported by the observation that Plasmodium infection leads to rapid hepatic failure and lethality in mice with a targeted disruption of the HO-1

67 gene.285 Thus, host survival in this context is dependent on the capacity to upregulate the

HO-1 enzyme.288 Ultimately, further studies will set a foundation for in-depth analyses of the triterpenoid scaffold and how this may be manipulated to generate potent and selective modulators of inflammation.

3.4 Experimental Section

Materials

C57BL/6J mice (wild-type) were purchased from Jackson Laboratory (Bar Harbor,

ME, USA), and the Nrf2-knockout (Nrf2-/-) mice on C57BL/6J background were purchased from RIKEN BioResource Center (Tsukuba, Japan). BA was isolated from the roots of Cucurbita pepo L. as previously reported,314 and OA was purchased from Tokyo

Chemical Industry (Tokyo, Japan). Stocks were made fresh in DMSO. Ursolic acid, betulinic acid, boswellic acid, glycyrrhetinic acid, DMSO, Cremophore EL, and LPS from E. coli were purchased from Sigma (St. Louis, MO, USA). All the primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and secondary antibodies from Southern Biotech (Birmingham, AL, USA). DMEM and

RPMI media were purchased from GIBCO (Grand Island, NY, USA) and then supplemented with low endotoxin FBS (<0.06 EU), obtained from Thermo Scientific

(Logan, UT, USA). The Trypan blue, penicillin/streptomycin, Griess assay reagents,

TRIZol reagent, PureLink RNA Mini Kit, SuperScript One-Step RT-PCR, SuperScript III

First-Strand Synthesis System for RT-PCR, RIPA buffer, 0.2 µm PVDF membrane,

Novex 4–20% tris glycine gels, and running and transfer buffers were all purchased from

Invitrogen (Grand Island, NY, USA). The protease cocktail inhibitor tablet was

68 purchased from Roche (Indianapolis, IN, USA), and MTT cell proliferation assay kit from ATCC (Manassas, VA, USA). TaqMan Fast Universal PCR Master Mix was obtained from Applied Biosystems (Foster City, CA, USA). Thioglycollate for primary macrophage stimulation was purchased from Becton Dickinson (Sparks, MD, USA) and

ECL Plus from Amersham (Buckinghamshire, UK). PBS was obtained from Cellgro by

Mediatech, Inc. (Manassas, VA, USA). Autoradiography film was purchased from

MidSci (St. Louis, MO, USA).

Cell Culture

The leukemic mouse macrophage cells (RAW 264.7) were obtained as a gift from

Dr. Michael Sporn (Dartmouth College, NH, USA), cultured in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin, and kept in culture at

37 °C in a 5% CO2 environment. Cells were kept in culture for no longer than a month and routinely checked for LPS responsiveness every few passages via detection of NO production measured using the Griess assay.

NO Measurement

RAW 264.7 cells were plated (1 × 105 cells/well) in a 96-well plate and allowed to attach for several hours before activation with 5 ng/mL LPS. LPS-activated cells were treated with varying concentrations of BA (1–1000 µM) for 24 h. NO levels were measured via Griess assay using 100 µL of Griess reagent with 100 µL of cell culture supernatant. Absorbance was read at 550 nm using the Sunrise plate reader by TECAN

69 (Mannedorf, Switzerland). IC50 values were calculated by fitting a nonlinear sigmoidal variant slope curve to the data using Prism 5.0 software by Graphpad Inc.

Toxicity Measurement

The MTT cell proliferation assay kit was used according to the manufacturer’s specifications to measure toxicity of BA-treated RAW 264.7 cells after 24 h. The Trypan blue exclusion test was used in Figure 3.11, p. 83.

RAW 264.7 Cell Treatment

RAW 264.7 cells were plated at 4 × 106 cells/60 mm plate and allowed to attach for 2 h. Cells were activated with 5 ng/mL LPS and immediately treated with varying concentrations of triterpenoids (maintaining 0.5% DMSO), triterpenoids alone without

LPS activation, or 0.5% DMSO control. Cells were harvested for immunoblot analysis after a 24 h treatment.

Primary Peritoneal Macrophage Isolation and Treatment

Prior to primary peritoneal macrophage harvest, 6–8-week-old C57BL/6J and

Nrf2-/- (C57BL/6J background) mice were injected with 2 mL of 4% thioglycollate via ip.

Primary peritoneal macrophages were collected with PBS, plated in 60 mm plates (3 ×

106 cells/plate) in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin, and allowed to attach for 2–3 h. Prior to LPS activation and BA treatment, cells were washed with PBS 3 or 4 times to remove non-macrophage cells.

70 Cells were treated with varying concentrations of BA (5, 10, 50, or 100 µM) or DMSO control. Cells were harvested for immunoblot analysis after a 48 h treatment.

Immunoblot Analysis

Cells and homogenized tissues were lysed with RIPA buffer containing protease inhibitors. Lysates were probed for HO-1 and iNOS using a 1:1000 primary antibody dilution for RAW 264.7 cells and primary macrophage cells and a 1:500 HO-1 antibody dilution for tissue lysates. Secondary antibodies (1:5000 dilution) were detected using

ECL plus with autoradiography.

RT-PCR Analysis

Total RNA from the treated cells was extracted and purified using TRIZol reagent. cDNA was synthesized and PCR reactions were performed using Superscript One-Step

RT-PCR. Primer sequences for RT-PCR analysis for iNOS and HO-1339 and CAT,

GCLC, GR, NQO1, and β-actin were adopted from a previous publication.340 The conditions were used accordingly: 55 °C for 30 min for reverse transcription, 94 °C for 2 min for predenaturation, followed by 30 cycles of 94 °C for 30 s for denaturing, 55 °C for

30 s for annealing, and 72 °C for 1 min for extension, followed by one cycle of 72 °C for

10 min for final extension.

Quantitative Real-Time PCR Analysis

Total RNA from treated cells was isolated with the PureLink RNA Mini Kit and converted to cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR.

71 TaqMan Fast Universal PCR Master Mix was used for real-time RT-PCR with mouse- specific Taqman gene expression assay from Applied Biosystems. The TaqMan PCR primers and probes used were as follows: iNOS (Mm01309902_m1), HO-1

(Mm00516007_m1), NQO1 (Mm00500821_m1), catalase (Mm00437992_m1), GCLC

(Mm00802655_m1), GR (Mm00833903_m1), and 18S rRNA (Hs99999901_s1) as a control. Amplification was performed using the 7500 Fast Real-Time PCR system and the 7500 Fast System SDS Software-Sequence Detection software version 1.3.1.21 by

Applied Biosystems. The assay used for the study was the relative quantification assay

(ΔΔCt) using the Run mode Fast 7500 profile (95 °C for 20 s, followed by 40 cycles of

95 °C for 3 s and 60 °C for 30 s).

In Vivo BA Administration

Both the C57BL/6J and the Nrf2-/- mice were treated with either a single dose of

500 mg/kg BA (dissolved in 80% PBS/10% DMSO/10% Cremophore) or vehicle control administered by ip injection. Mice were sacrificed, and liver tissue was harvested 8 h following BA administration. All experiments were performed in accordance with an approved protocol by the Institutional Animal Care and Use Committee at Case Western

Reserve University.

72 Figure 3.1. Bryonolic acid and oleanolic acid structures.

73 Figure 3.2. Bryonolic acid (BA) decreases NO levels and inhibits iNOS expression in

RAW 264.7 cells in a dose-dependent and time-dependent manner. RAW 264.7 cells were activated with 5 ng/mL LPS and treated with varying concentrations of BA for 24 h

(A–D) or varying time points (E). (A) Nitrite levels were measured via Griess assay in

LPS-activated cells treated with BA for 24 h. (B) RAW 264.7 cells were treated with varying concentrations of BA for 24 h, and the viability was measured by the MTT assay.

(C) iNOS protein levels were quantified through immunoblot analysis in LPS-activated cells treated with varying concentrations of BA for 24 h. (D) iNOS mRNA levels were measured by RT-PCR. (E) iNOS protein levels were quantified through immunoblot analysis in LPS-activated cells treated with 100 µM BA or DMSO control at various time points.

74 Figure 3.3. Bryonolic acid (BA) induces HO-1 expression in RAW 264.7 cells in an

LPS-independent manner. RAW 264.7 cells were activated either with 5 ng/mL LPS (A,

B) or without (C, D) with varying concentrations of BA for 24 h (A–D) or varying time points (E). (A, C, and E) Immunoblot analysis of HO-1 protein levels. (B and D) mRNA level measurement of HO-1 by RT-PCR.

75 Figure 3.4. Bryonolic acid (BA) induces Nrf2 and its target genes. RAW 264.7 cells were treated with BA at different time points. qRT-PCR was performed probing for Nrf2 and Nrf2 target genes including catalase (CAT), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione reductase (GR), heme oxygenase-1 (HO-1), and NAD(P)H dehydrogenase quinone 1 (NQO1) at varying time points in cells treated with 100 µM BA.

76 Figure 3.5. Activity comparison of bryonolic acid (BA) versus oleanolic acid (OA) at reducing nitrite levels and inducing HO-1. RAW 264.7 cells were treated with various concentrations of BA or OA for 24 h. (A) Nitrite levels were measured via the Griess assay. (B) Immunoblot analysis of HO-1 in the whole cell lysates of triterpenoid-treated cells.

77 Figure 3.6. Comparative analyses of induction of HO-1 expression by triterpenoids. (A)

Structurally similar triterpenoids were compared with bryonolic acid (BA) for their ability to induce HO-1 expression. RAW 264.7 cells were treated with 100 µM BA, oleanolic acid (OA), ursolic acid (UA), betulinic acid, boswellic acid, or glycyrrhetinic acid (glycyr. acid). (B) HO-1 protein levels were quantified through immunoblot analyses in cells treated with triterpenoids for 8 h. HO-1 mRNA levels were quantified via qRT-PCR in cells treated for 4 h (C) or 8 h (D).

78 Figure 3.7. Immunoblot analysis of cytoplasmic and nuclear Nrf2 in bryonolic acid

(BA)- and oleanolic acid (OA)-treated RAW 264.7 cells. RAW 264.7 cells were treated with 50 µM and 100 µM BA or OA at 1, 2 and 4 h and probed for nuclear and cytoplasmic Nrf2.

79 Figure 3.8. Bryonolic acid (BA) differentially induces Nrf2 target genes compared with oleanolic acid (OA). RAW 264.7 cells were treated with 100 µM BA or OA at different time points. qRT-PCR was performed to measure HO-1 (A), NQO1 (B), CAT (C),

GCLC (D), and GR (E) expression at various time points.

80 Figure 3.9. Bryonolic acid (BA) selectively regulates Nrf2 target genes. RAW 264.7 cells were treated with 100 µM BA at different time points. qRT-PCR was performed to measure β-actin and GAPDH.

81 Figure 3.10. Bryonolic acid (BA) induces HO-1 in primary peritoneal macrophages and liver and is dependent on the Nrf2 pathway. (A) Primary peritoneal macrophages harvested from Nrf2 wild-type (Nrf2+/+)or Nrf2 knockout (Nrf2-/-) (C57BL/6J background) mice were treated with varying concentrations of BA for 48 h. Immunoblot analysis of HO-1 protein levels in BA-treated primary macrophages. (B) Immunoblot analysis of HO-1 protein levels in liver tissues of Nrf2 wild-type (upper panel) or Nrf2 knockout (lower panel) mice treated with 500 mg/kg BA or vehicle for 8 h by ip.

82 Figure 3.11. Positive and negative control of bryonolic acid (BA)-treated RAW 264.7 cells. RAW 264.7 cells were treated with 5 ng/mL LPS or 100 µM BA for 24 h.

Immunoblot analysis of iNOS protein levels with β-actin as loading control (A) and cell viability measurement via tryphan blue exclusion test (B).

83 Chapter 4. Identification of Small Molecule Nrf2 Activators through Diversity-

Oriented Synthesis

Gatbonton-Schwager, T. N.; Han, Y; Ignatenko, V. A.; Letterio, J. J.; Tochtrop, G. P

4.1 Abstract

The immune system provides a cellular defense mechanism mediated by the Nrf2-

Keap1 signaling pathway against harmful agents. Among the most potent Nrf2 activators are synthetic oleanane triterpenoids (SOTs), which are potent antioxidant inflammation modulators (AIMs) that exhibit preventive and therapeutic properties against a variety of inflammation-mediated chronic diseases. Efforts in development of SOTs from the natural product oleanolic acid (OA) have been limited to iterative modifications around the triterpenoid skeleton. Therefore we wanted to explore the relationship between the triterpenoid skeleton and activity. Utilizing structural homologs of OA, bryonolic acid and lanosterol as a platform for Diversity Oriented Synthesis (DOS), we report efficient rearrangement of the carbocyclic triterpenoid skeleton, yielding structurally diverse molecules with a wide-range of anti-inflammatory activity quantified by reduction of nitric oxide (NO) levels in LPS-activated leukemic mouse macrophages. The linear derivatives are most active at suppressing NO production, with IC50 values in the micromolar range. Further A-ring modifications resulted in nanomolar anti-inflammatory activity. Mechanistic analyses demonstrated that the derivatives decreased NO production through transcriptional inhibition of iNOS expression and are effective Nrf2 activators. We demonstrated that the core triterpenoid skeletal structure plays an

84 important role in dictating activity, and that DOS is a valid approach for gaining access to effective structurally diverse Nrf2 activators, which would not have otherwise been attainable through the traditional medicinal chemistry approach.

4.2 Introduction

Inflammation is an important component of the body’s defense mechanism against endogenous and exogenous insults that give rise to tissue injury, stress or malfunction. This process is mediated by the transcription factor NF-κB that coordinates activation of immune cells and release of inflammatory mediators to rid the body of the inflammatory stimulus and return the tissue to homeostasis. Overabundance of inflammatory mediators such as reactive oxygen and nitrogen species (ROS/RNS) during dysregulation of the inflammatory process results in oxidative stress that leads to chronic inflammation, a condition which has been linked to neurodegenerative diseases,341 obesity,342 cardiovascular disease343 and cancer.344 Therefore, it is crucial to have a carefully regulated inflammatory response to properly defend against harmful insults and to prevent excessive damage.

Nrf2 is a transcription factor that regulates hundreds of anti-inflammatory, antioxidant, and cytoprotective genes including heme-oxygenase 1 (HO-1) and

NAD(P)H:quinone oxidoreductase 1 (NQO1). Under basal conditions, Nrf2 is sequestered in the cytoplasm by its transcriptional inhibitor, Keap1 and is targeted for ubiquitin-mediated degradation. During oxidative stress or in the presence of Nrf2 activators, modification of key cysteine residues in Keap1 results in the release of Nrf2, allowing it to translocate in the nucleus where it heterodimerizes with regulator proteins and binds to electrophilic response elements (EpRE) or antioxidant response elements

85 (ARE) located in the promoter region of anti-inflammatory, antioxidant and cytoprotective genes. The importance of the cytoprotective properties of Nrf2 has been demonstrated in mouse models of inflammation. For example, one study established that

DSS-induced colitis in Nrf2 knockout (Nrf2-/-) mice resulted in an increase of iNOS expression and overabundance of pro-inflammatory cytokines and chemokines.190, 191, 198

Many more studies on Nrf2-/- mice clearly demonstrate that deletion of Nrf2 results in increased susceptibility of mice to toxins,51-53 oxidative stress,54-56 inflammation,57, 58 and inflammation-mediated diseases such as cancer,59-61 thus demonstrating the important protective roles of Nrf2. Activation of Nrf2 by plant-derived and synthetic activators downregulates NF-κB-mediated inflammation via transcriptional suppression of inflammatory enzymes such as iNOS, and prevents the overproduction and secretion of pro-inflammatory cytokines.192, 193, 196, 199 Therefore, Nrf2 activation prevents disease progression, is an alternative approach to regulate inflammation, and is an important therapeutic target for prevention and amelioration of diseases driven by inflammation.

The molecular details of Nrf2 activation are rationalized by either oxidation or chemical adduction of cysteine residues of Keap 1 by oxidants or electrophiles generated during oxidative stress. One of the most promising classes of Nrf2 activators is the SOTs which have anti-inflammatory, anticancer and cytoprotective activities in a variety of tumor cells and animal models.53, 213, 241, 243-260 The initial therapeutic promise of synthetic triterpenoids led us to determine whether the carbocyclic triterpenoid structure is important for the therapeutic properties observed in this class of molecules. Derived from naturally occurring oleanane triterpenoids, iterative changes to the functionality around the pentacyclic ring structure lead to increased potency of Nrf2 activation. We

86 hypothesize that rearranging the core carbocyclic triterpenoid structure will yield novel small molecule Nrf2 activators and that triterpenoid activity is not limited to the steroid skeletal arrangement. To test our hypothesis, we utilized a DOS approach to rearrange the core skeletal triterpenoid structure in 3-4 synthetic steps to access chemical space, which would have not otherwise been attainable through a traditional medicinal chemistry approach. We chose lanosterol and bryonolic acid as platforms for our DOS approach because they contain a rare un-saturation between the B/C ring fusion that is a requirement for the oxidative/cleavage and aldol addition/condensation DOS strategy. A total of 27 skeletally diverse triterpenoids were synthesized, 14 from 5-membered ring bryonolic acid and 13 from 4-membered ring lanosterol. To initially assess their anti- inflammatory activity, we screened the derivatives for their ability to suppress nitric oxide production in an established in vitro model of LPS-activated leukemic mouse macrophage cell line.

The DOS strategy generated skeletally diverse molecules with a wide range of anti-inflammatory activity, with the linear derivative being the most active. This initial screen demonstrates that the core triterpenoid structure plays an important role in dictating the activity of triterpenoids. Several structures were chosen to further improve activity by utilizing chemical modifications on the A-ring previously demonstrated to increase potency of triterpenoid activity. Mechanistic analysis of the modified derivatives demonstrates that suppression of NO is due to their ability to suppress iNOS, which is the rate-limiting step of NO production. In addition, the derivatives induce transcription of Nrf2 target genes HO-1 and NQO1. Our results show that the therapeutic activity of triterpenoids is not limited to the steroid triterpenoid arrangement, but rather,

87 that there is also something inherently important about the core skeletal structure that contributes to activity. Furthermore, our work validates the DOS approach for identifying novel Nrf2 activators using few synthetic steps. Here we explored the utility of the DOS approach to gain access to chemical space, which would not have been attainable via the traditional medicinal chemistry approach.

4.3 Results and Discussion

Diversity Oriented Synthesis Chemistry approach on lanosterol and bryonolic acid

To determine whether the steroid skeleton is a requirement for triterpenoid activity, we rearranged the core carbocylic structure of bryonolic acid and lanosterol through a DOS strategy followed by biological analysis of anti-inflammatory and antioxidant activities of the skeletally diverse derivatives. Lanosterol and bryonolic acid were used as platforms for DOS approach because they contain a rare unsaturation between the B/C ring fusion that is a requirement for oxidative/cleavage and aldol addition/condensation DOS strategy. We protected lanosterol and bryonolic acid with acetylation of the hydroxyl group at C–3 and hydrogenation C–24/25 unsaturation of lanosterol (1) and methylation at C–29 and acetylation at C–3 of bryonolic acid (2)

(Figure 4.1, p. 98–99 and Figure 4.2, p. 100–101). C–7 and C–11 positions are oxidized345 and then subjected to oxidative cleavage–aldol addition/condensation sequence. Depending on the oxidation state of the molecule (1, 2, 3, 15, 16, or 17), the resultant decane–poly(one)s 4–6 and 18–20 will have different aldol addition and condensation products 7–14, and 21–29. Rearrangement of the ring structure resulted in diverse structures with a 6/5/7/5 ring system (7, 8, 10–12), 6/7/5/6/6 ring system (21, 22,

28), 6/5/7/6/6 (23, 25, 29) and linear derivatives (9, 13, 14, 24, 26).

Triterpenoid cell-based anti-inflammatory activity screen

During the inflammatory response, reactive oxygen species (ROS) such as NO are produced which facilitates the removal of the inflammatory stimulus such as in a bacterial-driven inflammation. To initially assess the activity of the skeletally diverse triterpenoids obtained from the DOS strategy, we used an established in vitro inflammation model of LPS-activated leukemic mouse macrophage, RAW 264.7 (RAW).

Activation of RAW cells produces NO, which spontaneously oxidizes to nitrites, therefore serving as an NO indicator346, which is quantifiable via spectrophotometrical measurement using a Griess assay. In an initial screen for activity, LPS-activated RAW cells were treated with triterpenoids and nitrite levels were measured from cell culture supernatants. Treatment with the skeletally diverse triterpenoids reduced nitrite levels, demonstrating a wide range of activity with IC50 values as low as 7.4 µM compared to inactive parent molecules 1 and 15 (Table 4.1, p. 102). With the exception of 2 derivatives, the majority of the derivatives are non-toxic as demonstrated by > 80% cell viability measured via MTT assay (Table 4.2, p. 103). Oxidation at C7 resulted in 2 and

16, which remained inactive, while further oxidation at C11, which produced diketone 3 from lanosterol, resulted in an active compound with an IC50 of 53.2 µM. Interestingly, diketone 17 derived from bryonolic acid remained inactive. Oxidative cleavage products di-, tri-, and tetra-decane–poly(one)s 4–6 from the lanosterol series had increasing activity correlated with an increasing number of oxidized carbon from IC50 = >100 µM,

11.7 µM, and 7.9 µM. While di- and tri- ketone decane–poly(one)s 18 and 19 from the

89 bryonolic acid series had little to no activity, tetra-ketone decane–poly(one)s 20 is the second most active from the oxidized/oxidative cleaved products with an IC50 = 11.5 µM.

Both the tetra-ketone decane–poly(one)s 6 and 20 (IC50 = 7.9 µM for 6 and IC50 = 11.5

µM for 20) are the most active out of the oxidation/oxidative cleavage production from the lanosterol and bryonolic acid series.

Aldol addition/condensation products from both series resulted in skeletally diverse molecules with a wide range of activity (7–14, 21–29). Aldol- addition/condensation products 7, 8, and 12 with a 6/5/7/5 ring system from the lanosterol series, resulted in active molecules (IC50 = 14.2 µM for 7, IC50 = 17.1 µM for 8, and IC50 = 10.1 µM for 12), which was derived from inactive 4. However, out of the aldol/addition products with the 6/5/7/5 ring system, 11 remained inactive despite a small difference in the position of unsaturation at C7/C8 (11) vs C8/C10 (12). Aldol addition products 9 and 10 had similar potency compared to their precursors 5 and 6. Aldol condensation of 9 to 13 resulted in a decrease in potency, while a repositioned ring unsaturation in 14 resulted in a 1.8-fold increase in potency compared to 9 (IC50 = 13.0

µM for 9, and IC50 = 7.4 µM for 14). A 3.4-fold difference in potency was observed between linear derivatives 13 to 14 despite the difference in the position of unsaturation

(IC50 = 25.0 µM for 13 and IC50 = 7.4 µM for 14).

Aldol addition/condensation of 19 resulted in active linear triterpenoids 24 and 26 with IC50 values of 17.5 µM for 24 and 30.3 µM for 26 in the bryonolic acid series.

Aldol addition/condensation of tetra-decane–poly(one)s 20 resulted in inactive (25) and active (27 with an IC50 = 8.3 µM) structures with 27 as the most potent of the aldol addition/condensation products. Aldol addition/condensation products from 18 yielded a

90 6/7/5/6/6 ring system (21, 22, and 28) as well as a 6/5/7/6/6 ring system (23 and 29) with a wide range of potency. Out of the 6/7/5/6/6 ring system structures, only 21 had activity with IC50 = 25.6 µM. The structural difference between 21, 22, and 28 that may be responsible for activity might be the stereochemistry of hydrogen between the B and C ring attached to C9. Aldol addition and condensation products 23 and 29 were similar in potency with IC50 = 46.3 µM for 23 and IC50 = 38.2 µM for 29. We can conclude that the core triterpenoid skeleton plays an important role in dictating activity as demonstrated by the wide range of activity in suppressing NO production in LPS-activated RAW cells.

The most potent of the derivatives is the linear derivative 14 with and IC50 of 7.4 µM, demonstrating the utility of the DOS approach in identifying structurally diverse active molecules.

A-ring modifications resulted in enhanced anti-inflammatory potency

A-ring modifications of 1-en-3-one and electron withdrawing carbonitrile on C-2 positions on synthetic triterpenoids have been previously demonstrated to improve potency (Figure 4.3, p. 104).245, 255, 347 We selected skeletally diverse aldol addition/condensation products for A-ring modification to improve potency and for further biological analyses based on these previous studies. We included the inactive parent steroid structures 1 and 15, 6/5/7/5 skeleton 7 and 11 and linear derivatives 14 and

26 for A-ring modification resulting in 1f, 7f, 11f, 14f, 15f, and 26f. While 1 and 15 were initially inactive at reducing NO production in LPS-activated RAW cells, modification of the A-ring resulted in active molecules 1f (IC50 = 1.681 µM) and 15f (IC50 = 619.9 nM)

(Table 4.3, p. 105 and Table 4.4, p. 106). A-ring functionalization of the aldol product 7

91 to 7f resulted in a 29-fold increase in potency from 14.2 µM to 490.8 nM, while inactive

11 gained activity after A-ring medication to 11f with an IC50 = 425.0 nM. Modification of 26 to 26f to resulted in 128-fold increase in potency from IC50 = 30.3 µM for 26 to

IC50 = 237.3 nM for 26f. The most active of these molecules is linear derivative 14f which increased 123-fold from ICs = 7.4 µM for 14 to 60.38 nM.

Mechanism for suppression of NO by triterpenoids

To determine the mechanism by which the triterpenoids suppress the production of NO following LPS exposure, we examined the expression of iNOS through immunoblot analysis in LPS-activated RAW cells. Treatment with 0.5 µM triterpenoids significantly reduced iNOS protein levels after an 18-hour treatment compared to

DMSO-treated control (Figure 4.4A, p. 106). Treatment with 1f slightly reduced iNOS protein levels, and treatment with 7f, 11f, and 26f significantly reduced iNOS protein levels. Treatment of LPS-activated cells with 14f resulted in complete loss of iNOS protein levels. To determine whether the decrease in iNOS protein is due to transcriptional suppression of the iNOS gene, we further analyzed the most active molecules 14f and 26f. As shown in Figure 4.4B, p. 106, treatment with 26f decreased iNOS mRNA level 2-fold while treatment of 14f decreased iNOS mRNA levels 10-fold after treatment of LPS-activated cells for 8 h. Thus, the decrease of NO production is mediated by the suppression of iNOS expression, as shown by the decrease in both protein and mRNA levels in the presence of triterpenoids.

Transcriptional activation of Nrf2 by linear triterpenoid derivatives

In addition to suppressing iNOS expression, triterpenoids have also been shown to activate Nrf2 and induce expression of anti-inflammatory and antioxidant enzymes such as HO-1 and NQO1. In order to determine whether the derivatives are able to activate

Nrf2, we treated RAW cells with the derivatives and performed immunoblot analysis on

Nrf2 target gene HO-1. Treatment of RAW cells with 0.5 µM 14f and 26f for 18 h increased HO-1 protein levels (Figure 4.5, p. 108) as illustrated by a robust induction of

HO-1 observed in cells treated with 1 µM triterpenoids. qRT-PCR analyses were performed to analyze transcriptional activation of Nrf2 target genes HO-1 and NQO1.

HO-1 mRNA levels were significantly induced by 14f 12-fold and 26f 14-fold after an 8 h treatment with 1 µM of each (Figure 4.5B, p. 108). NQO1 expression was increased

4.3-fold for 14f and 5.7-fold for 26f after 8 h with 1 µM (Figure 4.5C, p. 108). To demonstrate transcriptional activation of Nrf2 by the linear triterpenoids, HepG2 cells were transfected with an hNQO1-ARE luciferase reporter and treated with 5 µM 14f or

26f for 18 h. Treatment with 14f resulted in an 18-fold increase while treatment with 26f resulted in a 24-fold increase. These results demonstrate that linear derivatives 14f and

26f activate Nrf2.

These findings demonstrate that diversification of the triterpenoid skeletal structure through a unique approach of DOS resulted in skeletally diverse molecules with a wide spectrum of anti-inflammatory activity. In combination with optimization of the

A-ring, we were able to identify effective and structurally diverse Nrf2 activators. The significance of these studies validate the use of DOS in identifying novel effective Nrf2

93 activators which would not have been discovered through a traditional medicinal chemistry approach of iteratively changing functional groups around the same core structure. This study lends significant support to our initial hypothesis highlighting the importance of the skeletal structure in dictating activity and is clearly a valid and innovative approach for identifying novel Nrf2 modulators.

4.4 Experimental Section

Materials

Leukemic mouse macrophage cells (RAW 264.7) were obtained as a gift from Dr.

Michael Sporn (Darthmouth College, NH). DMEM media was purchased from GIBCO

(Grand Island, NY) and supplemented with low endotoxin FBS (< 0.06 EU) from

Thermo Scientific (Logan, UT). DMSO, Lipopolysaccharide (LPS) from E. coli, and non-enzymatic cell detachment solution were purchased from Sigma (St. Louis, MO).

Penicillin/streptomycin, Griess assay kit, RIPA Buffer, 0.2 µm PVDF membrane,

Novex® 4–20% Tris-glycine gels, running and transfer buffers, PureLinkTM RNA Mini

Kit, Superscript® III Reverse Transcriptase, TaqMan® Fast Universal PCR Master Mix, and Lipofectamine® 2000 Transfection Reagent were all purchased from Invitrogen

(Grand Island, NY). All the primary antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA) and secondary antibodies from Southern Biotech

(Birmingham, AL). Recombinant mouse M-CSF was purchased from Peprotech (Rocky

Hill, NJ). Protease cocktail inhibitor tablet was purchased from Roche (Indianapolis, IN) and PBS purchased from Cellgro by Mediatech, Inc. (Manassas, VA). The ECL plus was purchased from Amersham (Buckinghamshire, UK) and the autoradiography film was

94 from MidSci (St. Louis, MO). The MTT cell proliferation assay kit was purchased from

ATCC (Manassas, VA). The iNOS probe was purchased from Applied Biosystems

(Carlsbad, California) and the 18s rRNA probe was purchased from IDT (Coralville,

Iowa).

Diversity Oriented Synthesis Chemistry on Bryonolic Acid and Lanosterol and A- ring Functionalization

Given that the organic synthesis was performed by Dr. Vasily Ignatenko, and Dr.

Yong Han from the Tochtrop Lab, experimental details and synthetic characterization will be included in a subsequent publication. To this point, experimental details on application of DOS to bryonolic acid has been published in the Journal of Organic

Chemistry.348

Cell Culture

RAW 264.7 cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin, and kept in culture at 37 °C in a 5% CO environment. 2

Cells were kept in culture for no longer than a month and routinely checked for LPS responsiveness every few passages via detection of NO production measured using the

Griess assay.

NO Measurement

RAW 264.7 cells were plated at 1 × 105 cells/well in a 96-well plate and allowed to attach for several hours before activating with 10 ng/mL LPS and simultaneously

95 treating with triterpenoids or 0.5% DMSO control. NO levels were measured via Griess assay using 100 µL of Griess reagent with 100 µL of cell culture supernatant.

Absorbance was read at 550 nm using the Sunrise plate reader by TECAN (Mannedorf,

Switzerland). IC values were calculated by fitting a nonlinear sigmoidal variant slope 50 curve to the data using Prism 5.0 software by Graphpad Inc.

Toxicity Measurement

MTT cell proliferation assay kit was used according to the manufacturer’s protocol to measure toxicity in triterpenoid-treated, LPS-activated RAW 264.7 cells after

24 h.

Immunoblot Analysis

RAW 264.7 cells were plated at 4 × 106 cells/60 mm plate and allowed to adhere to the plate for 3 h. Cells were activated with 10 ng/mL LPS and immediately treated with varying concentrations of triterpenoids (maintaining 0.1% DMSO), DMSO control or LPS alone and harvested for immunoblot analysis and quantitative RT-PCR. Proteins were isolated by lysing the cells with RIPA Buffer containing protease inhibitors and equal amounts of proteins were loaded into a Novex 4–20% Tris-Glycine Gel. Proteins were transferred to a PVDF membrane, blocked with 5% non-fat dry skim milk in 0.5%

Tris-buffered saline containing Tween-20 (TBST), and probed with antibodies. Primary antibodies were diluted 1:500 to 1:1000 and secondary horse-radish peroxidase- conjugated antibodies were diluted 1:5000. Antibodies were detected using ECL plus with autoradiography.

Quantitative RT-PCR

Cells harvested for the immunoblot analysis were also harvested for quantitative

RT-PCR. Total RNA was extracted using the PureLink™ RNA Mini Kit followed by reverse transcription reaction using the Superscript® III Reverse Transcriptase. PCR was performed using TaqMan® Gene Expression Assay iNOS, HO-1, and NQO1 probes

(Assay ID: Mm01309902_m1, Mm00516007_m1, Mm00500821_m1, respectively),

PrimeTime qPCR assay (Assay ID: Mm.PT.42.122532.g) 18s rRNA control probe with

TaqMan® Fast Universal PCR Master Mix. Amplification was performed using the 7500

Fast Real-Time PCR system (95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s, and

60 °C for 30 s) and the 7500 Fast System SDS Software-Sequence Detection Software version 1.3.1.21 by Applied Biosystems.

Transient transfection and luciferase Reporter Assay

5 HepG2 cells were plated at 8 x 10 cells per well in a 24-well plate and incubated overnight. Cells were transfected with 500 ng hNQO1-ARE-luciferase construct and 300 ng Renilla using Invitrogen Lipofectamine® 2000 Transfection Reagent. Transfected cells were treated with 5 µM triterpenoids for 18 h. Luciferase activity was measured using Promega Dual Luciferase Assay Kit and a ML3000 Microtiter Plate Luminometer.

The hNQO1-ARE-luciferase construct were obtained as a gift from Dr. Jeffrey A.

Johnson (University of Wisconsin-Madison).

97 Figure 4.1. Reagents and conditions for DOS of lanosterol derivatives: (a) 2, 3, see

349 reference , CrO3 (4 equiv) 80 °C 10 min, BF3 (0.5 equiv), rt, 24 h 32% yield for 2, and

350 38% yield for 3; (b) 4, 5, and 6, see reference, RuCl3 (0.2 equiv)/NaIO4 (4 equiv),

CH3CN/CCl4/H2O 2/2/3, rt, time 24 h. 91% yield for 4, 86% yield for 5, and 92% yield

351 for 6; (c) 7, 9, 10 see reference, Basic Alumina (50 equiv), methanol, 50 °C, time 4 h.

98% yield from 7, 99% yield for 9, and 99% yield for 10. 8 (from 4), InCl3 (0.2 equiv),

Alumina (50 equiv) 50 °C, 24 h (d) 11, 12, 13, 14 SOCl2 (20 equiv.) rt 2 h.

99 Figure 4.2. Reagents and conditions for DOS of bryonolic acid derivatives: (a) 16, 17,

18, RuCl3 (0.2 equiv), NaIO4 (5 equiv), CH3CN/CCl4/H2O 2/2/3 rt, 24 h, 9% yield for 17, and 22% yield for 18; (b) 19, in situ, 38% yield; (c) 24, Al2O3 (100 equiv), DCM, rt, 24 h,

80% yield; (d) 26, Et3N, SOCl2, DCM, 0 °C to rt, 2 h, 98% yield; (e) 21, 22, 23, see

352 reference; (f) 28 (from substrate 22) and 29 (from substrate 23), BF3, Et2O, DCM, -

78 °C to rt, 18 h, 92% yield for 28, 22% yield for 29; (g) 20, RuCl3 (1 equiv), NaIO4 (10 equiv), CH3CN/CCl4/H2O 2/2/3 rt, 7 days, 18% yield; (h) 25 and 27, LDA (1,1 equiv),

THF, -78 °C to rt, 24 h, 9% yield for 25, and 64% yield for 27.

100 R = COOMe R = Starting Material 29 H E Total Synthetic Steps 11C D From Starting Material

3A B 7 = One Synthetic Step AcO H 15 = Two Synthetic Steps a = Three Synthetic Steps

R R R

H H H O O

H O O O AcO H 16 AcO H 17 AcO H 18 e b g

R R R H H H O O H O O H O H O AcO OH AcO AcO H H O 19 H O 20 21

h R c d R H H O H R R HO H O H O H H OH AcO OH H H AcO H O 22 H 23 H AcO H OH H H AcO O 24 O O 25 f f

R R R R H H O H O O H HO H AcO H O O H H AcO AcO H AcO O O 26 H OH 27 28 H 29

101 Table 4.1. Anti-inflammatory activity of skeletally diverse triterpenoids was measured by inhibition of NO production in LPS-activated RAW 264.7 cells. LPS-activated cells were treated with various concentrations of derivatives for 24 h. NO levels and cell viability were measured using the Griess assay and experiments were performed in triplicate.

102 Table 4.2. Cell viability measurement of LPS-induced RAW 264.7 cells treated with derivatives for 24 h. Viability were measured using the MTT assay and were performed in triplicate.

103 Figure 4.3. Reagents and conditions for A-ring functionalization. (a) LiOH or KOH.

(b) PCC or Jones reagent. (c) NaOMe, ethyl formate. (d) NH2OH. (e) NaOMe. (f)

DDQ.

104 Table 4.3. Anti-inflammatory activity of functionalized derivatives measured by inhibition of NO production in LPS-induced RAW 264.7 cells. Cells were simultaneously induced and treated with functionalized derivatives for 24 h. NO levels were measured using the Griess assay and performed in triplicate.

105 Table 4.4. Cell viability measurement of LPS-induced RAW 264.7 cells treated with functionalized derivatives for 24 h. Viability was measured using the MTT assay and performed in triplicate.

106 Figure 4.4. iNOS protein and mRNA levels in LPS-activated cells treated with triterpenoids. RAW 264.7 cells were induced with 10 ng/mL LPS and treated with derivatives at different time points. (A) Immunoblot analysis of iNOS enzyme in LPS- activated cells treated with 0.5 µM triterpenoids for 18 h. β-actin serves as protein loading control. (B) qRT-PCR analysis of iNOS mRNA in LPS-activated cells treated with 14f or 26f for 8 h.

107 Figure 4.5. Activation of Nrf2 by linear derivatives 14f and 26f. (A) Immunoblot analysis of HO-1 after 18 h (B) HO-1 mRNA and (C) NQO1 after 8 h. (D) HEPG2 cells were transfected with the hNrf2-ARE-luciferase reporter construct and treated with triterpenoids for 18 h. Nrf2 transactivation was quantified via measurement of luciferase activity.

108 Chapter 5. Identification of a Negative Feedback Loop in Biological Oxidant

Formation Regulated by 4-Hydroxy-2-(E)-Nonenal

Gatbonton-Schwager, T. N.; Sadhukhan, S.; Zhang, G.-F.; Letterio, J. J.; Tochtrop, G. P

5.1 Abstract

4-Hydroxy-2-(E)-nonenal (4-HNE) is a lipid peroxidation product that has become generally accepted as both causative and indicative of numerous disease states.

Here we report that 4-HNE modulates inducible nitric oxide production by activated macrophages via inhibition of iNOS expression. We demonstrate that 4-HNE activates the Nrf2 pathway and effectively suppresses nitric oxide (NO) production. Furthermore, we illustrate a proposed model of control of NO formation whereby at low concentrations of 4-HNE, a negative feedback loop maintains a constant level of NO production with an observed inflection at approximately 1 µM, while at higher 4-HNE concentrations positive feedback is observed. Taken together, the careful regulation of NO production by 4-HNE argues for a more fundamental role of this lipid peroxidation product in normal physiology.

5.2 Introduction

Polyunsaturated fatty acids (PUFAs) may undergo both enzymatic and nonenzymatic lipid peroxidation leading to unsaturated lipid hydroperoxides (LOOHs).

The aforementioned enzymatic processes are typically mediated by members of the lipoxygenase family and lead to the formation of a family of physiologic mediators of

109 inflammation, such as leukotrienes and prostaglandins.353, 354 The latter represents an altogether different and much less understood process whereby PUFAs, under conditions of oxidative stress, can spontaneously form peroxides at the allylic (or doubly allylic) positions of a number of physiologic lipids.355, 356 These lipid peroxides can subsequently undergo a variety of secondary reactions, some leading to stable oxygenated and polyoxygenated acyl chains, while others lead to chain cleavage and production of products containing either a methyl or carboxy terminus. 4-Hydroxy-2-(E)-nonenal (4-

HNE) is the most classically studied of the known lipid peroxidation (LPO) products, and has become accepted as a modulator of multiple disease states, including

Alzheimer’s disease,357, 358 atherosclerosis,359-361 and cancer.362 The pathogenicity of these LPO products has been rationalized through the formation of adducts with nucleophilic sites on proteins and DNA.363, 364 However, the recent view regarding 4-

HNE has evolved to appreciate the complex physiology and signaling aspects of this molecule.365-367

To better understand these signaling properties and to explore whether 4-HNE could impact the formation of biological oxidants, we initiated a series of experiments examining the relationship between 4-HNE concentrations and the ability of activated cultured macrophages to produce nitric oxide (NO). Our rationale behind these experiments was based on previous work showing that activation of the Nrf2-Keap1 signaling pathway effectively modulates the production of NO and the postulation that

4-HNE could activate this pathway due to its electrophilic nature.368-370 This coupled with our own work371 as well as others372, 373 showing that molecules which activate Nrf2

110 transcription also perturb inducible nitric oxide synthase (iNOS) expression set the basis for the experiments described herein.

5.3 Results and Discussion

Identification of a negative feedback loop for 4-HNE production.

Our initial experiment examined whether 4-HNE could modulate inducible NO formation in RAW 264.7 (RAW) culture macrophages stimulated with lipopolysaccharide (LPS). Using the standard Griess assay, we measured NO production after a 24 h exposure to LPS and various concentrations of 4-HNE to examine steady state levels of NO production. Surprisingly, we observed a dramatic decrease in NO production at 4-HNE concentrations greater than 1 µM (Figure 5.1A, p.

130). We interpreted this result as physiologically relevant given that the basal concentrations of 4-HNE found ubiquitously throughout mammalian tissues range from

100 nM to 1 µM.374, 375 This result becomes more significant considering the origins of

LPO products. The initial hydrogen abstraction step in LPO is generally ascribed to the chemical reactivity of oxidants such as superoxide and NO. Because 4-HNE is inhibiting the formation of NO, it is effectively inhibiting its own production by limiting the production of NO oxidant. We hypothesized that these observations could represent the discovery of a negative feedback loop for the production of 4-HNE through inhibition of oxidant formation. It is worth noting that the observed decrease in NO production is not gradual. NO levels drop dramatically over concentration ranges as small as 2.5-fold. Further, the observed effect cannot be attributed to toxicity (Figure

5.2, p. 131).

111 To examine whether 4-HNE has an effect on the constitutively expressed isoforms (neuronal nNOS and endothelial eNOS), we tested the effects of 4-HNE at various concentrations at earlier time-points before induction of iNOS could occur.

After 30 min of exposure to 4-HNE, no effect was observed in the nM to µM range, however at higher concentrations (9 mM to 10 mM, Figure 5.1B, p. 130) a dramatic increase in NO production was observed, with nitrite concentrations reaching greater than 20 µM. It is difficult to gauge whether this effect is due to a specific activation of nNOS/eNOS,376 or a secondary effect that is related to the toxicity inherent in these high concentrations of 4-HNE.377 Regardless, given our laboratory’s interests in iNOS, we decided to further explore the mechanistic underpinnings of the observed negative feedback profile.

Specificity of 4-hydroxy-2-(E)-alkenal C9–C10 derivatives on NO inhibition.

Our next experiment aimed to test whether the observed phenotype was specific for 4-HNE, or simply a non-specific effect derived from the electrophilic nature of the molecule. To test this, several 4-hydroxy-2-(E)-alkenal derivatives with varied chain lengths (C5–C12) were synthesized and evaluated for inhibition of NO production as above. 4-Hydroxy-2-(E)-pentenal (C5) was synthesized via a minor modification of the reported procedure (Figure 5.3A, p. 132).378 The other 4-hydroxy-2-(E)-alkenal derivatives (C6–C12) were synthesized using a new strategy inspired by Gardner et al.,379 which treats homo-allylic alcohols of appropriate chain length with m-CPBA to afford the 3,4-epoxyalcohols that are then oxidized with Dess-Martin periodinane. The

112 resulting 3,4-epoxyaldehydes undergo an in situ α-hydrogen elimination and concomitant epoxide opening forming of the desired 4-hydroxy-2-(E)-alkenals.

Evaluation of NO production in activated macrophages displayed a clear pattern whereby the C9 (4-HNE) and C10 alkenals showed the most pronounced decrease in NO production (Figure 5.3B, p. 132). This result was not due to toxicity

(Figure 5.4, p. 133), and points to the observed negative feedback being selective for 4-

HNE. The observation that C10 is slightly more potent is clear and repeatable across multiple concentrations. On this point, in our recent findings, we have found the presence of glutathionylated C10 in rat liver and heart tissues, and others have reported the presence of C10 in tissues.380 It is plausible that the similar molecular volume of

C9 and C10 effectively renders them identical in their effective modulation of signal transduction pathways controlling macrophages for NO production in vivo.

Mechanism of inhibition by 4-HNE of NO production.

From a mechanistic point of view, we proposed two possible explanations for the observed phenotypes. The first was that 4-HNE inhibited the enzymatic activity of iNOS, resulting in decreased NO production; the second explanation is rationalized as a consequence of decreased iNOS expression. To differentiate between these two possibilities we first investigated the effect of varying 4-HNE concentrations on recombinant murine iNOS. Activity was measured from the conversion of radiolabeled

L-arginine to citrulline using a standard assay and quantified using scintillation counts per minute (CPM).381 We tested the activity of recombinant iNOS at various 4-HNE concentrations, which correlated to the inhibitory concentrations observed in Figure

113 5.1A, p. 130. Between 1 µM and 1 mM no appreciable perturbation to iNOS activity was seen (Figure 5.5A, p. 134).

From these data, we concluded that 4-HNE is not a direct enzymatic inhibitor of iNOS. To explore our alternate hypothesis, we examined iNOS expression utilizing immunoblot and quantitative RT-PCR analysis of 4-HNE treated LPS-activated RAW macrophages. We observed that iNOS protein levels were not detectable in cells treated with 5 µM and above 4-HNE (Figure 5.5B, p. 134). This observation positively correlated with quantitative RT-PCR analysis (Figure 5.5C, p. 134) that showed a dose dependent inhibition in iNOS transcript by 4-HNE. Taken together, it is clear that the observed inhibition of oxidant formation is due to an inhibition of expression of iNOS.

Activation of Nrf2 signaling by 4-HNE suppresses NO production.

Previous reports have demonstrated that molecules which activate Nrf2 transcription also perturb iNOS expression.373 Based on these observations, we explored the hypothesis that 4-HNE inhibits iNOS expression through the activation of Nrf2.

Canonical activation of Nrf2 typically involves a small molecule activator that will specifically interact with the repressor protein, Keap1. When this occurs, Nrf2 dissociates from Keap1, translocates to the nucleus and affects the transcription of genes containing an antioxidant response element (ARE). To test whether 4-HNE affects nuclear translocation of Nrf2, we performed immunoblot analysis of cytoplasmic and nuclear Nrf2 protein in RAW macrophages treated with 4-HNE concentrations ranging from 1 µM to 10 µM. As seen in Figure 5.6A, p. 135, cytoplasmic Nrf2 protein levels

114 decreased in the presence of 1 µM 4-HNE, while nuclear Nrf2 protein levels increased in a dose-dependent manner after treatment for 5 min.

To further demonstrate Nrf2 activation by 4-HNE, we performed a series of quantitative RT-PCR analyses of the Nrf2 target genes: heme oxygenase (HO-1),

NAD(P)H:quinone (NQO1) and γ–glutamylcysteine synthetase (GCLC) in RAW macrophages. Figure 5.6B, p. 135 clearly shows marked increases in transcription for all three, with HO-1 and GCLC increasing 1.5-fold, while NQO1 gene expression increased

3-fold. At concentrations of 5 µM and above, HO-1, NQO1 and GCLC gene expression increased 2- to 3-fold relative to control. Taken together, these results clearly implicate activation of Nrf2 transcription as a probable mechanism to explain the observed changes in iNOS expression.

To confirm our proposed mechanism, we initiated a series of experiments using primary macrophages derived from either wild-type (wt), or Nrf2 deficient mice (Nrf2-/-).

We isolated bone marrow derived macrophages (BMDMs) from wt and Nrf2-/- mice and activated the cells with LPS. Activated BMDM were treated with 0.1 µM to 5 µM 4-

HNE for 48 h. In the presence of 5 µM 4-HNE, we observed the same decrease in NO as in the RAW macrophages, albeit with a slightly attenuated response (Figure 5.7A, p.

136). This decrease in NO was not due to toxicity (Figure 5.7B, p. 136). However, in

Nrf2-/- BMDMs, no appreciable differences were observed for NO production. Given the above experiment, we feel confident in ascribing the observed ability of 4-HNE to inhibit oxidant formation to be a direct result of Nrf2 activation.

Here, we have demonstrated that 4-HNE modulates the production of the biological oxidant, NO. Further, we had provided evidence that this effect is a result of

115 the activation of the Nrf2 transcription factor. While not definitively shown here, we would postulate that this activation is as a result of 4-HNE directly adducting one of the surface cysteine residues of the repressor protein, Keap1. We carefully considered alternate hypotheses including whether the observed effects could have arisen downstream of another process or as a result of a 4-HNE metabolite. To this end, we evaluated the state of reduced intracellular glutathione, and observed no appreciable difference (Figure 5.8, p. 137).382 Further, based on our own work investigating the metabolic fate of 4-HNE we concluded that it is highly probable that the active intracellular molecule is 4-HNE.383, 384

The significance of these observations is rooted in chemical processes that lead to the formation of LPO products. As mentioned earlier, the first step in this process is radical hydrogen abstraction of PUFA by reactive oxygen and reactive nitrogen substances (ROS and RNS, respectively), leading to the production of NO directly and the formation of 4-HNE in the presence of PUFAs. The finding that 4-

HNE, in turn, regulates the production of NO has the hallmark of a negative feedback pathway. We propose that these findings argue for an evolved feedback pathway to control the concentration of 4-HNE (and other LPO products) in tissues that express inflammatory mediators (such as iNOS) indirectly via the production of ROS and RNS.

The fact that the observed inhibitory concentrations of 4-HNE correspond to the observed physiologic concentrations lends further credence to our hypothesis that 4-HNE is an essential endogenous regulator of NO production. Taken together we have devised a model of the expression control of 4-HNE as shown in Figure 5.9, p. 138 that would argue for the production of 4-HNE as a carefully regulated process with an observed

116 negative feedback loop. High concentrations of 4-HNE have been observed during oxidative stress and in many disease states (blue region). However, at low concentrations over long time periods, there is an Nrf2-dependent negative feedback loop that inhibits LPS-induced nitric oxide production with an inflection at approximately 1 µM. This model stands in contrast to views in the field that see 4-HNE as a cytotoxic xenobiotic derived from physiology gone awry.

5.4 Experimental Section

Materials

The leukemic mouse macrophage cells (RAW 264.7) were obtained as a gift from

Dr. Michael Sporn (Darthmouth College, NH). DMEM media was purchased from

GIBCO (Grand Island, NY) and supplemented with low endotoxin FBS (< 0.06 EU) from

Thermo Scientific (Logan, UT). DMSO, Lipopolysaccharide (LPS) from E. coli, and non-enzymatic cell detachment solution were purchased from Sigma (St. Louis, MO).

Penicillin/streptomycin, Griess assay kit, RIPA Buffer, 0.2 µm PVDF membrane,

Novex® 4–20% Tris-glycine gels, running and transfer buffers, PureLinkTM RNA Mini

Kit, Superscript® III Reverse Transcriptase and TaqMan® Fast Universal PCR Master

Mix were all purchased from Invitrogen (Grand Island, NY). All the primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and secondary antibodies from Southern Biotech (Birmingham, AL). Recombinant mouse M-CSF was purchased from Peprotech (Rocky Hill, NJ). Protease cocktail inhibitor tablet was purchased from Roche (Indianapolis, IN) and PBS purchased from Cellgro by Mediatech,

Inc. (Manassas, VA). The ECL plus was purchased from Amersham (Buckinghamshire,

117 UK) and the autoradiography film was from MidSci (St. Louis, MO). The MTT cell proliferation assay kit was purchased from ATCC (Manassas, VA). The iNOS probe was purchased from Applied Biosystems (Carlsbad, California), and the 18s rRNA probe was purchased from IDT (Coralville, Iowa). The iNOS activity kit and the murine recombinant iNOS were purchased from Cayman Chemicals (Ann Arbor, MI) and the

[3H] Arginine Monohydrochloride from Perkin Elmer (Waltham, MA).

General methods for synthesis

Unless otherwise stated, the solvents and reagents were commercially available analytical grade quality and used without further purification. Flash chromatography was performed on silica gel (230–400 mesh) purchased from Dynamics Adsorbents (Atlanta,

GA). TLC was done on Hard Layer, Organic Binder TLC-plates with a fluorescent indicator and visualized by UV light (254 nm) purchased from Dynamics Adsorbents

(Atlanta, GA). 1H and 13C-NMR spectra were recorded on a Varian Inova spectrometer

(at the Department of Chemistry, Case Western Reserve University) operating at 400

MHz and 100 MHz for the 1H and 13C-NMR spectra, respectively. The internal

1 13 references were TMS (δ 0.00) and CDCl3 (δ 77.2) for H and C spectra, respectively.

NMR data are presented in the following order: chemical shift, peak multiplicity (b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet), coupling constant, and proton number.

118 4-Hydroxy-2-(E)-pentenal (3)

A solution of aldehyde, 2 (0.14 g, 1.076 mmol) in dry THF (6 mL) was cooled to

-78 °C, and 0.8 mL of MeLi (1.6 M solution in Et2O, 1.28 mmol) was added slowly. The solution was stirred for 30 min followed by work-up with saturated solution of NH4Cl at -

78 °C. The reaction mixture was then extracted with Et2O (3 × 10 mL), the combined organic layers were dried over Na2SO4, and the solvent was removed in vacuo to obtain the crude product. The later was then hydrolyzed with Amberlyst-15 as described for 2 to afford the crude 4-hydroxy-2-(E)-pentenal (3), which was then purified by column chromatography over silica (Hexane, 30% Ethyl acetate) to give 3 (0.083 g, 0.829 mmol,

1 77%). H-NMR (400 MHz, CDCl3): 1.39 (d, 3H, J = 6.8 Hz), 4.61 (m, 1H), 6.29 (ddd,

1H, J = 15.6 Hz, 7.6 Hz, 1.6 Hz), 6.84 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.57 (d, 1H, J = 7.6

13 Hz); C-NMR (100 MHz, CDCl3): 22.7, 67.3, 130.2, 160.1, 193.9. EI-HRMS (positive

+ mode): m/z Calcd. for C5H9O2 [MH ] 101.0602, found 101.0602.

Fumaraldehyde dimethylacetal (2)

Fumaraldehyde dimethylacetal, 2 was obtained by partial acid hydrolysis of fumaraldehyde bis(dimethylacetal), 1. Amberlyst-15 catalyst in acid form (0.06 g) was added to the bisacetal 1 (0.2 g, 1.135 mmol) in acetone (6 mL) and water (0.08 mL) under magnetic stirring at room temperature. Stirring was continued for 10 min (longer time resulted the hydrolysis of the second acetal group), then the reaction mixture was filtered through a bed of anhydrous sodium carbonate and sodium sulfate 1:1 (w:w) followed by the solvent evaporation in vacuo to get 2 (0.14 g, 1.076 mmol, 95% yield).

NMR data were in accord with the literature.385

119 General method for 4-Hydroxy-2-(E)-alkenals (3a–3g)

4-Hydroxy-2-(E)-alkenals, 3a–3g were synthesized using Gardner method.379 To a stirring solution of 3-alkenol, 4a–4g (1 mmol) in 4 mL CH2Cl2 was added a 1.5 molar excess of 70% m-chloroperoxybenzoic acid (0.37 g). The solution was stirred at room temperature for 1.5 h after which 4 mL of 10% NaHCO3 was added with vigorous stirring for 45 min. The reaction mixture was then extracted with CH2Cl2 (3 × 15 mL), the combined organic layers were dried over Na2SO4, and the solvent was removed in vacuo to obtain the crude product which was redissolved in 5 mL of CH2Cl2 and 6.6 mL of

Dess-Martin periodinane (0.3 M solution in CH2Cl2, 2 mmol) was added. The mixture was stirred for 2 h at room temperature and then 15 mL of Et2O and 8 mL of 1.3 M

NaOH was added into that and vigorously stirred for 1 min. The aqueous NaOH layer was removed, and an additional 10 mL of 1.3 M NaOH were added with vigorous stirring for 15 min. The aqueous layer was removed and the organic phase was washed with brine solution, the organic layer was dried over Na2SO4, and the solvent was removed in vacuo to obtain the crude 4-hydroxy-2-(E)-alkenal, which was then purified by column chromatography over silica (Hexane, 30–40% Ethyl acetate) to give pure 4-hydroxy-2-

(E)-alkenal, 3a–3g.

4-Hydroxy-2-(E)-hexenal (3a)

1 Overall yield: 42.1% (0.048 g, 0.421 mmol); H-NMR (400 MHz, CDCl3): 0.98

(t, 3H, J = 7.6 Hz), 1.57-1.74 (m, 2H), 4.36 (m, 1H), 6.29 (ddd, 1H, J = 15.6 Hz, 8.0 Hz,

1.6 Hz), 6.82 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.55 (d, 1H, J = 8.0 Hz); 13C-NMR (100

120 MHz, CDCl3): 9.6, 29.5, 72.3, 130.9, 159.3, 193.9. EI-HRMS (positive mode): m/z Calcd.

+ for C6H11O2 [MH ] 115.0756, found 115.0756.

4-Hydroxy-2-(E)-heptenal (3b)

1 Overall yield: 40% (0.051 g, 0.40 mmol); H-NMR (400 MHz, CDCl3): 0.92 (t,

3H, J = 7.6 Hz), 1.32-1.61 (m, 4H), 4.41 (m, 1H), 6.27 (ddd, 1H, J = 15.6 Hz, 8.0 Hz, 1.6

Hz), 6.82 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.52 (d, 1H, J = 8.0 Hz); 13C-NMR (100 MHz,

CDCl3): 14.1, 18.7, 38.6, 70.9, 130.6, 160.1, 194.2. EI-HRMS (positive mode): m/z Calcd.

+ for C7H13O2 [MH ] 129.0915, found 129.0919.

4-Hydroxy-2-(E)-octenal (3c)

1 Overall yield: 43% (0.061 g, 0.43 mmol); H-NMR (400 MHz, CDCl3): 0.89 (t,

3H, J = 7.6 Hz), 1.28-1.66 (m, 6H), 4.40 (m, 1H), 6.28 (ddd, 1H, J = 15.6 Hz, 8.0 Hz, 1.6

Hz), 6.82 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.53 (d, 1H, J = 8.0 Hz); 13C-NMR (100 MHz,

CDCl3): 14.1, 22.7, 27.5, 36.3, 71.2, 130.7, 159.9, 194.1. EI-HRMS (positive mode): m/z

+ Calcd. for C8H15O2 [MH ] 143.1072, found 143.1078.

4-Hydroxy-2-(E)-nonenal (3d)

1 Overall yield: 46% (0.072 g, 0.46 mmol); H-NMR (400 MHz, CDCl3): 0.94 (t,

3H, J = 7.6 Hz), 1.29-1.68 (m, 8H), 4.41 (m, 1H), 6.27 (ddd, 1H, J = 15.6 Hz, 8.0 Hz, 1.6

Hz), 6.82 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.51 (d, 1H, J = 8.0 Hz); 13C-NMR (100 MHz,

CDCl3): 14.2, 22.7, 25.1, 31.8, 36.6, 71.3, 130.7, 159.6, 194.0. EI-HRMS (positive

+ mode): m/z Calcd. for C9H17O2 [MH ] 157.1228, found 157.1231.

121 4-Hydroxy-2-(E)-decenal (3e)

1 Overall yield: 39% (0.066 g, 0.39 mmol); H-NMR (400 MHz, CDCl3): 0.90 (t,

3H, J = 7.6 Hz), 1.29-1.65 (m, 10H), 4.44 (m, 1H), 6.31 (ddd, 1H, J = 15.6 Hz, 8.0 Hz,

1.6 Hz), 6.84 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.57 (d, 1H, J = 8.0 Hz); 13C-NMR (100

MHz, CDCl3): 14.2, 22.7, 25.3, 29.2, 31.8, 36.6, 71.3, 130.7, 159.5, 194.0. EI-HRMS

+ (positive mode): m/z Calcd. for C10H19O2 [MH ] 171.1385, found 171.1388.

4-Hydroxy-2-(E)-undecenal (3f)

1 Overall yield: 41% (0.076 g, 0.41 mmol); H-NMR (400 MHz, CDCl3): 0.90 (t,

3H, J = 7.6 Hz), 1.28-1.67 (m, 12H), 4.43 (m, 1H), 6.32 (ddd, 1H, J = 15.6 Hz, 8.0 Hz,

1.6 Hz), 6.83 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.58 (d, 1H, J = 8.0 Hz); 13C-NMR (100

MHz, CDCl3): 14.2, 22.8, 25.4, 29.3, 29.5, 31.9, 36.7, 71.3, 130.8, 159.4, 193.9. EI-

+ HRMS (positive mode): m/z Calcd. for C11H21O2 [MH ] 181.1541, found 181.1541.

4-Hydroxy-2-(E)-dodecenal (3g)

1 Overall yield: 44% (0.087 g, 0.44 mmol); H-NMR (400 MHz, CDCl3): 0.92 (t,

3H, J = 7.6 Hz), 1.25-1.66 (m, 14H), 4.44 (m, 1H), 6.31 (ddd, 1H, J = 15.6 Hz, 8.0 Hz,

1.6 Hz), 6.84 (dd, 1H, J = 15.6 Hz, 4.8 Hz), 9.58 (d, 1H, J = 8.0 Hz); 13C-NMR (100

MHz, CDCl3): 14.3, 22.8, 25.4, 29.4, 29.6, 29.7, 32.0, 36.7, 71.3, 130.8, 159.3, 193.8. EI-

+ HRMS (positive mode): m/z Calcd. for C12H23O2 [MH ] 199.1698, found 199.1702.

3-Alken-1-ol (4a-4g)

122 3-Alken-1-ol 4a–4d were purchased from TCI America. The cis-3-decen-1-ol

(4e) was obtained by catalytic semi hydrogenation (1 atm) of a solution of 3-decyn-1-ol.

3-decyn-1-ol (1 g, 6.483 mmol) was dissolved in dry Et2O (25 mL) in a small round bottom flask. Lindlar catalyst (50 mg, Pd on CaCO3) and 1 g of quinoline were added and hydrogen was supplied from balloons. After completion of the reaction (confirmed by NMR) the mixture was filtered to remove the catalyst and the solvent removed on a rotary evaporator. Dichloromethane was added to the residue and washed with 1M acetic acid, brine, and water, and dried with Na2SO4. The crude was then purified by column chromatography over silica (Dichloromethane, 1% Methanol) to afford cis-3-decen-1-ol,

1 4e (0.932 g, 5.964 mmol, 92%). H-NMR (400 MHz, CDCl3): 0.89 (t, 3H, J = 6.8 Hz),

1.23-1.55 (m, 8H), 2.08 (td, 2H, J = 6.8 Hz, 6.6 Hz), 2.37 (td, 2H, J = 6.6 Hz, 6.4 Hz),

3.63 (td, 2H, J = 5.8 Hz, 5.6 Hz), 5.31-5.39 (m, 1H), 5.51-5.63 (m, 1H); 13C-NMR (100

MHz, CDCl3): 14.1, 22.6, 27.3, 29.0, 29.6, 30.8, 31.8, 62.4, 124.8, 133.8.

Trans-3-undecen-1-ol (4f) and trans-3-dodecen-1-ol (4g) were synthesized using

386 knoevenagel condensation of nonanal or decanal and malonic acid followed by LiAlH4 reduction. Malonic acid (2.03 g, 19.5 mmol) was dissolved in triethylamine (2.97 g,

29.27 mmol) in a 2-neck round-bottom flask fitted with a magnetic stirring bar and a reflux condenser. Nonanal (2.77 g, 19.5 mmol) was added very slowly under inert atmosphere at room temperature. The reaction mixture was then heated to 80 °C and maintained at this temperature for 3 h. The product was then acidified with 1M HC1 and extracted with Et2O. Organic layers were thoroughly washed with brine and water and dried over Na2SO4. Solvent was removed in vacuo to obtain trans-3-undecenoic acid

(2.91 g, 15.8 mmol, 81%). The crude product was pure enough to do the next step. The

123 LiAlH4 (0.93 g, 24.5 mmol) was dissolved in 15 mL dry THF, stirred for 5 min under inert atmosphere. Trans-3-undecenoic acid (1.5 g, 8.15 mmol) was dissolved in 5 mL

THF and added slowly. The reaction was allowed to continue for 4 h under inert atmosphere, then 2 mL H2O was added to quench the reaction, and 1M HCl was added until pH < 3. Most of the THF was removed in vacuo, and the residue was diluted with

10 mL H2O, and extracted with CH2Cl2 (3 × 30 mL). The combined organic layers were dried with Na2SO4, and the solvent was removed in vacuo. The resulting residue was purified by column chromatography over silica (Hexane, 30% Ethyl acetate) to give pure trans-3-undecen-1-ol 4f (1.3 g, 7.7 mmol, 94%). The trans-3-dodecen-1-ol 4g was also synthesis following the same method as for 4f and comparable yield.

1 trans-3-Undecen-1-ol (4f): H-NMR (400 MHz, CDCl3): 0.88 (t, 3H, J = 6.8 Hz),

1.27-1.39 (m, 10H), 2.01 (q, 2H, J = 6.4 Hz), 2.26 (q, 2H, J = 6.4 Hz), 3.61 (t, 2H, J = 6.4

Hz), 5.37 (dt, 1H, J = 15.2 Hz, 7.2 Hz), 5.55 (dt, 1H, J = 15.2 Hz, 6.8 Hz); 13C-NMR

(100 MHz, CDCl3): 14.2, 22.8, 29.3, 29.4, 29.6, 32.0, 32.8, 36.1, 62.2, 125.8, 134.4.

1 trans-3-Dodecen-1-ol (4g): H-NMR (400 MHz, CDCl3): 0.88 (t, 3H, J = 7.2 Hz),

1.22-1.37 (m, 12H), 2.01 (q, 2H, J = 6.4 Hz), 2.26 (q, 2H, J = 6.4 Hz), 3.62 (t, 2H, J = 6.4

Hz), 5.37 (dt, 1H, J = 15.2 Hz, 7.2 Hz), 5.56 (dt, 1H, J = 15.2 Hz, 6.8 Hz); 13C-NMR

(100 MHz, CDCl3): 14.3, 22.8, 29.4, 29.5, 29.7, 29.7, 32.1, 32.8, 36.1, 62.2, 125.8, 134.6.

Cell culture

RAW 264.7 cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin and kept in culture at 37 °C in a 5% CO2 environment.

Cells were kept in culture for no longer than a month and routinely checked for LPS

124 responsiveness every few passages via detection of nitrite production measured using the

Griess Reagent Kit.

Cell viability measurement

Remaining cells from the Griess Assay were used for viability measurement using the MTT Cell Proliferation assay kit from ATCC following manufacturer’s instruction.

Absorbance was read at 550 nm using the SunriseTM plate reader by TECAN. Percent viability of treated cells was calculated relative to the LPS-activated treated with DMSO control as 100% viability.

Nitrite level measurement

RAW 264.7 cells were plated at 1 × 106 cells/well in a 96-well plate and allowed to attach for 3 h before simultaneous activation with 10 ng/mL LPS and treated with 4- hydroxy-2-(E)-alkenal derivatives (C5–C12, C9 being 4-HNE) or 0.25% DMSO control.

4-Hydroxy-2-(E)-alkenal derivatives were freshly made in DMSO. LPS-activated cells were treated with varying concentrations of 4-HNE for either 30 min or for 24 h. For studies of other 4-hydroxy-2-(E)-alkenal derivatives, LPS-activated cells were treated with 1 µM derivatives for 24 h. Nitrite levels were measured via Griess assay according to manufacturer’s specifications using 100 µL Griess reagent with 100 µL sample supernatant. Absorbance was read at 550 nm using the SunriseTM plate reader by

TECAN (Mannedorf, Switzerland).

125 Immunoblot Analysis

6 RAW 264.7 cells were plated at 4 × 10 cells/60 mm plate and allowed to attach for 3 h. Cells were activated with 10 ng/mL LPS and treated with varying concentrations of 4-HNE, DMSO control, or LPS with DMSO control and harvested for immunoblot analysis and quantitative RT-PCR. Proteins were isolated via lysing the cells with RIPA

Buffer containing protease inhibitors and equal amounts of proteins were loaded into a

Novex 4–20% Tris-Glycine Gel. Proteins were transferred to a PVDF membrane, blocked with 5% non-fat dry skim milk in 0.5% Tris-buffered saline containing Tween-

20 (TBST), and probed with antibodies. Primary antibodies were diluted 1:500 to 1:1000 and secondary horse-radish peroxidase antibodies were diluted 1:5000. Antibodies were detected using ECL plus with autoradiography.

Quantitative RT-PCR Cells harvested for the immunoblot analysis were also harvested for quantitative

Mm00802655_m1, respectively), PrimeTime qPCR assay (Assay ID:

Mm.PT.42.122532.g) 18s rRNA control probe with TaqMan® Fast Universal PCR

Master Mix. Amplification was performed using the 7500 Fast Real-Time PCR system

(95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s, and 60 °C for 30 s) and the 7500

Fast System SDS Software-Sequence Detection Software version 1.3.1.21 by Applied

Biosystems.

126

iNOS activity assay

iNOS activity was measured using the iNOS activity kit via detection of radiolabeled arginine (Arginine Monohydrochloride L-[2,3,4-3H]) conversion to L- citrulline. Reactions were prepared according to manufacturer’s instructions using the iNOS (murine recombinant) enzyme. Each reaction was treated with various 4-HNE concentrations and allowed to proceed at room temperature for 1 h. Radioactivity was quantified by counts per minute (CPM) with 1450 MicroBeta TriLux Microplate

Scintillation Counter. The positive control contained iNOS enzyme, and the negative control contained the iNOS enzyme treated with a NOS competitive inhibitor L-NG-

Nitroarginine (L-NNA).

Bone marrow derived macrophage isolation and treatment

Bone marrow derived macrophages (BMDM) from 6–8 week old C57BL/J6 mice

(wild-type) and Nrf2 knockout mice (Nrf2-/-) were isolated and plated in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin and M-CSF. Fresh medium containing M-CSF was added after 3 days of incubation. Cells were washed with PBS 2–

3 times to remove non-macrophage cells and a non-enzymatic detaching solution was used to collect BMDM. Cells were plated at 4 × 106 cells/60 mm plate in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, and cultured overnight. BMDM were activated with 500 ng/mL LPS and treated with varying concentrations of 4-HNE or 0.05% DMSO control. Cells were harvested for Griess assay and MTT analysis after a 48 h treatment. All experiments performed were in accordance

127 with an approved protocol by the Institutional Animal Care and Use Committee at Case

Western Reserve University.

Conjugated 4-HNE-GSH LC-MS measurement

4-hydroxydecanenal-GSH, and 4-HNE-GSH Michael addition adducts were assayed from our previously published methods, with modification.383, 384, 387, 388 Briefly, media (1 mL) or cell lysates (32.5 µL) from 4-HNE treated RAW 264.7 cells for 15 min or 18 h were spiked with 0.25 nmol of 4-hydroxydecanenal-GSH (internal standard).

Media from culture and cell lysates were treated with 4 mL or 0.2 mL acetonitrile, respectively, to precipitate proteins. After centrifugation at 4 °C, the supernatant was dried under N2 gas. After dissolving the dried residue in 100 µL Milli-Q water, 40 µL was injected into a Thermo Scientific Hypersil GOLD C18 column (150 × 2.1 mm) with a guard column (Hypersil GOLD C18 5 µm, 10 × 2.1 mm) into a Dionex-UltiMate 3000

HPLC combined with a 4000 Qtrap mass spectrometer (Applied Biosystems, Foster City,

CA, USA). The chromatographic method was developed at 0.2 mL/min (i) from 0 to 25 min with a 1–45% gradient of buffer B (95% acetonitrile, 5% water, and 0.25% formic acid) in buffer A (95% water, 5% acetonitrile, and 0.25% formic acid), (ii) from 25 to 26 min with a 45–90% gradient of buffer B in buffer A, (iii) from 26 to 31 min with 90% buffer B in buffer A, and (iv) from 31 to 32 min with a 90–1% gradient of buffer B in buffer A, with 10 min of equilibration with 99% buffer A before the next injection. The

4000 QTrap mass spectrometer was operated under positive ionization mode with the following source settings: turbo-ion-spray source at 600 °C, N2 nebulization at 65 psi, N2 heater gas at 55 psi, curtain gas at 30 psi, collision-activated dissociation gas pressure

128 held at high, turbo-ion-spray voltage at 5500 V, declustering potential at 90 V, entrance potential at 10 V, collision energy at 50 V, and collision cell exit potential at 10 V. Data acquisition was performed in multiple reaction monitoring mode monitoring the transition of [M+H]+ m/z 464 in Q1 to [MH−156]+ m/z 308 (protonated GSH) in Q3 as quantifier. The internal standard precursor ion and product ion were at m/z 475 and 308, respectively.

129 Figure 5.1. Nitrite levels in 4-HNE treated LPS-activated RAW 264.7 cells. Nitrite levels were measured via Griess assay in cells simultaneously activated with 10 ng/mL

LPS and treated with varying concentrations of 4-HNE for (A) 24 h and (B) 30 min.

0.25% DMSO was used as control. Experiments were performed at least 3 times and error bars represent standard error of mean. *P < 0.05; ***P < 0.001.

130 Figure 5.2. Viability of LPS-activated RAW 264.7 cells treated with increasing concentrations of 4-HNE for 24 h. Experiments were performed at least 3 times and error bars represent standard error of mean.

131 Figure 5.3. Synthesis and activity of C5–C12 4-hydroxy-2-(E)-alkenal derivatives. (A)

Synthesis of 4-hydroxy-2-(E)-alkenal derivatives C5–C12. Compound 3 is described as

C5 in the text and compounds 3a–3g represents C6 to C12 where n = 0–6 respectively.

(B) Nitrite levels from LPS-activated macrophage treated with 1 µM 4-hydroxy-2-(E)- alkenal derivatives C5–C12 for 24 h. Experiments were performed at least 3 times and error bars represent standard error of mean. **P < 0.01.

132 Figure 5.4. Viability of LPS-activated RAW 264.7 cells treated with 1 µM C5–C12 4- hydroxy-2-(E)-alkenal derivatives for 24 h. Experiments were performed at least 3 times and error bars represent standard error of mean.

133 Figure 5.5. (A) iNOS activity was measured via quantification of radiolabeled L- arginine to citrulline conversion. iNOS enzyme alone served as positive control while iNOS enzyme treated with Nω-nitro-L-Arginine (L-NNA) inhibitor served as negative control. (B) Immunoblot analysis of iNOS protein levels in 4-HNE treated LPS- activated macrophage treated for 18 h. DMSO treated cells were used as negative control while DMSO treated LPS-activated cells were used as positive control. β-actin was used as protein loading control. (C) mRNA levels of 4-HNE treated LPS-activated macrophage treated for 18 h. Fold change in expression were calculated relative to

DMSO treated LPS-activated iNOS expression which was normalized to 18s rRNA expression. Experiments were performed at least 3 times and error bars represent standard error of mean. ***P < 0.001.

134 Figure 5.6. (A) Immunoblot analysis of Nrf2 protein levels in the cytoplasm and nucleus. RAW 264.7 cells were treated with increasing concentrations of 4-HNE for 5 min. β–actin and lamin B1 were used as loading controls. (B) mRNA levels of 4-

HNE treated macrophage were treated for 4 h. Fold change in expression was calculated relative to DMSO treated HO-1, NQO1, and GCLC expression which was normalized to

18s rRNA expression. Experiments were performed at least 3 times and error bars represent standard error of mean. **P <0.01.

135 Figure 5.7. Nitrite levels and viability in 4-HNE treated activated primary macrophage cells. (A) Nitrite levels were measured via Griess assay in cells simultaneously activated with 500 ng/mL LPS and treated with varying concentrations of 4-HNE for 48 h. (B) Cell viability was measured using MTT assay. LPS-activated cells treated with DMSO were used as control. Experiments were performed at least 3 times and error bars represent standard error of mean. *P < 0.01.

136 Figure 5.8. Conjugated 4-HNE-GSH measurement from LPS-activated RAW 264.7 cells treated with 4-HNE. LPS-activated cells were treated with increasing concentrations of 4-HNE for 15 min and 18 h. Extracellular and intracellular 4-HNE-GSH concentrations were measured via LC-MS.

137 Figure 5.9. A model for 4-HNE control of induced nitric oxide production. At low 4-

HNE concentrations over long time periods, there is an Nrf2-dependent negative feedback loop that inhibits LPS-induced nitric oxide production with an inflection at approximately 1 µM (yellow region). At higher concentrations a positive feedback loop is present whereby increasing concentrations of 4-HNE in turn elicits higher concentrations of nitric oxide during a short time period (blue region).

138 Chapter 6. Summary and Future Directions

The goal of my thesis project was to biologically and chemically analyze small molecule activators of Nrf2-Keap1 Signaling with a focus on two classes of Nrf2 activators: (1) exogenously derived triterpenoids, and (2) an endogenous electrophile, 4-

HNE. The aims for studying triterpenoid Nrf2 activators were to determine whether the core triterpenoid skeletal structure is a requirement for activation of Nrf2-Keap1 signaling and whether triterpenoid skeletal diversification through DOS is a valid approach to obtain novel and effective Nrf2 modulators. The objective of the 4-HNE study was to understand how this electrophile signals through Nrf2-Keap1. In order to determine the structural requirements for triterpenoid activity, DOS was applied to two naturally occurring triterpenoids to diversify the skeletal structure. Bryonolic acid and lanosterol were chosen due to a rare unsaturation between the B/C ring fusion. Since bryonolic acid is not commercially available, we developed a large-scale isolation method from roots of germinated seeds of Cucurbita pepo L. (common zucchini)

(Chapter 2).389 Biological and mechanistic analysis demonstrated that bryonolic acid exerts transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo (Chapter 3).371 Diversification of the triterpenoid skeletal structure through DOS resulted in molecules with a wide spectrum of anti-inflammatory activity, and in combination with optimization of the A-ring, resulted in the identification of effective and structurally diverse Nrf2 activators (Chapter 4). These studies validate the use of DOS for the identification of novel and effective Nrf2 activators, which would not have been discovered through a traditional medicinal chemistry approach of iteratively changing functional groups around the same core structure. This study confirms our

139 initial hypothesis highlighting the importance of the skeletal structure in dictating activity.

Our findings on the utility of DOS in accessing unexplored chemical space can be extended to additional structures with therapeutic potential. We anticipate that application of DOS on cyclic structures such as the flavonoids and polyphenols would also yield diverse and effective Nrf2 modulators. In addition to our triterpenoid studies we extended our interest in determining the role of 4-HNE in modulating Nrf2-Keap1 signaling (Chapter 5). During oxidative stress, oxidation of polyunsaturated fatty acids is catalyzed by biological oxidants such as ROS leading to LPOs such 4-HNE.390, 391 We hypothesized that activation of Nrf2 by 4-HNE suppresses biological oxidant formation, such as NO leading to inhibition of lipid peroxidation, and thus 4-HNE. Our studies demonstrate that 4-HNE modulates inducible NO production by activated macrophages via inhibition of iNOS expression. Nrf2 activation by 4-HNE effectively suppressed NO production illustrating a feedback control for LPOs. The careful regulation of NO production by 4-HNE argues for a more fundamental role of this lipid peroxidation product in normal physiology. The summary of our findings are listed below:

• Developed large-scale isolation for bryonolic acid from Cucurbita pepo L.

• Characterized a mechanism for bryonolic acid anti-inflammatory and

antioxidant activity.

• Identified that the core skeletal structure of triterpenoids is important for

activity.

• Validated the DOS approach in identifying effective Nrf2 modulators.

• Illustrated a feedback control for the production of 4-HNE.

140 The overall significance of these studies is that understanding structural requirements for activation of Nrf2 would facilitate the identification of novel Nrf2 activators. In addition, determining how markers of oxidative stress are regulated can lead to new approaches for controlling their production to limit the extent of their damage.

6.1. Summary and Future Directions - Chapter 2. Bryonolic Acid: A Large-Scale

Isolation and Evaluation of Heme Oxygenase 1 Expression in Activated

Macrophages

The objective of this study was to develop a large-scale isolation method for bryonolic acid for future in vitro and in vivo analysis and for application of the DOS strategy to determine structural requirements for triterpenoid activity. Large-scale isolation yielded 1.34% bryonolic acid based on dry weight (200 mg of BA from 14.9 g of dry roots), which is 7-fold greater than previously reported isolation from roots of

Benincasa cerifera Savi (winter melon).293 Our initial analysis of activity demonstrated that bryonolic acid robustly induces HO-1 protein in leukemic mouse macrophage. The induction of HO-1 has direct clinical relevance to the treatment of human diseases such as malaria, which is caused by Plasmodium falciparum parasite. Induction of HO-1 suppressed pathogenesis of malaria and prevented accumulation of cytotoxic heme.392

Breakdown of cytotoxic heme by HO-1 has been implicated as a mechanism of protection against the severe form of malaria, cerebral malaria. In vivo studies comparing wild-type and HO-1 knockout mice demonstrated that HO-1 expression protects against the development of the cerebral form of malaria in Plasmodium-infected mice.393 With our study identifying Cucurbita pepo L as a rich source for HO-1 inducing bryonolic acid,

141 it is plausible to integrate roots from bryonolic acid producing plants in the diet in areas where malaria is prevalent such as in Sub-Saharan Africa. An important future study would be to determine whether plants from the Cucurbitaceae family locally grown in developing countries where malaria is widespread contain physiologically relevant amounts of bryonolic acid or additional active triterpenoids that can induce HO-1. Our studies on identifying abundant amounts of biologically active triterpenoids in the root system should be motivation to reanalyze root systems of plants for previously uncharacterized triterpenoids that could yield biologically active molecules such as bryonolic acid.

6.2. Summary and Future Directions - Chapter 3. Bryonolic Acid

Transcriptional Control of Anti-inflammatory and Antioxidant Genes in

Macrophages in Vitro and in Vivo

The rationale for this study was to characterize activity and validate the use of bryonolic acid as a platform for DOS to determine the structural requirements for triterpenoid activity. Utilizing an established in vitro model of inflammation, we discovered that bryonolic acid inhibits production of an important inflammatory mediator,

NO via transcriptional suppression of iNOS in an LPS-activated macrophage cell line.371

The most striking activity that we observed in bryonolic acid treated macrophages and mice was the induction of the antioxidant and anti-inflammatory HO-1. Anti-allergic and anti-inflammatory properties of bryonolic acid have been demonstrated in lab animals where treatment with bryonolic acid inhibited homologous passive cutaneous anaphylaxis in rats and delayed hypersensitivity in mice.394, 395 However, a mechanistic analysis of

142 this activity had not previously been performed. Our work provides mechanistic insights for the previously reported in vivo anti-inflammatory activity of bryonolic acid through induction of anti-inflammatory HO-1.185-187, 396-398

We have demonstrated that bryonolic acid activates Nrf2, as evidenced by Nrf2 translocation in the nucleus and induction of Nrf2 target genes. Modification of key cysteine residues in Keap1 via Michael addition is an established mechanism for Nrf2 activation.50 However, bryonolic acid does not contain a thiol-reactive group such as an

α,β-unsaturated carbonyl carbon for a Michael reaction with cysteine thiols. Future studies should include determining whether bryonolic acid undergoes biotransformation via phase I metabolism into a reactive Michael acceptor. It is possible that modifications of cysteine residues in Keap1 are not a prerequisite for Nrf2 activation by bryonolic acid.

In order to test this, Keap1 constructs expressing cysteine to alanine mutations and Nrf2-

-/- -/- ARE-luciferase reporters can be co-transfected in Keap1 and Nrf2 mouse embryonic fibroblasts (MEFs).76 Given that there are 27 reactive cysteine residues on human Keap1, we propose to initially mutate the seven most reactive residues - Cys151, Cys257, Cys273,

Cys288, Cys297, Cys434, and Cys613.50, 75, 76 Transfected cells expressing mutated key

Keap1 cysteine residues would be treated with bryonolic acid and Nrf2 activation analyzed via luciferase reporter assay. If reactivity with Keap1 cysteine residues is not a prerequisite for Nrf2 activation, then bryonolic acid should be able to induce Nrf2 in the presence of Keap1 lacking key cysteine residues. If bryonolic acid is still able to induce expression of Nrf2 target genes in the absence of key cysteine residues, then a new mechanism of Nrf2 activation could be postulated. We hypothesize that there is a binding pocket on Keap1 that would allow for triterpenoid-Keap1 interaction that would

143 result in a conformational change to release Nrf2 from Keap1 allowing it to translocate into the nucleus to activate target genes. Further studies are required to determine the mechanism of activation by bryonolic acid or triterpenoids lacking reactive groups that allows for Keap1 cysteine residue modifications.

Given that bryonolic acid is active in vivo, it is important to test whether it is able to ameliorate inflammatory-based diseases. The EAE (experimental autoimmune encephalomyelitis) mouse is a model of multiple sclerosis, which is an autoimmune disease that results in inflammation of the central nervous system (CNS). The disease causes infiltration of inflammatory cells in the CNS that results in the demyelination of axons. We chose to do our initial testing in the EAE model due to the availability and the relative short period of time to carry out the entire experiment. EAE was induced with immunization of myelin antigen adjuvant, allowing approximately two weeks for the disease to develop before treatment with bryonolic acid. Symptoms of the disease were monitored using the EAE clinical scoring system (see legend to Fig. 6.1, p. XXX) to determine improvement or regression of symptoms during treatment. Mice were treated with bryonolic acid at an initial concentration of 200 nanomole every other day by gavage for a total of 9 treatments. Since there was no improvement in clinical score, the

200 nanomole treatment was stopped for a week. The bryonolic acid dose was then increased to 400 nanomole and mice were treated 7 more times every other day by gavage. At a dose of 200 nanomole, bryonolic acid did not have a significant effect in decreasing the clinical score of treated mice (Figure 6.1, p. 150). However, upon increasing the dose from 200 nanomole to 400 nanomole bryonolic acid, the clinical score of treated mice decreased immediately after the treatment. This data indicates that

144 in vivo bryonolic acid activity is translatable to ameliorating inflammation driven diseases in mice. Future studies include performing immunoblot analysis on CNS tissues from these mice to determine if markers of inflammation such as IFN-γ, TNFα, IL-1 and iNOS are decreased in bryonolic acid treated mice compared to wild-type. Although bryonolic acid slightly decreased the clinical score of the mice, it would be interesting to determine if there is a greater decrease in clinical score at a higher concentration.

Bryonolic acid does not dissolve well in the gavage solution of 7.5% TBST. Therefore, future experiments would include dissolving bryonolic acid in

10%Cremophor/10%DMSO/80%TBST which may improve solubility and thus increase bryonolic acid bioactivity.

6.3. Summary and Future Directions - Chapter 4. Identification of Small

Molecule Nrf2 Activators Through Diversity-Oriented Synthesis

Successful large-scale isolation (Chapter 2) and careful biological characterization of bryonolic acid (Chapter 3) are important precursors to the application of DOS on bryonolic acid. Motivated by the therapeutic properties of SOTs, our goal was to determine whether the core triterpenoid skeletal structure is a requirement for activation of Nrf2-Keap1 signaling and whether triterpenoid skeletal diversification through DOS is a valid approach to obtain novel and effective Nrf2 modulators. In a collaboration with chemists from the Tochtrop Lab, Dr. Yong Han and Dr. Vaisily Ignatenko, structurally diverse molecules were synthesized using DOS on structural homologs of SOTs bryonolic acid and lanosterol (Figure 4.1, p. 98 and Figure 4.2, p. 101). Our study demonstrates that rearrangement of the triterpenoid core carbocyclic structure via DOS

145 modulates the activity, resulting in increased anti-inflammatory activity compared to the parent molecules bryonolic acid and lanosterol as measured by suppression of NO (Table

4.1, p. 102). The key finding of our study is that the structurally diverse derivatives exhibit a wide spectrum of anti-inflammatory activity, demonstrating that the triterpenoid skeleton dictates activity. We can rationalize that anti-inflammatory activity of the derivatives is mediated by Nrf2-dependent activation of HO-1 as demonstrated by bryonolic acid (Figure 3.3, p. 75), its derivatives (Figure 6.2, p. 151), and lanosterol derivatives (Figure 6.3, p. 152).371, 389

One of the interesting discoveries during our studies is the differential regulation of anti-inflammatory and antioxidant genes exerted by triterpenoids. Previous studies have demonstrated an inverse correlation between expression of the inflammatory gene iNOS and phase 2 genes in triterpenoid-treated cells.244 Based on these studies, we predicted that there would be a clear inverse relationship between inflammatory enzymes iNOS and COX-2 proteins and anti-inflammatory HO-1 protein. To our surprise, we observed a differential regulation of HO-1, iNOS and COX-2 in LPS-activated RAW cells treated with the skeletally diverse triterpenoids (Figure 6.2 – 6.4, p. 151 – 153).

Treatment of cells with 26f and 7f resulted in a decrease in iNOS, which correlated with an increase of HO-1 protein level (Figure 6.2, p. 151). However, in cells treated with

14f, a decrease in iNOS protein levels did not correlate with an increase in HO-1 protein levels at this time point. In comparing 11f versus 14f, an increase in HO-1 protein by 11f but not by 14f did not correlate with a greater decrease of iNOS protein by 11f. In our initial activity analysis of lanosterol derivatives, we discovered that the degree to which iNOS, COX-2, and/or HO-1 protein levels are changed varies between the compounds

146 (Figure 6.3, p. 152). For example, 5 decreased iNOS protein levels to a greater degree compared to 10 without a significant difference in increasing HO-1 protein in both treatments. The pattern of protein level changes does not follow what has been shown previously for the triterpenoids (increase in iNOS and COX-2 and decrease in HO-1). To determine whether these observed changes are significant, iNOS, COX-2 and HO-1 protein levels were quantified through densitometry. The different protein level pattern changes were categorized as follows: decrease in iNOS and increase in COX-2 and HO-

1; decrease in iNOS and COX-2 and increase in HO-1; selective decrease in iNOS; decrease in iNOS and increase in HO-1; decrease in COX-2 and increase in HO-1 and selective increase in HO-1 protein level (Figure 6.4, p. 153). These data support our hypothesis that changing the core triterpenoid skeleton leads to changes in activity and exerts differential regulation of iNOS, COX-2 and HO-1. We anticipate that time- dependent expression analysis of Nrf2 target genes by the derivatives will result in varying patterns of Nrf2 target induction similar to what we reported in comparing bryonolic acid and oleanolic acid (Figure 3.8, p. 80). We hypothesize that these different expression patterns can be explained by the skeletally diverse triterpenoid binding to

Keap1 and exerting different regulation of Nrf2 activation. Given that there are different mechanisms for the regulation of Nrf2 activation,84-90 we can stipulate that depending on the triterpenoid structure binding to Keap1, the mechanism for Nrf2 activation will vary.

Two models had been proposed for Nrf2 activation - the ‘hinge and latch’ model and the

Keap1-Cul3 dissociation model. In the ‘hinge and latch’ model, the low affinity binding

‘latch’ DLG motif is dissociated from Keap1 while the higher affinity ETGE motif or

‘hinge’ stays bound during Nrf2 activation. 84 Newly synthesized Nrf2 accumulates and

147 translocates in the nucleus allowing for transcription of Nrf2-target genes.85, 86 In the second model for Nrf2 activation, Keap1 and Cul3 are dissociated from each other, allowing for Nrf2 stabilization and translocation into the nucleus.87, 88 We can predict that as a consequence of different Nrf2 activation methods, there will be variations of co- activator and/or co-repressor binding resulting in differential regulation of Nrf2 target genes.

We improved the potency of select molecules by functionalization of the A-ring with a carbonitrile and a ketone group, which have been previously reported to increase the potency of triterpenoids. Functionalization of the derivatives resulted in increased anti-inflammatory potency with an EC50 of 60.4 nM for the most potent linearized structure, 14f, then followed by the second most potent, 26f (Table 4.3, p. 105). The mechanism of inhibition of NO production by these derivatives is through inhibition of iNOS expression. In addition to their anti-inflammatory activity, these derivatives activate the Nrf2-Keap1 pathway as shown by the induction of anti-inflammatory and antioxidant Nrf2 target genes and transcriptional activation of Nrf2-ARE-luciferase reporter.

Given that the linear derivatives 14f and 26f exerted effective inhibition of LPS- activated macrophages in vitro, the next logical step is to determine whether these derivatives are active in vivo. I performed a pilot study where mice were pretreated with

0.1 mg/kg, 1 mg/kg, or 10 mg/kg, 14f via i.p. 24 h prior to LPS treatment. Concurrent with LPS treatment, the mice received a second dose of 14f. Blood was collected via cardiac puncture 6 h after LPS and triterpenoid treatment and NO was measured using a

Griess Assay. Our preliminary results show that treatment with 10 mg/kg linear

148 triterpenoid 14f resulted in a 38% decrease of NO compared to LPS control (Figure 6.5, p. 154). A more careful analysis is required to determine whether 14f as well as 26f reduces NO levels via suppression of iNOS as well as whether both are able to activate

Nrf2 in vivo. Additional in vivo activity can be assessed via qRT-PCR analysis of expression of inflammatory enzymes and cytokines, as well as induction of Nrf2 target genes.

149

Figure 6.1. Treatment of EAE mouse model with bryonolic acid. EAE was induced with immunization of myelin antigen adjuvant, allowing approximately two weeks for disease development before treatment with bryonolic acid (green line). Bryonolic acid

(BA) was administered via gavage at 200 nanomole and 400 nanomole every other day

(green arrows). Symptoms of the disease were monitored using the following clinical scoring system: 0-no signs of disease; 1-numbness of limb; 2-limp tail, paralyzed tail (tail flat on the floor, no curve/tone); 3-moderate hind-limb weakness, limping not normal walking (numbness of hind legs when pinched); 4-severe hind-limb weakness (one hind leg paralysis); 5-Complete hind limb paralysis (both legs paralyzed); 6- quadriplegia or pre-morbid state; death.

150 Figure 6.2. Triterpenoids reduce iNOS protein levels and increase HO-1 expression in

LPS-activated RAW 264.7 cells. iNOS and HO-1 protein levels were quantified through immunoblot analyses in RAW 264.7 cells induced with 10 ng/mL LPS and treated with

0.5 µM triterpenoids for 18 h. β-actin serves as protein loading control.

151 Figure 6.3. RAW 264.7 cells were induced with 10 ng/mL LPS and treated with 25 µM triterpenoid for 24 h. Immunoblot analysis was performed probing for iNOS, COX-2, and HO-1.

152 Figure 6.4. Categorization of differential regulation by lanosterol derivatives. COX-2 suppressor (light blue), iNOS suppressor (red), HO-1 inducer (green), and COX-2 inducer (dark blue).

153 Figure 6.5. Mice were pretreated with 0.1 mg/kg, 1 mg/kg, or 10 mg/kg, 14f via i.p. 24 h prior to LPS treatment. Concurrent with 0.5 mg/kg LPS treatment, mice received a second dose of 14f. Blood was collected via cardiac puncture 6 h after LPS and triterpenoid treatment and nitric oxide (NO) was measured using a Griess Assay. *P <

0.05.

154 1 A1.1. H-NMR (400 MHz, pyridine-d5) of bryonolic acid.

155 13 A1.2. C-NMR (100 MHz, pyridine-d5) of bryonolic acid, 40-180 ppm.

156 13 A1.3. C NMR (100 MHz, pyridine-d5) of bryonolic acid, 15-55ppm.

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