A High Throughput Ultrafiltration LC-MS Platform for the Discovery of Vitamin D Receptor Ligands

Jerry James White

B.S. (University of California at Riverside) 2006

THESIS

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medicinal Chemistry in the Graduate College of the Univeristy of Illinois at Chicago, 2012

Chicago, Illinois

Defense Committee:

Richard B. van Breemen, Advisor and Chair Dejan Nikolic Pavel Petukhov Brian Murphy Adam Negrusz, Biopharmaceutical Sciences Copyright

Jerry James White

2012

ACKNOWLEDGEMENTS

This dissertation would not have been completed without the support of family, friends, fellow graduate students, the medicinal chemistry faculty, and my advisor, Dr. Richard B. van Breemen. To Dr. van Breemen I would like to state my appreciation for fostering my scientific development in his lab, through his highly technical knowledge, practical advice and his patient disposition.

I thank my dissertation committee members, Dr. Brian Murphy, Dr. Dejan Nikolic, Dr. Adam Negursz, and Dr. Pavel Pethukov, for their support and guidance with my research project.

I would like to thank Mr. Rich Morrissy for his advice both in the laboratory and outside of the lab. Drs. Dejan Nikolic, Carrie Crot, and Yongsoo Choi I would also like to thank for their constant guidance during the course of my graduate education.

There have been many graduate students that have stimulated intellectual debates that have shaped my research. I would like to recognize, Drs. Jeff Dahl and Shunyun Mo, Ms. Yang Yuan, Xi Qiu, Linlin Dong, Kevin Krock, and Jay Kalin.

Finally, I would like to thank my wife for her love and support and by simply being patient when life looked bleak at times.

iii TABLE OF CONTENTS

CHAPTER PAGE

1 INTRODUCTION ...... 1 1.1 Endogenous Effectors of the VDR Endocrine System ...... 1 1.1.1 The Steroid Hormone 1,25-Dihydroxycholecalciferol ...... 1 1.1.2 The Bile Salt Lithocholic Acid ...... 4 1.2 Nuclear Receptors ...... 7 1.2.1 Vitamin D Receptor ...... 9 1.2.2 Structure of the Vitamin D Receptor ...... 11 1.3 Classical Vitamin D Functions ...... 13 1.4 Non-classical Systems and Potential for Treatment Options . . 15 1.4.1 Immune Disorders ...... 16 1.4.2 Cardiovascular Health ...... 17 1.4.3 Brain Development and Function ...... 19 1.4.4 Maternal Health and Fetal Development ...... 19 1.4.5 Neoplastic Diseases ...... 20

2 QUANTITATIVE ANALYSIS OF CHEMOPREVENTATIVE AGENTS IN RAT TISSUE USING LC-MS-MS ...... 24 2.1 Introduction ...... 24 2.2 Materials and Methods ...... 26 2.2.1 Reagents ...... 26 2.2.2 Animals ...... 26 2.2.3 Sample Preparation ...... 27 2.2.4 LC-MS-MS of AM6-36 ...... 27 2.2.5 LC-MS-MS of PVN-1-48 ...... 31 2.2.6 LC-MS-MS of Casimiroin ...... 34 2.2.7 LC-MS-MS of SB-I-46 ...... 37 2.2.8 LC-MS-MS of 4-Styrylaniline ...... 40 2.2.9 LC-MS-MS of Abyssinone ...... 42 2.2.10 Validation ...... 44 2.3 Results and Discussion ...... 46 2.4 Conclusions ...... 56

3 DEVELOPMENT FO AN ULTRAFILTRATION LC-MS PLATFORM FOR VDR ...... 58 3.1 Introduction ...... 58 3.2 Materials and Methods ...... 61 3.2.1 Chemical and Reagents ...... 61 3.2.2 Ultrafiltration Screening Conditions ...... 61 3.2.3 LC-MS Conditions for the Proof of Principle Assay ...... 63 3.2.4 LC-MS Conditions for Screening of AM6-36 ...... 64 3.4 Results and Discussion ...... 65 3.5 Conclusions ...... 69

iv TABLE OF CONTENTS (Continued)

CHAPTER PAGE

4 HIGH THROUGHPUT ULTRAFILTRATION LC-MS ...... 70 4.1 Introduction ...... 70 4.2 Experimental Section ...... 72 4.2.1 Chemicals and Reagents ...... 72 4.2.2 Sample Preparation ...... 72 4.2.3 Screening Protocol ...... 74 4.2.4 LC-MS Conditions ...... 76 4.4 Results and Discussion ...... 76 4.5 Conclusions ...... 80

5 METABOLOMICS GUIDED PROCESSING OF COMPLEX ULTRA- FILTRATION DATA SETS FOR LIGANDS WITH THERAPEUTIC VALUE 83

5.1 Introduction ...... 83 5.1.1 Metabolomics Software ...... 84 5.1.2 Hypercalcemic Effects of Lead Structures ...... 85 5.2 Materials and Methods ...... 86 5.2.1 Chemicals and Reagents ...... 86 5.2.2 Extracts and Pure Compounds ...... 87 5.2.3 High Throughput Screening against VDR and ERp57 ...... 88 5.2.4 LC-MS Conditions ...... 88 5.2.5 Profiling Solutions ...... 89 5.3 Results and Discussion ...... 89 5.4 Conclusions ...... 111

6 CONCLUSIONS AND FUTURE DIRECTIONS ...... 113

CITED LITERATURE ...... 115

VITA ...... 130

v LIST OF TABLES

TABLE PAGE

TABLE 2.1 INTRADAY VALIDATION FOR PVN-1-48, CASIMIROIN, AM6-36, SB-I-46, ABYSSINONE, AND 4-STYRYLANILNE ...... 45

TABLE 2.2 BIODISTRIBUTION OF AM6-36 IN RAT ...... 48

TABLE 2.3 BIODISTRIBUTION OF PVN-1-48 IN RAT ...... 49

TABLE 2.4 BIODISTRIBUTION OF CASIMIROIN IN RAT ...... 50

TABLE 2.5 BIODISTRIBUTION OF SB-I46 IN RAT ...... 51

TABLE 2.6 BIODISTRIBUTION OF 4-STYRYLANILINE IN RAT ...... 52

TABLE 2.7 BIODISTRIBUTION OF ABYSSINONE IN RAT ...... 53

TABLE 4.1 COMPARISON OF NORMALIZED RESPONSES WITH RESPECT TO THE MOST POTENT LIGAND 8PN ...... 81

TABLE 5.1 COMPARISON OF NORMALIZED RESPONSES WITH RESPECT TO THE MOST POTENT LIGAND 8PN ...... 94

vi LIST OF FIGURES

FIGURE PAGE

Figure 1.1 Naturally occuring secosteroids ...... 2

Figure 1.2 Activation of vitamin D ...... 5

Figure 1.3 Major Inactivation Pathway for vitamin D through 23 or 24 Hydroxylation ...... 6

Figure 1.4 Common bile acids ...... 8

Figure 1.5 Classical actions of vitamin D ...... 14

Figure 1.6 Common immunomodulatory effects of vitamin D ...... 18

Figure 1.7 Common pathways observed in cancer ...... 22

Figure 2.1 Positive ion LC-MS-MS (API 4000) analysis of rat serum spiked with AM6-36 and internal standard UNC-01 using CID and SRM . . . . . 29

Figure 2.2 Positive ion electrospray CID product ion tandem mass spectrum of protonated AM-6-36 ...... 30

Figure 2.3 Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of PVN-1-48 and internal standard casimiroin spiked into rat serum . . . 32

Figure 2.4 Positive ion electrospray CID product ion tandem mass spectrum of protonated PVN-1-48 ...... 33

Figure 2.5 Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of casimiroin and internal standard PVN-1-48 spiked into rat serum . . . 35

Figure 2.6 Positive ion electrospray CID product ion tandem mass spectrum of protonated casimiroin ...... 36

Figure 2.7 Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of SB-I-46 and internal standard PVN-1-48 spiked into rat serum ...... 38

Figure 2.8 Positive ion electrospray CID product ion tandem mass spectrum of protonated SB-I-46 ...... 39

Figure 2.9 Positive ion LC-MS-MS (TSQ Quantum) analysis of rat serum spiked with 4-styryla niline and internal standard 8PN using CID and SRM . . . . 41

vii LIST OF FIGURES (continued)

FIGURE PAGE

Figure 2.10 Negative ion electrospray LC-MS-MS (TSQ Quantum) with CID and SRM of Abyssinone and internal standard 8PN spiked into rat serum . . . 43

Figure 2.11 LC-MS-MS standard curve for AM6-36 spiked into rat serum. UNC-01 was used as an internal standard ...... 48

Figure 2.12 LC-MS-MS standard curve for PVN-1-48 spiked into rat serum. Casimiroin was used as an internal standard ...... 49

Figure 2.13 LC-MS-MS standard curve for casimiroin spiked into rat serum. PVN-1-48 was used as an internal standard ...... 50

Figure 2.14 LC-MS-MS standard curve forSB-I-46 spiked into rat serum. PVN-1-48 was used as an internal standard ...... 51

Figure 2.15 LC-MS-MS standard curve for 4-styrylaniline spiked into rat serum. 8PN was used as an internal standard ...... 52

Figure 2.16 LC-MS-MS standard curve for abyssinone spiked into rat serum. 8PN was used as an internal standard ...... 53

Figure 3.1 Conceptual diagram of ultrafiltration LC-MS ...... 60

Figure 3.2 Structures of compounds utilized during the proof of princple stage of ultrafiltration LC-MS screening for the VDR ...... 62

Figure 3.3 Positive ion APCI of standards with calcitriol eluting at 4.0 min and vitamin D eluting at 5.4 min. The accurate mass spectra for calcitriol is displayed (below). Note the in-source fragment ions corresponding to the losses of water from the protonated molecule ...... 66

Figure 3.4 Positive ion APCI, ultrafiltraion LC-MS screening of calcitriol (retention time 4.0 min) for binding to the VDR (ligand binding domain). Calcitriol peak enhancement can be seen relative to the normalized control indicating specific bindind to the VDR ...... 68

Figure 4.1 Structures of compounds used a internal standards (IS), positive controls, and novel analytes that display binding to the VDR ...... 73

Figure 4.2 Evolution of ultrafiltration LC-MS ...... 75

viii LIST OF FIGURES (continued)

FIGURE PAGE

Figure 4.3 Positive ion electrospray UHPLC-MS analysis of ultrafiltraes for the ultrafiltration-MS screening of a libreary of 100 compounds for ligands to VDR. A) resolution of standard compounds, B) resultant overlayed ultrafiltration experiment, C) adverse feature of ultrafiltration . . . . . 78

Figure 5.1 Initial screening of 50 indenoisoquinoline analogs against VDR using positive ion UHPLC-MS. The concentration of VDR was 1 μM while the concentration of each indenoisoquinoline was 5 μM ...... 92

Figure 5.2 Structures of the indenoisoquinolines that displayed affinity to VDR during ultrafiltration UHPLC-MS screening in figure 5.1 ...... 93

Figure 5.3 Competitive ultrafiltration UHPLC-MS profiling of a mixture of 10 indenoisoquinoline ligands for relative affinities to VDR ...... 94

Figure 5.4 Structures of indenoisoquinoline analogs with affinities for both RXR and VDR ...... 95

Figure 5.5 Computer reconstructed mass chromatograms of VDR ligands discovered during ultrafiltration UHPLC-MS screening of 100 compounds . . . . . 97

Figure 5.6 Structures of compounds that displayed binding towards VDR during ultrafiltration UHPLC-MS as shown in figure 5.5 ...... 98

Figure 5.7 Screening of a 100-compound library for ligand binding to VDR . . . . . 100

Figure 5.8 Structures of compounds that displayed binding towards VDR during ultrafiltration UHPLC-MS screening shown in figure 5.7 ...... 101

Figure 5.9 Ultrafiltration UHPLC-MS screening of a directed library of reseveratrol analogs for ligands to the VDR. These computer reconstructed chromatograms show hits for approximately half of the directed library. See figure 5.10 for hits from the rest of the library ...... 103

Figure 5.10 Ultrafiltration UHPLC-MS screening of a library of resveratrol analogs for ligands to the VDR. Part 2 of 2 (see figure 5.9 for part 1) ...... 104

Figure 5.11 Chemical structures of resveratrol analogs that displayed binding towards VDR during ultrafiltration UHPLC-MS screening in figure 5.9 ...... 105

Figure 5.12 Chemical structures of resveratrol analogs that showed binding to VDR (Part 2 of 2. See part 1 in figure 5.11) ...... 106

Figure 5.13 Ultrafiltration UHPLC-MS Screening computer reconstructed chromatograms showing hits from a 100 compound library ...... 108

ix LIST OF FIGURES (Continued)

FIGURE PAGE

Figure 5.14 Structures of compounds that displayed binding towards VDR as displayed in the chromatogram in figure 5.13 ...... 109

Figure 15.5 Proof-of-principle binding of calcitriol to ERp57 obtained using a new ultrafiltration UHPLC-MS assay. Chromatograms for m/z 399 and m/z 381, which correspond to the sequential losses of water form calcitrol are shown. The dotted lines represent chromatograms for the negative control (nonspecific binding). Specific binding of calcitriol to ERp57 is indicated by enhancement of the peakat 0.5 min in the experimental ultrafiltrates comparedwith the negative control . . . . . 110

x LIST OF ABBREVIATIONS

7-DHC 7-dehydrocholesterol

6PN 6-prenylnaringenin

8PN 8-prenylnaringenin

ACN acetonitrile

ADME absorption, distribution, metabolism, excretion

APCI atmospheric pressure chemical ionization

AR androgen receptor

CA cholic acid

CaBP calcium binding proteins

CAR constitutive androstane receptor

CID collision-induced dissociation

CD clusters of differentiation

CDCA chenodeoxcholic acid

COX2 cyclooxygenase 2

CUR curcumin

DBD DNA binding domain

DCA deoxycholic acid

DNA deoxyribonucleic acid

CDK cyclin dependent kinase

CDKI cyclin dependent kinase inhibitor

EC50 half maximal effective concentration EIBPC extracted base peak chromatogram

ER estrogen receptor

ERE estrogen receptor element

ESI electrospray ionization

FDA federal drug and food administration

xi LIST OF ABBREVIATIONS (continued)

FGF23 fibroblast growth factor 23

FIA flow injection analysis

FXR farnesoid X receptor

GR glucocorticoid receptor hCAP cathelicidins

HPLC high pressure liquid chromatography

HTS-PUF high throughput screening pulsed ultrafiltration filtration

IC50 half maximal inhibitory concentration IFN-γ interferon gamma

IGF-1 insulin-like growth factor 1

IL Interleukin

IOM Institute of Medicine

ITTOF ion trap time of flight

IX isoxanthohumol kb kilobase

LBD ligand binding domain

LCA lithocholic acid

LC-MS liquid chromatography mass spectrometry

LOD limit of detection

LOQ limit of quantitation

LXR liver X receptor

MARRS membrane associated rapid response

MHC major histocompatibility complex

MMP metalloprotease

MWCO molecular weight cutoff

NCoR nuclear receptor co-repressors

xii LIST OF ABBREVIATIONS (continued)

NO nitric oxide

NR nuclear receptor

NSAID nonsteroid anti-inflammatory drug

ODC ornithine decarboxylase

OPG osteoprotegerin

Pdia3 protein disulfide isomerase family, member 3

PTH parathyroid hormone

PR progesterone receptor

PXR pregnane X receptor

QR quinone reductase

RA retinoic acid

RAR retinoic acid receptor

Rb retinoblastoma pocket proteins

RNA ribonucleic acid

RANKL receptor activator of nuclear factor kappa-β ligand

RXR retinoid X receptor

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SIM selected ion monitoring

SMRT silent mediator for retinoid

SOP standard operating procedure

SRM selected reaction monitoring

TFA trifluoroacetic acid

TGF- β1 transforming growth factor-β1 Th helper T cells

TLR toll like receptor

TR thyroid hormone receptor

xiii LIST OF ABBREVIATIONS (continued)

Treg regulatory T cells

TRPV transient receptor potential vanilloid

TNF α tumor necrosis factor-α

UHPLC ultra high pressure liquid chromatography

UIC University of Illinois at Chicago

UV ultraviolet

VDR vitamin D receptor

VDRE vitamin D response element

xiv PERMISSIONS

Figure 1.5 was reprinted with permission Holick, M. F. (2007) Vitamin D Deficiency, New England Journal of Medicine 357, 266-281. Copyright the New England Journal of Medicine doi:10.1056/NEJMra070553 http://www.nejm.org/doi/full/10.1056/NEJMra070553

Figure 1.6 was reprinted with permission Hart, P. H., Gorman, S., and Finlay-Jones, J. J. (2011) Modulation of the immune system by UV radiation: more than just the effects of vitamin D?,Nature Reviews Immunology 11, 584- 596. Copyright Nature Publishing Group, a division of Macmillan Publishers Limited doi:10.1038/nri3045 http://dx.doi.org/10.1038/nri3045

Figure 1.7 was reprinted with permission Deeb, K. K., Trump, D. L., and Johnson, C. S. (2007) Vitamin D signalling pathways in cancer: potential for anticancer therapeutics, Nature Reviews Cancer 7, 684-700. Copyright Nature Publishing Group, a division of Macmillan Publishers Limited doi:10.1038/nrc2196 http://www.nature.com/nrc/journal/v7/n9/full/nrc2196.html

xv SUMMARY

The essential qualities of vitamin D as the antirachitc factor have been well described during the past century. However, vitamin D is not a true vitamin, since the primary source of this essential nutrient in humans is the epidermis. The unique photochemical reaction relies on a specific wavelength of UV light, which is only produced in the summer months at northern latitudes. The relationship between vitamin D, UV light, and geographical location has giving rise to many epidemiological studies that have correlated vitamin D deficiency with a myriad of diseases. Vitamin D supplementation for many of these diseased processes requires very large doses of this calcitropic compound, resulting in a toxic condition known as hypercalcemia. Therefore, the purpose of this dissertation has been to discover ligands that elicit the desired effects against specific disease states without the adverse calcemic response.

The initial discovery of ligands that bind to the nuclear receptor associated with vitamin D (vitamin D receptor) came about with the quantitative analysis of chemopreventative agents described in chapter 2. Then, based on our ultrafiltration liquid chromatography mass spectrometry approach to solution phase screening of purified compounds and extracts a vitamin D receptor assay was developed. Chapter 3 describes this process and demonstrates the binding of the first nonsecosteroid compound discovered by our lab.

Our laboratory has expanded our screening efforts and is actively receiving many more synthetic compounds and extracts than ever before. Chapter 4 describes a high throughput approach to the ultrafiltration platform that has expanded the sampling capacity of all ultrafiltraton screening targets by 100-fold. Chapter 5 merges the topics of chapter 3 and 4 by employing the newly developed high throughput ultrafiltration approach to the VDR assay. An effort was made to screen as many samples as possible. In order to process a vast number of data sets a metabolomics based software package was evaluated for ultrafiltration data comparison.

xvi CHAPTER 1

INTRODUCTION

1.1 Endogenous Effectors of the VDR Endocrine System

The history of vitamin D displays a cyclic quality with periods of innovative research leading to the discovery of unique qualities associated with vitamin D and its endocrine response interspersed with long periods of marginal advancements. Vitamin D was named pragmatically assigning it the fourth letter of the alphabet, since it was the four vitamin discovered. The therapeutic value of vitamin D was realized with the discovery of the antirachitic factor found within cod liver oil and the subsequent determination of the involvement of UV (ultraviolet) light with the generation of this essential nutrient (1, 2). As a direct consequence of these ground breaking discoveries, the prevalence of rickets diminished greatly in developed countries. Research concerning vitamin D tapered off when supplementation was implicated as the root cause of idiopathic hypercalcemia (3). With the discovery of the vitamin D constituent that produces the endocrine response and the cloning of the VDR in the 1980s, there was a revitalized interest in vitamin D and its pleiotropic nature (4, 5). Current recommendations for vitamin D supplementation that promote adequate bone health were provided by the Institute of Medicine (IOM), which recommends a daily supplement of 600 IU/d for all individuals between the 1-70 years and 800 IU/day for individuals over 70 yr (6). However, these recommendations are considered insufficient by those who contend that greater amounts are needed to produce many of the proposed auxiliary responses (7, 8).

1.1.1 The Steroid Hormone 1,25-Dihydroxycholecalciferol

Cholecalciferol (vitamin D3) serves as the pro-hormonal form of the calciotropic hormone 1α,25-dihydroxycholecalciferol (calcitriol) in humans, however several other sterols structurally similar to vitamin D3 have been identified (9). In particular, ergocalciferol (vitamin 2D ) is derived from fungus/yeast and is of interest due to its prevalence in nutritional supplements, (see figure

1 2

Figure 1.1. Naturally occuring secosteroids

H H

HO HO HO Vitamin D Lumersterol 2 Vitamin D3

H H H

HO HO HO Vitamin D 4 Vitamin D5 Vitamin D6 3

1.1 for structures of common natural vitamin D analogs). Considerable debate over the clinical effectiveness of vitamin 2D is in the current literature (10-12). The main concerns about vitamin D2 include increased metabolic clearance, disparity of binding affinities towards all essential targets, and the stability and bioavailability of various formulations (13).

The photochemical conversion of 7-dehydrocholesterol (7-DHC) which is found primarily in keratinocytes concentrated in the two inner most layers of the epidermis serves as the most effective route to acquire vitamin D. During peak periods of UV-B light (290-315 nm) production e.g. summer months an individual can generate sufficient levels of vitamin D within a 13 minute period (14). There are many confounding factors which limit the endogenous skin production of vitamin D, which include latitude, season, aging, melatoin content, regional attire, and an increase in indoor activities (15). Consumption of nutrient rich dietary sources (fatty fish, sun dried mushrooms, fortified dairy products) can compensate for a deficit acquired during periods of low UV-B exposure (e.g., winter months) (16). Mechanistically, irradiation of 7-DHC with UV-B light results in a spontaneous electrocyclic reversion and subsequent thermal isomerization of the B ring conferring the characteristic secosteroid structure of vitamin D (17). Photo irradiation with

UV-B is a self-regulating process where the formation of pre-vitamin D3 can isomerize to vitamin

D3 or back to 7-DHC, similarly pre-vitamin D2 will shift back to inactive products lumisterol and tachysterol. Additional regulatory measures are for vitamin D3 to enter circulation or absorb excess UV-B light and isomerize to a variety of products including, suprasterol I, suprasterol II, and

5,6-transvitamin D3 (18, 19). The photolytic conversion of 7-DHC to vitamin D is shown in figure 2. Newly produced vitamin D circulates systemically with a ~4-6 h half-life, ultimately ending up at the liver where its structure is modified further (20).

When it was recognized that vitamin D must be metabolically activated in order to elicit a response, the structures of the most important vitamin D metabolites, 25-dihydroxycholecalciferol (calcidiol) (21) and the hormonal form calcitriol (22), were elucidated. These seminal discoveries along with many subsequent studies demonstrated the vast array of oxidized metabolites of 4

vitamin D that are formed in vivo by the cytochrome P450 enzymes. A search for the mixed function oxidases capable of forming these hydroxylated products was initiated (23). Metabolic activation of vitamin D was found to be accomplished through the sequential hydroxylation of carbons 25 and 1, respectively.

Hepatic tissue contains at least four microsomal cytochrome P450 isozymes (CYP2D11, CYP2D25, CYP3A4, and CYP2R1) that are able to hydroxylate carbon 25 of the seco-steriod with CYP2R1 displaying the greatest affinity towards vitamin D (24). The 25-hydroxylase is constitu- tively active and seemingly without regulation generating the most prevalent vitamin D species found in serum, calcidiol. A plasma protein, structurally similar to albumin (vitamin D binding protein) binds this metabolite with high affinity and significantly enhances its circulating lifetime to approximately 2-3 weeks (25). In contrast, only one known enzyme possess the 1α-hydroxylase reactivity hydroxylation (CYP27B1) capabilities which performs its action in the mitochondria of the proximal convoluted and straight tubules of the kidney. Within renal cells, CYP27B1 function is tightly mediated by Ca2+ and Pi as well as by a host of regulatory hormones demonstrating control through stimulatory (PTH, calcitonin, GH, and IGF-I) or inhibitory (FGF23 and calcitriol) actions (26). More than 30 metabolites of vitamin D have been identified usingin vitro and in vivo systems, with the majority formed by a single enzyme CYP24A1. Multiple catabolic staging steps produce many metabolites from the substrates calcidiol and calcitriol. Calcitroic acid formation is the end product after an initial 24-hydroxylation, while the 23-hydroxylation side chain lactone product serves as the major detoxification pathways (27). The principal metabolic activation and inactivation pathways of vitamin D are summarized in figures 1.2 and 1.3, respectively.

1.1.2 The Bile Salt Lithocholic Acid

Bile is produced by hepatocytes and primarily consists of water, cholesterol, phospholip- ids, bilirubin, and bile acids. The gallbladder stores bile and releases it into the duodenum after ingesting of food, where it acts as a surfactant, emulsifying fats contained within the consumed material. This process increases the absorption of lipid soluble nutrients including vitamins D,E, 5

Figure 1.2. Activation of vitamin D

UVB 270-300 nM

CH3 Electrocyclic Reaction [1,7] Hydride Shift

7-Dehydrocholesterol Pre-vitamin D Vitamin D

OH OH

CYP 2R1 CYP 27B1 Liver Kidney

HO HO OH Calcidiol Calcitriol 6

Figure 1.3. Major Inactivation Pathway for vitamin D through 23 or 24 Hydroxylation

OH OH

H H

CYP24A1

HO HO

CYP27B1

OH OH OH

H H CYP24A1 CYP24A1

HO OH HO OH HO OH

CYP24A1 CYP24A1

O OH O

O H H

HO OH HO OH 7

K, and A (28). Bile acids are then absorbed in the terminal portion of the ileum through passive and active transport and recirculated through an enterohepatic mechanism back to the liver. Nuclear Receptors (NRs), FXR, PXR and VDR are stimulated by bile acids, which in turn activate a wide range of signaling pathways that mediate the synthetic and enterohepatic mechanisms associated with self-regulation, triglycerides, cholesterol, energy and glucose homeostatsis. Bile acids are generated through the principle catabolic pathway for cholesterol. Modification of the fused ring structure of cholesterol, oxidation and conjugation, as well as the truncation of the side chain constitutes the biosynthetic pathway of bile acids. In humans, cholic acid (CA) and chenodeoxycholic acid (CDCA) are the most abundant primary bile acids, while their associated secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA) are also generated in appreciable amounts by microbial enzymes present in the colon (figure 1.4) (29). LCA and 3-keto-

LCA activate VDR and display effective concentrations (EC50) of 8 µM and 3 µM, respectively. These finding suggest that VDR-mediated actions have evolved with the ability to sense toxic levels of this secondary bile acid and mitigate its clearance in a proactive manner before adverse effects are seen (30, 31).

1.2 Nuclear Receptors

The nuclear receptor (NR) superfamily comprise a distinct grouping of evolutionarily related transcription factors mediated through ligand activated molecular complexes, which directly interact with specific DNA sequences (32). Certain NRs are localized to the nuclear portion of the cell irrespective of ligand presence, while other NRs, such as the androgen receptor (NR3C4, AR), glucocorticoid receptor (NR3C1, GR), and progesterone receptor (NR3C3, PR) are found within the cyptoplasm during periods of ligand absence (1). A host of biological actions are directly influenced by a single binding event, controlling cellular processes involved in differentiation, development, homeostasis and behavior. Each NR is capable of generating a myriad of essential and non-redundant biological responses stimulated by the interactions of specific small lipophilic molecules that are derived from both endogenous and external sources including, for 8

Figure 1.4. Common bile acids

H H

HO Cholesterol

O O OH

H OH O

H H H H

H H HO OH O O Cholic Acid Chenodeoxycholic acid

O OH O

OH OH

H H H H

HO HO Deoxycholic acid Lithocholic acid 9 example, the class of steroid hormones, thyroid hormone, and bile salts. Currently, 48 members of the NR superfamily have been identified through sequencing the human genome have been organized into 6 families consisting of NR1, NR2, NR3, etc. (7). However, only half of the known NRs have defined ligands giving rise to a subset designated as orphan receptors (33).

NRs profoundly influence the transcriptional output of a cell when acted upon by the appropriate substrate through either stimulatory or inhibitory mechanisms. The complexity of NR signaling expands exponentially when all aspects of each ligand protein complex are examined; which avenue will the ligand mediated response take depends on its mode of ligand-dependent or independency, genomic or non-genomic, gene repression, activation, or trans-repression (34). Taken together all of these pathways provide many avenues that can be exploited for drug discovery purposes; however the converse posit is also valid confounding the prospect of selective compounds (35).

1.2.1 Vitamin D Receptor

In the mid-1970s, a series of experiments demonstrated that a macromolecular protein, contained within the chromatin fraction of chick intestine was capable of binding the putative ligand calcitriol (36, 37). This protein is known today as the vitamin D receptor, VDR. The VDR is expressed in all nucleated cells with a low expression rate of a few copies per cell up to 25,000 copies per cell. VDR belongs to the NR1I subfamily of NRs, which were classically thought to be retained to the nuclear portion of the cell regardless of ligand binding status. Ligand-activated complexes of the NR1 sub-grouping hetero-dimerize with un-liganded retinoid X receptors

(RXR, α,β,γ; NR2B1, 2B2, and 2B3) (38). Specific vitamin D mediated actions pertaining to rapid response mechanisms have been reported to be localized to an ancillary position near the plasma membrane (39). Recently, some groups have contended that a separate membrane associated rapid response (MARRS) protein (11, 40) is involved with calcitriol mediated actions, while others maintain that VDR itself generates these actions through ligand conformational changes in conjunction with overlapping binding pockets (41, 42). 10

VDR displays a high degree of structural homology, ligand binding, and functionality within all mammals, bird, amphibians, and fish that have a calcified bone structure. A grouping of NRs (PXR, CAR, and FXR and the oxy-steroid receptors LXR α and β) producing metabolic actions share the greatest similarity in structure with VDR. VDR, PXR, and CAR have all demonstrated varying abilities to bind to bile acid metabolites and or activate CYP enzymes involved with the detoxification of xenobiotics (43). Un-liganded VDR associates with a variety of co-repressors (silent mediator for retinoid (SMRT) and thyroid hormone receptors (TR), NR co-repressors (NCoR), Alien or sin3) keeping this potent NR transcriptionally silent. When calcitriol binds to VDR, it is phosphorylated at specific serine residues, which in turn triggers a conformational change (dramatic rearrangement of H12 among others) allowing for the release of the associated co-repressor species (16). In addition, the binding event stimulates the association of VDR to its dimerization partner which then binds to its corresponding vitamin D response elements (VDRE). VDREs are generally comprised of two hexa-nucleotide half elements arranged as direct repeats with a three nucleotide spacer (DR3) or as an inverted palindrome sequence spaced six nucleotides (ER6) (44). The optimum VDRE was determined experimentally through random- ized oligonucleotide binding to the VDR-RXR complex, and was found to be a DR3 variety with a base sequence AGGTCA similar to the estrogen response element (ERE) (45). Binding of the VDR-RXR to positive regulating VDREs displays a specific polarity with VDR binding to the 3’ half site while RXR gravitates towards the 5’ site (46). A gene regulated by the actions of vitamin D can contain multiple VDREs in their promoter sequence, which may not be in close proximity to the coding region (47, 48). Binding of calcitriol leading to the VDR-RXR heterodimer is not the only modulatory factor which mediates gene expression through DNA interactions. An array of specialized co-activators are recruited by the dimeric species ultimately forming a multifaceted complex consisting of chromatin modulators, basal transcription factors, and RNA polymerase II, in addition to a host of accessory and unrelated proteins displaying co-activator roles (49, 50).

The location of the human VDR gene was determined through a series of molecular biology techniques elucidating its precise location on chromosome 12q 13-14. The nuclear recep- 11 tor family shares a common structural organization with the coding regions of their genes. The VDR gene contains approximately 75 kilobases (kb) of genomic DNA arranged with in a complex structure of 11 coding exon sequences interspersed between sequences of introns ranging from 0.2 to 13 kbs in length (51). Currently, three forms of human VDR have been identified, and a certain proportion of the expressed VDR products are alternatively spliced (52). In particular, a 50 amino acid-extended VDRB1 variant originates from alternative posttranscriptional splicing and the use of a more upstream in-frame start codon which can be found in most humans cells (53). A variety of gene polymorphisms associated with the VDR protein have been identified including the commonly studied restriction fragments Bsml, ApaI, and TaqI, further complicating the link between VDRs biological responses and disease onset (54). Ultimately, a messenger RNA is produced and translated into a protein consisting of 427 amino acid residues and a molecular mass of approximately 50 kDa, with an overall structure relatedness to all members of the NR superfamily. A varying degree of sequence similarity can be seen, however, five distinct sub-domains following the general designations: A/B, C, D, E, and F are consistent throughout its members (55).

1.2.2 Structure of the Vitamin D Receptor

An analysis of the crystallographic evidence and homology models for VDR indicates a three layer system consisting of 12 α-helices and a short β-sheet of three strands (56, 57). The N- terminus A/B domain extends to the DNA binding domain (DBD) and is a short region displaying hyper-variability. VDR’s atypically small A/B domain consists of only 20 amino acid residues, which is in contrast to the glucocorticoid receptor (GR) containing over 200 residues. The function of VDR has been shown to be independent of the A/B domain as the deletion of all 20 residues will not interrupt VDR mediated transcription in this system (58). The most conserved domain among nuclear receptors is the DBD (C region), which contains nine cysteines found ubiquitously throughout the family (59). The first eight cysteines have proven to be essential to the function of VDR as determined through direct mutagenesis studies. These cysteine residues form a DNA binding motif known as zinc fingers via the coordination of two zinc atoms with four cysteine 12 residues. This region is also important for the nuclear translocation of VDR (60, 61). A hinge region (region D) connects the DBD with the ligand binding domain (LBD) and contains an amino acid sequence not homologous to other nuclear receptors with approximately 50 more residues than other receptors. Additionally, region D in human VDR displays little homology to the VDRs of other species. This region seems to serve as a highly flexible linker between regions C and E im- parting a high degree of rotational freedom allowing for the binding of a variety of DNA response elements and the mediation of nuclear receptors with select co-repressor proteins.

In order to generate high quality crystals of VDR, a flexible extension of the LBD found between helix 1(H1) and helix 3 (H3) must be omitted. Therefore, a construct minus the extra region of the LBD was assembled, and the structure of VDR co-crystallized with calcitriol was determined (57). Using this crystal structure, an extensive assessment of the ligand binding dynamics was conducted. A large binding pocket of approximately 700 Å3 consisting primarily of hydrophobic amino acid residues was found to be the characteristic feature of the VDR LBD. Calcitriol curves around H3 with its A-ring interacting with the C-terminus of H5 and the 25-hydroxyl end positioned near H7 and H11. The 1-hydroxyl forms two molecular connections with the amino acid backbone via hydrogen bonds with Ser-237 (H3) and Arg-274 (H5), while the 3-hydroxyl forms two hydrogen bonds with Ser-278 (H5) and Tyr-143. The remnants of the steroid B ring forms a conjugated diene that is held in position by the stabilizing planar interactions of Trp-286. The α-face of the C ring contacts Trp-286, whereas the C18 methyl group points toward Val-234 (H3). The D ring aliphatic side chain adopts an extended conformation while surrounded by hydrophobic residues. The 25-hydroxyl group is hydrogen bonded to His-305 (H6) and His- 397 (H11). Based on the molecular nature of the binding pocket, it is also observed that His-305 would be a hydrogen bond acceptor, whereas His-397 would be a donor. H12 displays multiple conformations based on ligand binding and is crucial for co-activator binding, transactivation absorption in the intestine, and reabsorption within the kidney, as well as bone remodeling properties (42, 62). 13

1.3 Classical Actions

VDR stimulated by calcitriol has been shown to control the expression of at least 11 genes (SPP1, TRPV6, LRP5, BGP, RANKL, OPG, CYP24A1, PTH, FGF23, PHEX, and klotho) all of which serve as effectors for bone and mineral homeostasis (63). Intestinal (duodenum) and renal calcium transfer consists of a passive concentration gradient dependent para-cellular transport and an active ATP-dependent trans-cellular transport. The latter is largely regulated by calcitriol and is critical when calcium supply is low. Calcium transported in a trans-cellular manner involves three sequential steps all of which are controlled by the actions of calcitriol. An influx of calcium into the epithelial cells mediated by apical calcium channels of the transient receptor potential vanilloid (TRPV) family and promoted by steep electrochemical gradient across the apical membrane (64). Calcium is then transported through the cytosol bound to calcium binding proteins, calbindins (CaBPs). Finally, the extrusion of calcium across the baso-lateral membrane into the extracellular fluid is performed by an energy requiring process (65). Figure 5 displays calcitriols regulatory actions in the intestine.

Another classical site of action for calcitriol is bone where its actions have profound effects on the remodeling of this tissue. Calcitriol with the concurrent presence of PTH stimulates bone resorption by activating the receptor mediated response in osteoblasts, presumably by stimulat- ing a cytokine signal within these cells. This action increases the recruitment, differentiation and fusion of precursors into active osteoclasts able to perform the bone resorption process. Bone formation is also dependent on vitamin D, which mediates the normal mineralization of newly formed osteoid along the clacification front. In individuals with a vitamin D deficiency, excess osteoid accumulates from lack of calcitriol repression of collagen synthesis by osteoblasts leading to a weakened bone (66). Calcitriols mediation of bone remodeling processes is shown in figure 1.5, reprinted from the New England Journal of Medicine with permission (67).

The molecular action of the VDR with calcitriol has proven to be a dynamic process with the regulation of approximately 3% of the human geneome by the vitamin D endocrine response. 14

Figure 1.5. Classical actions of vitamin D 15

With the virtually ubiquitous expression of VDR and diverse cellular response to calcitriol exposure, this molecular interaction has only recently begun to demonstrate its pleotropic nature (68). Calcitriol mediates a variety of biological functions, and these effects can be slow to develop via the nuclear transcription pathway or rapid by inducing a cascade of signal transduction pathways. The well-defined homeostatic role with balancing calcium and phosphate stores based on its actions in the kidney, bone, and intestine has only recently been exposed as a small subset of its diverse actions. This has spurred investigators to probe the actions of calcitriol and VDR within a diverse set of tissue and diseases, including the malignancies that occur within these tissues (69).

1.4 Non-Classical Systems and Potential for Treatment Options

In 1981, serum levels of calcitriol were found to be elevated amongst 10% of patients presenting with granuloma-forming diseases such as sarcoidosis (70). The concept of extrarenal expression of the metabolic enzyme (1α-hydroxylase) was established in diseased macrophages converting large amounts of circulating calcidiol in calcitriol. Consequently, a host of subsequent studies have explored the potential immunomodulatory properties of calcitriol and its metabolites (71). In 1986, Rook et al., described additional qualities of vitamin D with cultured human macrophages which demonstrated that calcitriol inhibits the growth of Mycobacterium tuberculosis (72).

The anti-proliferative properties of calcitriol have been explored in considerable detail with respect to its potential use as therapy for various common cancers. The major limitation with this strategy stems from the potent hypercalcemic properties of calcitriol associated with its classical endocrine role. As a result, significant effort has been devoted to generate synthetic analogs of calcitriol that retain the antiproliferative activity while diminishing the calciotropic response (73). Similar strategies have also predominated to advance further immunological applications for vitamin D (16). 16

1.4.1 Immune Disorders

Dendritic (DC) and macrophage immune cells are derived from myeloid progenitors and possess constituently active VDR as well as CYP 27B1 and 2R1. Lymphocytes B and T cells develop from the lymphoid progenitor and maintain constitutive CYP27B1 activity with inducible VDR expression. A distinct mechanism outside of the calcitrotropic controls regulates the production of 1α-hydroxylase within these cells, namely interferon (IFN)-γ and lipopolysaccharides (LPS) (74). The VDR mediated response acting on immune cells results in an overall inhibitory effect on adaptive immunity while bolstering the innate response as well as demonstrating an essential role in prevention of bacterial infections (75).

Calcitriol aids the maturation process of macrophages by promoting monocytic precursors into a mature differentiated state. Vitamin D deficiency has been shown to blunt the maturation of macrophages; in addition, these macrophages display a diminished ability to express the major histocompatibility complex class II (MHC-II) (76). Macrophages treated with calcitriol display an enhanced expression of macrophage specific surface antigens as well as the lysosomal enzyme acid phosphatase. This process is essential for antimicrobial actions resulting in an oxidative burst, which stimulates chemotaxis and phagocytosis. Additionally, calcitriol acts through an inhibitory mechanism to regulate macrophage secretions of inflammatory cytokines and chemo- kines (77). Toll like receptors (TLR) on the macrophage periphery bind to bacterial lipo-peptides and stimulate the expression of CYP27B1 and VDR (78). Calcitriol enhances the antibacterial actions of myeloid cells through the generation of short cationic peptides known as cathelicidins (hCAP) or to a lesser extent β-defensins when stimulated through TLRs (72). The generation of these antibacterial peptides as well as the suppression of metalloproteases (MMP) 7 and 10, and the induced secretion of interleukin (IL) 10 and prostaglandin E2 by calcitriol display preventative effects against Mycobacterium tuberculosis (79).

DCs are antigen presenting cells and are negatively regulated by calcitriol through a variety of mechanisms. Calcitriol influences the differentiation, maturation and immune-stimulatory 17

ability of DCs through decreased expression of MHC II, the clusters of differentiation (CD) 40, 80, and 86 (61)Cz, and the maturation proteins CD1a and CD83 (80). Additionally, calcitriol negatively regulates the synthesis of the pro-inflammatory cytokines IL-6, IL-12, and IL-23. The net effect of calcitriol on DC cells is the diminished antigen presenting compactly toward T cells (67).

At a specific threshold, calcitriol mediates B cells by inhibiting proliferation through apoptotic mechanisms. Signs of cell cycle arrest can be seen within calcitriol treated B cells, with the up-regulation of p27, and the decreased mRNA of CDK4, CDK6, and cyclin D. Activated T cells display a significant increase in VDR levels upon activation and vitamin D targets both Th1 and Th2 cell types. Calcitriol tailors the T cell response by attenuating Th1 actions while supporting the development of a Th2 dominant environment. This is accomplished through the adulteration of effecter molecule secretion patterns including the inhibition of IL-2, IFN-γ, tumour necrosis factor-α and IL-5 in Th1 cells. Conversely, transforming growth factor-β1 (TGF- β1) and IL-4 serve as an immunosuppressive and promoting Th2 phenotype. Calictriol administration also stimulates the development of regulatory T cells (Treg) as well as Th17 cells, which potentiate the Th2 response. The VDR stimulation by calcitriol serves as a treatment option for Th1 mediated autoimmune diseases including, systemic lupus erythematosus, type-1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and psoriasis (81). Figure 1.6 displays common immune effects and was obtained from Nature Reviews Immunology with permission from (82).

1.4.2 Cardiovascular health

The cardiovascular system incompasses a variety of anatomical structures and is com- posed of multiple tissue types. Vitamin D has been shown to affect the vascular endothelium through its mediation of proper vascular tone of endothelial cells. Significant inhibition of an- giogenesis occurs with calcitriol treatment of endothelial cells (83), and through the regulation of pro-inflammatory cytokine production, a marked decrease in vascular inflammation can be seen

(84, 85). The VDR and 1α-hydroxylase are localized in cardiomyocytes and fibroblasts which give rise to investigations into the roles of vitamin D and cardiac development as well as tissue spe- 18

Figure 1.6. Common immunomodulatory eﬀects of vitamin D 19 cific diseases (86). High doses of vitamin D result in an increase incidence of arteriosclerosis and vascular calcification (87); however appropriate amounts within the normal physiological range show the converse protective effect (88). The homeostatic role of the rennin-angiotensin system is attenuated by the VDR, resulting in lowered hypertension and decreased overall cardiac risk (89).

1.4.3 Brain Development and Function

Calcidiol has been shown to penetrate the blood brain barrier similarly to retinoic acid (65), and radiolabeled calcitriol was found to localize in particular segments of the brain (90). The VDR was identified in brain tissue when antibodies were created allowing for the immunohisto- chemical detection of this protein (91). Neuro-protective effects of vitamin D include regulation of extracellular calcium (91), mitigation of pro-inflammatory cytokines, and control of glucocor- ticoids (92) that can cause neuronal atrophy. Epidemiological studies implicate vitamin D status in a wide range of neuropsychiatric disorders such as schizophrenia (93), depression (94), and cognitive impairment (95). Vitamin D insufficiency has also been implicated as a neuro-protective agent for dopamine neurons mediating abnormal signaling in Parkinson disease models (96).

1.4.4 Maternal Health and Fetal Development

Maternal vitamin D inadequacy has been shown to be highly prevalent amongst women during pregnancy up through delivery and involve a variety of ethnic groups as well as dwelling latitudes. Although, prenatal vitamins are ubiquitously prescribed, the representative values hy- povitaminosis D as defined by the systemic calcidiol concentration (<25-50 nmol/L) vary widely amongst literature sources. Vitamin D insufficiency ranges from 5-20% in women displaying light complexion, while women with darker skin show a markedly increase incidence of 30-70% (97). Given the many disease states associated with vitamin D deficiency, many researchers have postulated that the role of vitamin D in maternal health and fetal development is well beyond proper bone development. Additionally, it has been determined that the sole source of calcidiol for the 20 fetus is obtained through maternal (98).

Vitamin D deficiency has been shown to reduce the fertility of VDR or CYP27B1 null animal models. Implications have arisen for a number of serious consequences to both mother and fetus that manifest when suboptimal circulating maternal and consequentially fetal vitamin D concentrations are reported. Several complications that arise during pregnancy have been associated with hypo-vitaminosis D including preeclampsia (99), gestational diabetes (100), intrahepatic cholestasis of pregnancy (101), incidence of cesarean section (102), and bacterial vaginosis (103). While the onset of type 1 diabetes (104), inflammatory and atopic disorders (105) as well as schizophrenia (96) have been linked to vitamin D insufficiencies during gestation. The previously described immunological roles associated with vitamin D are assumed to be part of the underlying processes for many of these disorders.

1.4.5 Neoplastic Diseases

Nearly all nucleated cells within the body express the VDR although concentrations can be exceedingly low. Additionally, the molecular machinery utilized for bio-activation of calcidiol (1α-hydroxylase) and the catabolic enzyme 24-hydroxylase are present and inducible. The response of these cells to (supra)physiological concentrations of calcitriol can involve inhibition of cell proliferation, typically characterized by a G0/G1 cell cycle arrest (106). This action of calcitriol has been extensively studied and described within malignant and normal cell lines. Calcitriol mediation of cell cycle arrest is less pronounced than classical chemotherapeutic agents, and its growth inhibition actions are greatly influenced by cellular sensitivity and specific cell type (107).

An investigation into the mechanisms associated with the underlying molecular actions within malignant cells reveals a complex network of signaling pathways. In line with a G0/G1 cell cycle arrest, calcitriol was found to affect the phosphorylation state of the retinoblastoma

(Rb) pocket proteins (especially p107 and 130) (49). Their involvement in cycle cell regulation as gatekeeper proteins is a crucial restriction point at the end of the G1-phase where cells are either 21 allowed or disallowed from proceeding into S-phase. VDRs actions in cell cycle progression have been implicated in the down regulation of several cyclins and CDK inhibitors (CDKI), and the upregulation of cyclin dependent kinases (CDK); p19, p21 and p27) or by other unknown mechanisms (68). These actions allow for calcitriol mediation of the phosphorylation state of Rb through direct or indirect means. Consequently, Rb binds and inactivates E2F transcription factors which are crucial for cell cycle progression since it has been demonstrate that a large number of E2F- target genes are involved in cell cycle progression and control. They can act as a master switch to regulate a variety of genes responsible for cell cycle progression, DNA replication and repair, etc. Additionally, calcitriol can have many other effects on the cell cycle through the up-regulation of Tiff and IGF-BP3 and by down-regulating the EGF-R receptor, c-myc, jun and fos (106). Calcitriol abrogates the action mediated by prostaglandins thereby decreasing its stimulation of cellular growth (108). Figure 1.7 shows biological pathways implicated in cancer processes, reprinted from Nature Reviews Cancer (49) with permission. 22

Figure 1.7. Common pathways observed in cancer 23

Several large scale epidemiological studies (109) have revealed an inverse relationship between vitamin D and UV-B exposure with the incidence of colorectal (110), breast (111) or prostate cancer (112). Several VDR gene polymorphisms have also been implicated with an increased prevalence of cancer (113). UV-B exposure as a predictor of vitamin D status has proven to be unreliable. Sampling of serum for the levels of calcidiol has proven to be the most reliable method for drawing correlations between the occurrence of cancer and other diseases (114). Several in- tervention studies which provided vitamin D supplementation have been conducted; however, no causal relation with cancer incidence can be confirmed. Calcitriol and a variety of synthetic analogs have been able to significantly reduce tumor growth in animal models of colon, breast, prostate and other cancer types (73). In addition, combination therapies with vitamin D analogs and classical chemotherapeutic agents have displayed at least additive effects (107). It has been postulated that the typical dosing regimen of 400 IU/d is significantly undervalued especially when global vitamin D deficiency has been reported to be a common occurrence. Many questions have yet to be resolved with this potential regulator of malignant processes, and multiple areas within this field need further investigation. CHAPTER 2

QUANTITATIVE ANALYSIS OF CHEMOPREVENTATIVE AGENTS IN RAT TISSUE

USING LC-MS-MS

2.1 Introduction

Cancer can be described broadly as the aberrant cellular growth and division without regulation which is implicated in a wide variety of disease states. To date, approximately 200 varieties of cancer have been classified according to pathology, molecular biology, anatomic and physiologic characteristics as well as numerous other factors all of which further complicate the diagnosis and treatment process (115). In previous generations, life expectancy limited the prevalence of cancer; however, a consequence of improved public health and medical advancement is that an estimated 1 out of 3 individuals in the United States will be diagnosed with a malignancy by the age of 70 (116). As such, the demand for selective chemical and biological agents that possess effective chemotherapeutic and/or chemopreventative properties has reached a critical level. The incidence of cancer arising from the epithelium (lung, breast, colon, prostate, etc.) has been shown to be disproportionally abundant; therefore our chemoprevention project focuses on these cancer types (117).

Chemoprevention is a term used in parallel with chemotherapy, while the goals for each term are obvious contrasting tactics they are similar with regard to the approaches used for the discovery of treatment options. The transformation of normal cellular function into neoplastic phenotypes requires the progression of specific staging events typically at specific genetic markers. This process evolves over exteneded periods and consists of iterative initiation, promotion and progression stages, ultimately manifesting in mutagenesis of cellular machinery. Genetic pre- disposition and a host of environmental factors including dietary choices, combine a risk of cancer incidence for an individual. There are entire classes of compounds (flavonoids, carotenoids,

24 25 phenolic acids, organosulfides, etc.) found within botanicals that elicit the biological detoxification machinery removing carcinogens for the body (118). This is the premise of our chemoprevention project for which we have expanded our scope to include marine and bacterial sources in addition to plant extracts and synthetically derived compounds in pursuit of chemopreventative agents.

Our long standing multifaceted approach for the identification of potential chemompre- vention agents utilizes the expertise of several collaborating groups that combine synthetic tools with a myriad of in vivo and in vitro assays. By targeting well described biological processes that are implicated in cancer, with our unique and extensive library of compounds collected from synthetic, marine and botanical sources potent novel structures that have relevant therapeutic value are likely to be identified. Periodically, compounds that display promising results within a particular pathway are subjected to additional ADME characterization, therefore a complete preliminary assessment for each compound is generated and its ultimate disposition determined.

A resent collection of compounds submitted for analytical measurements included abyssinone, styrylaniline, AM6-36, casirimoin, PVN-I-48, and SB-I-46. These compounds have shown activity in a variety of in vitro and in vivo assays related to several common neoplastic conditions. (2S)-Abyssinone II is a prenylated flavonoid derived from the Chinese plant Broussonetia papyr- ifera and shows promise as an aromatase inhibitor. AM6-36 is one of a series of synthetic indenoisoquinoline derivatives initially developed as camptothecin-based topoisomerase I inhibitors. SB-I-46 and styrylaniline are a few synthetic resveratrol structural analogs with pleotropic qualities, and casirimion and PV-I-48 were designed to target ribosyldihydronicotinamide dehydro- genase (quinone reductase 2) sharing a common quinolin-2-one structural motif (119-123). As part of the collaborative nature of this chemoprevention project, our laboratory routinely carries out preliminary ADME analyses, since we possess the requiste analytical instrumentation and the technical expertise for the rapid determination of these properties. Among these promising chemoprevention agents, AM6-36 was subsequently shown as part of this dissertation to be a ligand for the VDR. 26

2.2 Materials and Methods

2.2.1 Reagents

3-Amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36), PVN-I-48, casimiroin, and 4-(3,5-dimethoxystyryl)aniline (SB-I-46) were synthesized and provided by Dr. Mark Cushman of Purdue University. 7-Hydroxystaurosporine (UCN-01) was purchased from Sigma-Aldrich (St. Louis, MO) and 8-prenylnaringenin (8PN) was obtained from the UIC/NIH Center for Botanical Dietary Supplements Research (124). Several milligrams of each compound were dissolved in dry DMSO, resulting in a 1 mg/mL stock solution of each compound received and were stored at -20 °C until analysis. Deionized water was purified for LC-MS analysis using a Milli-Q Integral Water Purification System (Millipore, Billerica, MA). All other solvents were purchased from Fisher Scientific (Hanover, IL) and were designated Optima LC-MS grade.

2.2.2 Animals

Female Sprague-Dawley (Indianapolis, IN) rats were utilized for a preliminary assessment of the biodistribution for the chemoprevention candidates. For each compound, abyssinone, styrylaniline, AM6-36, casirimoin, PVN-I-48, and SB-I-46 rats were administered 40 mg/kg of body weight for 3 consecutive days by oral gavage, sample collection and serum and tissue pre- processing were carried out by Dr. Clinton Grubbs of the University of Alabama at Birmingham (Birmingham, AL). Blood was collected on days 1 and 3, and tissues were collected after sacrifice on day 4.

2.2.3 Sample Preparation

Rat serum and tissue (liver, mammary, and perirenal fat) samples were shipped on dry ice and promptly stored at -80 °C until immediately prior to the analytical analysis. Tissue samples were homogenized using a Kinematica (Bohemia, NY) Polytron. Rat liver was homogenized in 27

0.05 M phosphate buffer (pH 7.4), while tissues with greater adiposity (mammary gland and perirenal fat) required the addition of an organic modifier for proper dissolution and were homogenized in a mixture of phosphate buffer and methanol (50:50; v/v) yielding tissue homogenates containing 0.2 g tissue/ml. Aliquots of 100 µL of each tissue type were partitioned into Eppendorf microcentrifuge tubes, and the proteins were precipitated with four volumes of ice-cold acetonitrile. After vortex mixing each sample for 15 s and sonicating for 5 min, each sample was placed on ice for an additional 15 min. Pelleting of the protein was accomplished by centrifugation at 3,000 × g for 5 min. The resulting supernatant was decanted into a separate Eppendorf tubes and an appropriate internal standard was added yielding a final concentration of 100 nM. The entire solution volume was evaporated to dryness using a steady stream of nitrogen. Each sample was reconstituted in 100 µL of the initial HPLC moble phase and then analyzed using LC-MS-MS.

Blank tissue and serum samples were used for evaluation of extraction efficiency and also testing for matrix interference during LC-MS-MS. First, the efficiency of the extraction protocol was determined by spiking blank matrix with each analyte before and after extraction followed by quantitative analysis using LC-MS-MS. Second, possible LC-MS-MS signal suppression or enhancement due to co-eluting constituents of each blank matrix were evaluated. Calibration curves were generated for each analyte spiked into each blank matrix.

2.2.4 LC-MS-MS of AM6-36

LC-MS-MS analysis of AM6-36 was carried out using a Shimadzu (Kyoto, Japan) LC-20AD Prominence HPLC system interfaced to an Applied Biosystems (Foster City, CA) API 4000 triple quadrupole mass spectrometer controlled by Analyst version 1.5 software. Chromatographic separation of AM6-36, internal standard UNC-01, and potential adverse matrix constituents was achieved by employing a 10 min linear gradient of 10-80% ACN in 0.1% aqueous formic acid at

250 µl/min. An 10 µl aliquot was injected onto a Waters (Milford, MA ) XTerra MS C18 column

(2.1 mm × 100 mm, 3.5 μm) in a temperature controlled column oven at 30 °C. Following positive ion electrospray and collision-induced dissociation (CID) using nitrogen gas the product ion tan- 28 dem mass spectrum was recorded, and then selected reaction monitoring (SRM) was employed to record the protonated molecule of AM6-36, m/z 320 to 303 and the internal standard (UNC-01) m/z 483 to 130. Mass spectrometer parameters were optimized by employing automated sequential flow injection analysis (FIA) controlled by the software; source temperature, 400 °C; spray voltage, 4000 V; declustering potential, -60 V; curtain gas, 10; gas 1, 40; entrance potential, 10 V; capillary exit potential, -20 V; collision energy, -20; and a dwell time of 500 ms per SRM transition.

An LC-MS-MS SRM chromatogram for the analysis of AM6-36 is shown in figure 2.1. AM6-36 eluted at 7.3 min, and internal standard UNC-01 eluted at 8.9 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of AM6-36 (figure 2.2) showed fragment ions ofm/z 303, 275, 217, and 190. The base peak of m/z 303 corresponded to cleavage of the terminal aliphatic nitrogen, resulting in a neutral loss of NH3 and was selected for use during SRM. The abundant fragment ion of m/z 130, corresponding to the loss from the protonated molecule of the internal standard UNC-01 was also produced using SRM, product ion data not shown. 29

Figure 2.1. Positive ion LC-MS-MS (API 4000) analysis of rat serum spiked with AM6-36 and internal standard UNC-01 using CID and SRM

1.4e4

AM6-36, SRM transition m/z 320 303 1.2e4

8500.0

UNC-01, SRM transition m/z 483 130

LC-MS-MS response LC-MS-MS 5500.0

2500.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention time (min) H N O HO O

N NH2 H N 2 N N O O AM6-36 H UNC-01 O

HN 30

Figure 2.2. Positive ion electrospray CID product ion tandem mass spectrum of protonated AM-6-36

303.0 [M+H]+ 320.0 1.6e6

1.4e6

1.2e6

1.0e6

8.0e5 MS-MS response

6.0e5 245.0

4.0e5 275.0

2.0e5 190.0 217.2 262.0 163.2 205.0 0.0 90 110 130 150 170 190 210 230 250 270 290 310 m/z 31

2.2.5 LC-MS-MS of PVN-1-48

A similar LC-MS-MS quantitative method was developed for PVN-1-48, except for the following adjustments due to compound dependent parameters. Positive ion electrospray was used for ionization, and the SRM transitions used for quantitative analysis during LC-MS-MS for PVN-1-48 and internal standard (casimiroin) were m/z 264 to 234 and m/z 234 to 219, respectively. Mass spectrometer parameters were optimized by employing automated sequential flow injection analysis (FIA) controlled by the software; source temperature, 400° C; spray voltage, 5000 V; declustering potential, -120 V; curtain gas, 10; gas 1, 40; entrance potential, 10 V; capillary exit potential, -16 V; collision energy, -35; and a dwell time of 500 ms per SRM transition.

An LC-MS-MS SRM chromatogram for the analysis of PVN-1-48 is shown in figure 2.3. PVN-1-48 eluted at 5.3 min, and internal standard casimiroin eluted at 5.6 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of PVN-1-48 (figure 2.4) showed fragment ions ofm/z 248, 234, and 205. The base peak of m/z 234 corresponded to the cleavage of two methyl radical species and was selected for use during SRM. The abundant fragment ion of m/z 219, corresponding to the loss of a radical methyl group from the protonated molecule of the internal standard casimiroin was also produced and selected for SRM LC-MS-MS analysis. 32

Figure 2.3. Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of PVN-1-48 and internal standard casimiroin spiked into rat serum

3.0e4

PVN-1-48, SRM transition m/z 264 234 2.6e4

2.2e4

1.8e4 Casimiroin, SRM transition m/z 234 219 1.4e4 LC-MS-MS response LC-MS-MS

1.0e4

6000.0

2000.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Retention time (min)

O O

N O N O O O O PVN-1-48 Casimiroin 33

Figure 2.4. Positive ion electrospray CID product ion tandem mass spectrum of protonated PVN-1-48

[M+H]+ 264.0

7.5e5

6.5e5

5.5e5

4.5e5 234.0

3.5e5 MS-MS response

2.5e5

1.5e5 134.0 206.0 163.0 191.0 249.0 175.0 5.0e4 219.0 0.0 90 110 130 150 170 190 210 230 250 m/z 34

2.2.6 LC-MS-MS of Casimiroin

A similar LC-MS-MS quantitative method was developed for casmiroin, except for the following adjustments due to compound dependent parameters. Positive ion electrospray was used for ionization, and the SRM transitions used for quantitative analysis during LC-MS-MS for casimiroin and internal standard (PVN-1-48) were m/z 234 to 219 and m/z 264 to 234, respectively. Mass spectrometer parameters were optimized by employing automated sequential flow injection analysis (FIA) controlled by the software; source temperature, 400° C; spray voltage, 5000 V; declustering potential, -120 V; curtain gas, 10; gas 1, 40; entrance potential, 10 V; capillary exit potential, -16 V; collision energy, -35; and a dwell time of 500 ms per SRM transition.

An LC-MS-MS SRM chromatogram for the analysis of casimiroin is shown in figure 2.5. casimiroin eluted at 5.6 min, and internal standard PVN-1-48 eluted at 5.3 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of casimiroin (figure 2.6) showed fragment ions ofm/z 219 and 191. The base peak of m/z 219 corresponded to the loss of a methyl radical and was selected for use during SRM. The abundant fragment ion of m/z 234, corresponding to the loss of two separate methyl radical species from the protonated molecule of the internal standard PVN-1-48 was also produced using SRM LC-MS- MS analysis. 35

Figure 2.5. Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of casimiroin and internal standard PVN-1-48 spiked into rat serum

1.05e5

9.50e4

8.50e4

Casimiroin, SRM transition 7.50e4 m/z 234 219

6.50e4

5.50e4 PVN-1-48, SRM transition 4.50e4 m/z 264 234 LC-MS-MS response LC-MS-MS

3.50e4

2.50e4

1.50e4

5000.00

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Retention time (min)

O O

N O N O O O O PVN-1-48 Casimiroin 36

Figure 2.6. Positive ion electrospray CID product ion tandem mass spectrum of protonated casimiroin

[M+H]+ 234.0 1.4e6

1.2e6

1.0e6

8.0e5

6.0e5 MS-MS response

4.0e5

219.0 2.0e5 191.2 103.2 89.0 132.2 116.2 145.4 161.4 176.4 0.0 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 m/z 37

2.2.7 LC-MS-MS of SB-I-46

A similar LC-MS-MS quantitative method was developed for SB-I-46, except for the following adjustments due to compound dependent parameters. Positive ion electrospray was used for ionization, and the SRM transitions used for quantitative analysis during LC-MS-MS for PVN-1-48 and internal standard (casimiroin) were m/z 256 to 179 and m/z 264 to 234, respectively. Mass spectrometer parameters were optimized by employing automated sequential flow injection analysis (FIA) controlled by the software; source temperature, 400 °C; spray voltage, 4000 V; declustering potential, -50 V; curtain gas, 12; gas 1, 30; entrance potential, 10 V; capillary exit potential, -20 V; collision energy, -25; and a dwell time of 500 ms per SRM transition.

An LC-MS-MS SRM chromatogram for the analysis of SB-I-46 is shown in figure 2.7. SB-I- 46 eluted at 6.9 min, and internal standard PVN-1-48 eluted at 5.3 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of SB-I-46 (figure 2.8) showed fragment ions of m/z 179, 118, and 101. The base peak of m/z 179 corresponded to the cleavage of all the heteroatom substituents on the stilbene base structure (-NH3, -2 X CH2O) and was selected for use during SRM. The abundant fragment ion of m/z 234 was again due to the loss of two methyl radicals from the internal standard PVN-1-48 and was produced using SRM LC-MS-MS analysis.

Figure 2.7. Positive ion electrospray LC-MS-MS (API 4000) with CID and SRM of SB-I-46 and internal standard PVN-1-48 spiked into rat serum

3800 SB-I-46, SRM transition 3400 m/z 256 179

3000

2600

2200

1800 LC-MS-MS response LC-MS-MS PVN-1-48, SRM transition 1400 m/z 264 234

1000

600

200

1.0 3.0 5.0 7.0 9.0 11.0 Retention time (min)

NH2 O

N O O O O SB-I-46 PVN-1-48 39

Figure 2.8. Positive ion electrospray CID product ion tandem mass spectrum of protonated SB-I-46

1.4e7 101.0

1.2e7

179.0 1.0e7

8.0e6

6.0e6 MS-MS response

4.0e6

[M+H]+ 256.0 2.0e6

118.2 132.0 152.2 165.2 0.0 100 120 140 160 180 200 220 240 260 m/z 40

2.2.8 LC-MS-MS of 4-Styrylaniline

LC-MS-MS analysis of 4-styrylaniline was carried out using a Waters (Milford, MA) 2694 HPLC system interfaced to an Thermo (San Jose, CA) TSQ Quantum triple quadruple mass spectrometer controlled by Xcaliber version 2.07 sofware. Chromatographic separation of 4-styrylaniline, internal standard 8PN, and potential adverse matrix constituents was achieved by employing a 15 min linear gradient of 40-100 % methanol in 0.1% aqueous formic acid at 200 µl/min. An

10 µl aliquot was injected onto a Waters (Milford, MA ) XTerra MS C18 column (2.1 mm × 100 mm,

3.5 μm) in a temperature controlled column oven at 30 °C. Following positive ion electrospray and collision-induced dissociation (CID) using argon gas the product ion tandem mass spectrum was recorded, and then selected reaction monitoring (SRM) was employed to record the protonated molecule of 4-styrylaniline, m/z 196 to 118 and the internal standard (8PN) m/z 341 to 165. Mass spectrometer parameters were optimized by a syringe pump infusion of 1 µM standards controlled by the software; spray voltage, 3,500 V; sheath gas pressure, 55; capillary temperature, 240 °C; collision energy, 30 V; collision pressure, 1 mTorr.

An LC-MS-MS SRM chromatogram for the analysis of 4-styrylaniline is shown in figure 2.9. 4-styrylaniline eluted at 5.1 min, and internal standard 8PN eluted at 9.2 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of 4-styrylaniline was not record due to optimization parameters within the tune file setting. The base peak as selected by the software of m/z 118 corresponded to cleavage of the alkene linker of the stilbene and was selected for use during SRM. The abundant fragment ion of m/z 165, corresponding to the cleavage of the centeral flavone ring resulting in a reverse Diels Alder product of 8PN and the addition loss of the prenyl side chain was also produced using SRM, product ion data not shown.

Figure 2.9. Positive ion LC-MS-MS (TSQ Quantum) analysis of rat serum spiked with 4-styrylaniline and internal standard 8PN using CID and SRM

90 Styrylaniline, SRM transition m/z 196 118 70

8PN, SRM transition 70 m/z 341 165 Relative LC-MS-MS Response Relative

1 2 3 4 5 6 7 8 9 10 11 12 13 Retention time (min)

OH NH2

HO O

4-styrylaniline 8PN

OH O 42

2.2.9 LC-MS-MS of Abyssinone

LC-MS-MS analysis of abyssinone was carried out using a Waters (Milford, MA) 2694 HPLC system interfaced to an Thermo (San Jose, CA) TSQ Quantum triple quadruple mass spectrometer controlled by Xcaliber version 2.07 software. Chromatographic separation of abyssinone, internal standard 8PN, and potential adverse matrix constituents was achieved by employing a 20 min isocratic run of 34 % methanol in 0.1% aqueous formic acid at 200 µl/min. An 10 µl aliquot was injected onto a Waters (Milford, MA) XTerra MS C18 column (2.1 mm × 100 mm, 3.5

μm) in a temperature controlled column oven at 30 °C. Following negative ion electrospray and collision-induced dissociation (CID) using argon gas the product ion tandem mass spectrum was recorded, and then selected reaction monitoring (SRM) was employed to record the protonated molecule of abyssinone, m/z 323 to 187 and the internal standard (8PN) m/z 339 to 219. Mass spectrometer parameters were optimized by a syringe pump infusion of 1 µM standards controlled by the software; spray voltage, -4,500 V; sheath gas pressure, 55; capillary temperature, 200 °C; collision energy, 35 V; collision pressure, 1 mTorr.

An LC-MS-MS SRM chromatogram for the analysis of abyssinone is shown in figure 2.10. abyssinone eluted at 8.9 min, and internal standard 8PN eluted at 9.2 min. No interfering compounds from the rat serum matrix were observed. The product ion tandem mass spectrum of abyssinone was not record due to optimization parameters within the tune file setting. The base peak as selected by the software ofm/z 187 corresponded to the cleavage of the central ring of the flavone, resulting in a neutral loss corresponding to the cleavage of dihydro-2H-pyran-4(3H)-one ring and was selected for use during SRM. The abundant fragment ion of m/z 219, corresponding to the cleavage of the dihydro-2H-pyran-4(3H)-one ring of 8PN and the addition loss of the prenyl side chain was also produced using SRM, product ion data not shown. 43

Figure 2.10. Negative ion electrospray LC-MS-MS (TSQ Quantum) with CID and SRM of Abyssinone and internal standard 8PN spiked into rat serum

TSQ Quantum, SRM, Negative Ion Electrospray 90

Abyssinone, SRM transition 70 m/z 323 187

90 8PN, SRM transition m/z 339 219 70 Relative LC-MS-MS response Relative

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Retention time (min)

OH OH

HO O HO O

O OH O 8PN Abyssinone 44

2.2.10 Validation

The standard operating procedure (SOP) for quantitative method validation for our laboratory can be found within the FDAs guidelines for bioanalytical method validation (125). Fol- lowing the protocols outlined within this guide, a partial method validation was developed for PVN-1-48, casimiroin, SB-I-46, styrylaniline, abyssinone and AM6-36 separately. Due to time con- straints placed on these quantitative measurements and the laborious nature of the stipulated requirements for full validation of a method, a procedure that relies solely on intraday accuracy and precision was developed. Interday analysis is important for characterizing the robustness of the analytical system as well as the operator error; however this procedure is not essential for a highly accurate and precise method. As part of the FDA method for partial validation, an intraday protocol employing 3 replicates of a 6 point calibration curve for each analyte within all four blank matrices was conducted. Calibration curves demonstrated linearity over at least 3 orders of magnitude, ranging from 0.5-500 ng/mL. Requirements for interday measurements include performing the same set of analyses over a period spanning 3 separate days. Additional quality control samples were also prepared in triplicate at low, medium, and high concentrations with respect to the standard curve (see table 2.1). 45

TABLE 2.1

INTRADAY VALIDATION FOR PVN-1-48, CASIMIROIN, AM6-36, SB-I-46, ABYSSINONE, AND 4-STYRYLANILNE

PVN-1-48 Intraday LOD LLOQ Concentration (ng/100µL) (n=4) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 0.25 0.21 12.2 84.0 25 2.52 10.4 101.0 0.1 0.25 50 24.6 5.24 98.4 Casimiroin Intraday LOD LLOQ Concentration (ng/100µL) (n=4) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 0.25 0.23 11.8 92.0 25 2.48 4.28 99.2 0.05 0.25 50 25.65 4.01 103.0 AM6-36 Intraday (n=4) LOD LLOQ Concentration (ng/100µL) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 1 0.93 11.8 92.0 10 2.48 4.28 99.2 0.5 1.0 40 25.65 4.01 103.0 SB-I-46 Intraday (n=4) LOD LLOQ Concentration (ng/100µL) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 0.25 0.20 14.1 80.0 25 2.47 7.46 98.8 0.1 0.25 50 25.2 6.61 101.0 4-Styrylaniline Intraday LOD LLOQ Concentration (ng/100µL) (n=4) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 0.25 0.26 8.16 104.0 25 2.48 6.88 99.2 0.05 0.15 50 24.7 7.39 98.8 Abyssinone Intraday LOD LLOQ Concentration (ng/100µL) (n=4) (ng/100µL) (ng/100µL) Expected Measured (mean, n= 4) %CV Accuracy % 0.25 0.22 10.0 88.0 25 2.47 11.1 99.0 0.05 0.20 50 25.3 6.74 101.2

2.3 Results and Discussion

Rapid and accurate assessment of the in vivo distribution in rats for 6 investigational chemoprevention agents was assessed in serum and several tissues. Calibration curves for all analytes were linear over the intended concentration range, with r2 > 0.99. The linearity of the SRM response was determined by plotting the peak areas of the analytes to the internal standards on the ordinate versus the nominal analyte concentration on the abscissa as spike into blank serum or homogenized tissue. No weighting was utilized for the construction of calibration curves and were obtained using linear regression analysis. Figures 4.13-4.19 display calibration curves for all four analytes produced from spiking blank serum. The limit of quantitation (LOQ) was determined by using the response ratio signal-to-noise of 10:1, while the limit of detection (LOD) was determined by a 3:1 ratio. Values for all analytes were found to be similar with a LOQ of ~2 ng/mL and LOD of ~0.5 ng/mL, see table 2.1 for specific values of all 6 analytes. Sample recovery was found to be greater than 85% for all compounds in all matrix types. Matrix effects were of little consequence for this analysis, resulting in insignificant variation between quality control samples.

Quantitative LC-MS-MS measurements of the 6 chemoprevention compounds in rat tissues were carried out with Dr. Yongsoo Choi. Additional assessment of each these compounds involved a battery of standard preclinical experiments including; caco-2 permeability studies conducted by Dr. Soyoun Ahn, metabolic stability performed by Dr. Jing Hu and Ms. Linan Chen, and plasma binding was determined by Mr. Ke Huang. The cumulative results provide early insight into the absorption, metabolism, and distribution of each potential chemoprevention agent.

Selection of an appropriate internal standard for each analyte is crucial for generating accurate and precise standard curves and determining biological sample concentrations. Internal standards are meant to be a mimic of the analytes chemical nature, with synthetic stable isotope labeled compounds as the gold standard. Stable isotope labels are extremely effective with regard to normalized deviations acquired with sample processing or ionization variability: however 47 they are costly and can require a significant time investment for synthetic production. In order to expedite the quantitative analysis, compounds that elute within 1 min of the target analyte were selected as internal standards. Fortunately, PVN-1-48 and casimirion are structurally similar allowing each compound to serve a dual purpose as analyte of interest and internal standard. Other internal standards were selected from among the many commercial and botanical compounds available in the van Breemen laboratory.

The amount of perirenal fat and mammary tissue obtained from each rat in this study was determined to be in insufficient amounts for quantitative analysis of the test compound. There- fore, perirenal fat from 3 rats was combined into a single sample for analysis, and mammary tissue from 3 rats was combined into a single sample. Liver samples collected from rats treated with 4-styrylaniline and abyssinone were also combined due to inadequate amounts of tissue. Tables 2.2, 2.3, 2.4, and 2.5 display the concentrations of each analyte tested within the respective tissue type, and these data are discussed below.

Detectable amounts of AM6-36, caimiroin, abyssinone, SB-I-46, PVN-1-48 and 4 styrylaniline were measured in rat serum as well as in all rat tissues analyzed following administration of 40 mg/kg of material by gavage for up to 3 days. The concentrations of AM6-36 and 4 styrylanline displayed a similar trend for rat serum and proved constant on day 1 and day 3 to be (~0.8 and 0.4 µg/mL for AM6-36 and 4-styrylaniline, respectively). These compounds were concentrated in the liver with a mean value of 4.28 and 4.19 µg/g, respectively. AM6-36 and 4-styrylaniline were also measured in significant amounts in mammary tissue and perirenal fat (see tables 2.2 and 2.7). 48

Figure 2.11. LC-MS-MS standard curve for AM6-36 spiked into rat serum. UNC-01 was used as an internal standard

y = 0.9108x - 0.3687 5 R2 = 0.9968

1 Spiked Peak Area (AM6-36/UNC-01) Spiked Peak

0 10 20 30 40 50 60 [AM6-36]/[UNC-01]

TABLE 2.2

BIODISTRIBUTION OF AM6-36 IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) AM6-36 (µg/mL), n=9 (µg/mL), n=9 n=9 Pooled, n=3 Pooled, n=3 Rat 4 0.84 ± (0.01) 0.82 ± (0.003) 3.72 ± (0.07) N/A N/A Rat 5 0.84 ± (0.01) 0.84 ± (0.007) 5.03 ± (0.23) N/A N/A Rat 6 0.82 ± (0.004) 0.82 ± (0.002) 3.75 ± (0.13) N/A N/A Total 0.83 ± (0.01) 0.83 ± (0.01) 4.28 ± (1.66) 0.29 ± (0.01) 0.28 ± (0.01) 49

Figure 2.12. LC-MS-MS standard curve for PVN-1-48 spiked into rat serum. Casimiroin was used as an internal standard

5 y = 0.4257x + 0.0315 R² = 0.9996 4

1 Spiked Peak Area (PVN-1-48/Casimiroin) Spiked Peak 0 10 20 30 40 50 60 [PVN-1-48]/[Casimiroin]

TABLE 2.3

BIODISTRIBUTION OF PVN-1-48 IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) PVN-1-48 (µg/mL), n=9 (µg/mL), n=9 n=9 Pooled, n=3 Pooled, n=3 Rat 4 0.28 ± (0.013) 0.27 ± (0.002) 4.81 ± (0.36) N/A N/A Rat 5 0.73 ± (0.105) 0.06 ± (0.003) 2.00 ± (0.18) N/A N/A Rat 6 0.21 ± (0.002) 0.02 ± (0.002) 1.06 ± (0.12) N/A N/A Total 0.41 ± (0.25) 0.12 ± (0.12) 2.60 ± (1.66) 0.73 ± (0.05) 1.14 ± (0.06) 50

Figure 2.13. LC-MS-MS standard curve for casimiroin spiked into rat serum. PVN-1-48 was used as an internal standard

5 y = 0.4628x + 0.0639 4 R² = 0.9994

1 Spiked Peak Area (Casimiroin/PVN-1-48) Spiked Peak 0 10 20 30 40 50 60 [Casimiroin]/[PVN-1-48]

TABLE 2.4

BIODISTRIBUTION OF CASIMIROIN IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) Casimiroin (µg/mL), n=9 (µg/mL), n=9 n=9 Pooled, n=3 Pooled, n=3 Rat 4 5.38 ± (0.23) 0.84 ± (0.03) 1.87 ± (0.17) N/A N/A Rat 5 8.17 ± (0.45) 1.07 ± (0.08) 2.34± (0.08) N/A N/A Rat 6 3.55 ± (0.07) 0.18 ± (0.01) 4.00 ± (0.05) N/A N/A Total 6.05 ± (2.13) 0.76 ± (0.38) 1.54 ± (0.88) 0.78 ± (0.05) 1.18 ± (0.02)

Figure 2.14. LC-MS-MS standard curve for SB-I-46 spiked into rat serum. PVN-1-48 was used as an internal standard

5 y = 0.386x + 0.0853 R² = 0.9994 4

Spiked Peak Area (SB-I-46/PVN-1-48) Spiked Peak

10 20 30 40 50 60 [SB-I-46]/[PVN-1-48]

TABLE 2.5

BIODISTRIBUTION OF SB-I46 IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) SB-I-46 (µg/mL), n=9 (µg/mL), n=9 n=9 Pooled, n=3 Pooled, n=3 Rat 1 2.99 ± (0.12) 1.04 ± (0.005) 4.68 ± (0.57) N/A N/A Rat 2 2.90 ± (0.09) 0.67 ± (0.005) 9.88 ± (0.57) N/A N/A Rat 3 4.00 ± (0.06) 0.21 ± (0.001) 5.61 ± (0.91) N/A N/A Total 3.21 ± (0.45) 0.64 ± (0.034) 6.28 ± (2.36) 0.51 ± (0.01) 0.69 ± (0.04) 52

Figure 2.15. LC-MS-MS standard curve for 4-styrylaniline spiked into rat serum. 8PN was used as an internal standard

5 y = 0.4302x + 0.0843 R² = 0.9995 4

1 Spiked Peak Area (4-styrylaniline/8PN) Spiked Peak 0 10 20 30 40 50 60 [4-styrylaniline]/[8PN]

TABLE 2.6

BIODISTRIBUTION OF 4-STYRYLANILINE IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) Styrylaniline (µg/mL), n=6 (µg/mL), n=6 Pooled, n=3 Pooled, n=3 Pooled, n=3 Total 0.38 ± (0.097) 0.40 ± (0.07) 4.19 ± (0.11) 0.366 ± (0.09) 0.648 ± (0.15)

Figure 2.16. LC-MS-MS standard curve for abyssinone spiked into rat serum. 8PN was used as an internal standard

5 y = 0.3896x + 0.1386 R² = 0.9992 4

1 Spiked Peak Area (Abyssinone/8PN) Spiked Peak

0 10 20 30 40 50 60 [Abyssinone]/[8PN]

TABLE 2.7

BIODISTRIBUTION OF ABYSSINONE IN RAT

40 mg/kg Serum Day 1 Serum Day 3 Liver (µg/g) Mammary (µg/g) Perirenal Fat (µg/g) abyssinone (µg/mL), n=6 (µg/mL), n=6 Pooled, n=3 Pooled, n=3 Pooled, n=3 Total 0.007 ± (0.006)

Serum levels of casimiroin and SB‐I‐46 were high on day 1 (mean values of 6.05 and 3.21

μg/ml, respectively; tables 2.4 and 2.5), but a marked reduction was observed by day 3 (mean values of 0.64 and 0.76 μg/ml, respectively) was seen. An abundance of SB‐I‐46 and casimiroin was found in rat liver with mean concentrations of 6.28 and 1.54 μg/g. The concentration of SB‐

I‐46 and casimiroin in mammary gland and perirenal fat, were the highest of all compounds tested. PVN‐1‐46 displayed consistently low serum concentrations, a high amount held within the liver, and relatively high amounts in mammary and perirenal fat. Abyssinone was barely detected in rat serum, concentrated like all other compounds in the liver, and displayed the lowest amount in mammary and perirenal fat samples. These data are consistent with the oral absorption of each compound predicted by the in vitro Caco‐2 cell monolayer studies.

Additionally, microsomal incubation studies of AM6‐36 reveal moderate metabolic stability

(126).

AM6‐36 has displayed an affinity for the retinoid X receptor alpha (RXRα) formally an orphan receptor subtype of the ligand activated members of the nuclear receptor superfamily.

The isoforms of RXR (RXRα, RXRβ, and RXRγ) possess a high degree of sequence homology to the retinoic acid receptors (RARs) with geometric isomers as ligands 9‐cis‐retinoic acid and all trans‐retinoic acid (vitamin A metabolites), respectively. RXRs ligand activated responses lead to conformational changes promoting the dissociation of co‐repressors while initializing contacts with co‐activating species. RXR once activated triggers the transcriptional machinery and directly interacts with specific regions of genes known as hormone response elements

(HREs). The RXR ligand mediated responses associated with HREs have demonstrated the ability to influence metabolic processes, cellular differentiation and apoptotic mechanisms. RXR selective compounds have shown clinical relevance as a treatment option for certain forms of cancer, such as bexarotene (LGD1069) use within cutaneous T‐cell lymphoma and is currently in clinical trials for breast and lung cancer (127‐129).

As a primary screen for RXR ligands our chemoprevention drug discovery project utilizes a RXR‐specific response element (RXRE) reporter gene assay for the determination of

RXR‐mediated transcriptional responses in addition to demonstrating binding affinity towards

RXR (126). An RXR based ultrafiltration LC‐MS assay serves as a secondary assay for the

55 conformation of binding events (130). In the breast cancer cell line (MCF7) AM6‐36 promoted cellular inhibition with an IC50 = 0.47 μM, while displaying markers of cell cycle arrest as well as up‐regulated RXRα‐mediated transcriptional products. The demonstrated ligand activated responses of RXRα elicited by AM6‐36 display significant structural dissimilarity to the endogenous ligands, also the limited number of known compounds that act in this manner.

Since AM6‐36 was found to generate an analogous response to that of the natural ligand 9‐cis‐

RA, this compound represents a promising structurally distinct chemopreventive agent (126).

The pleiotropic nature of resveratrol has resulted in numerous in vivo and in vitro studies describing the therapeutic value of this polyphenol in a host of disease states (131‐133) The chemopreventative properties of resveratrol were first realized with the discovery in our own department that resveratrol chemoprevention actions in skin cancer, however large doses must be administered before a significant response can be seen (132). Structural analogs of resveratrol were subsequently synthesized in order to improve upon its sensitivity and selectivity for several common cancer targets (119, 122). The resveratrol analog SB‐I‐46 was identified through this process and has been found to be a relatively potent aromatase inhibitor (AI) with an IC50 of 0.31 μM (9). A similar level of AI inhibition can be found with the prescribed pharmaceutical

Cytadren, a 2‐aminoglutethimide with a modest IC50 value of 0.2 μM (134). Aromatase is a member of the CYP 450 family and is primarily responsible for the conversion of androgens to estrogen through sequential oxidation and elimination steps. AIs produce a state of hypo‐ estrogenism, which has proven to be an effective treatment option for patients with estrogen receptor positive breast and ovarian cancer. The aberrant cellular growth in these diseases can be highly dependent on estrogen providing an avenue for cancer prevention inhibition. SB‐I‐46 promotes is also a potent inhibitor (96%) of ornithine decarboxylase (ODC) as well as a moderate inhibitor of NF‐kB (IC50 = 22.54 μM), and stimulates the production of the detoxifying enzyme such as quinine reductase 1 (NQR1) (119, 135).

4‐Styrylalanine, the other resveratrol analog investigated, displays comparable levels of activity against neoplastic processes to its counterpart SB‐I‐46. Compared with SB‐I‐46, 4‐ styrylaniline is a more potent inhibitor of NF‐kB (IC50 = 1.1 μM) and an inducer of chemoprevention enzymes such as QR1 (IR = 3.3, and CD = 33 μM) (119). 4‐Styrylaniline also

56 has been shown to decrease nitric oxide (NO) production as well as retard growth of MCF‐7 breast cancer cells. NO displays contrasting roles depending on cellular concentrations within cancer cells. Normally, NO production promotes cell cycle arrest and suppresses cellular proliferation at levels typically found in cells, but carcinoma cells can show significant enhancement of NO formation (136). Based on these observations, agents that target processes reducing intracellular concentrations of NO could be chemopreventive agents. In the MCF‐7 cell model, 4‐styrylalanine displayed inhibition of iNOS (IC50 = 3.6 μM) and aromatase (IC50 = 22

μM), accompanied by the suppression of cell proliferation (119).

2.4 Conclusions

Our multidisciplinary chemoprevention drug discovery and development program screens natural products due to their inherent chemical diversity, allowing for the identification of novel structures with the potential for multifaceted treatment mechanisms (133, 137). The development of compounds from these drug discovery assays requires numerous stages of preclinical and clinical investigation for an evaluation of safety and efficacy of each lead structure. Lead optimization through synthetic means affords large scale production of sufficient quantities of material for in vitro studies and subsequent progression into animal models and eventually clinical trials.

In vitro activity of lead compounds rarely translates to efficacious in vivo models. For example, an effective drug must reach the target tissue, and activity in vitro does not guarantee appropriate bioavailability and biodistribution. Many factors can influence a compounds biological response, such as the mode of delivery, formulation, metabolic activity, and barriers associated with specific tissue types. Therefore preclinical in vivo studies supported by analytical measurements are necessary to assess the biodistribution of each compound, before it can advance to the next stage of development. Quantitative LC‐MS‐MS assays of lead chemoprevention compounds in serum and tissues were developed and used to assess biodistribution within this chapter. These data were evaluated in conjunction with the Caco‐2 absorption, metabolism, and serum binding studies, resulting in the promotion of AM6‐36, a

57 compound in the indenoisoquinoline class as a candidate for further investigation as a chemoprevention agent.

CHAPTER 3

DEVELOPMENT OF AN ULTRAFILTRATION LC-MS PLATFORM FOR VDR

3.1 Introduction

The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily which binds the endogenous secosteriod 1α, 25-dihydroxyvitamin D3 (calcitriol) with high affinity (kd ~0.1 nM), producing a myriad of biological responses within a diverse range of tissue classes (138). In addition to the routine homeostatic roles acting on bone, kidney and intestine as a

2+ regulator of Ca and Pi transport and bone remodeling, the VDRs presence in many non-classical cellular types has greatly expanded the role of this nuclear receptor. These systems include several constituents involved in immune responses (T and B cells, macrophages, and monocytes), reproductive organs (uterus, testis, ovary, prostate, placenta, and mammary glands), muscle, endocrine system (pancreas, pituitary, thyroid, and adrenal cortex), as well as brain, liver, and skin (68). These recently discovered non-classical actions have given rise to a number of potential disease states at least partially regulated through the active vitamin D metabolite, calcitriol (16). The ability of calcitriol to promote differentiation and inhibit proliferation of normal and malignant cells has suggested a multifaceted response in cancer prevention and treatment (49). The correlations drawn between vitamin D deficiency and a multitude of neoplastic conditions, autoimmune diseases, cardiovascular dysfunction and diabetes have been supported in part by numerous epidemiological studies, which present a compelling case between exposure to UV light and the incidence of a variety disease states (82, 109, 139, 140). In vitro and cell based models have generated promising results, however a direct causal link implicating vitamin D deficiency to most conditions remains elusive based on the current collection of in vivo studies. Complicating the treatment protocol for calcitriol and its derivatives, hypercalcemic responses typically manifest after the administration of doses usually required for the prevention or treatment of

58 59 specific conditions (107). Therefore, novel structural analogs that diminish these adverse effects are desirable as a treatment option for a multitude of biological conditions.

Our laboratory has merged a simple yet highly effective ultrafiltration sample preparation technique with liquid chromatography mass spectrometry (LC-MS) and has continued to optimize this process for over a decade (141). This platform has become an indispensable selective and sensitive screening tool for the identification of ligands found within complex marine and botanical natural product extracts to receptors and enzymes of pharmacological significance (135, 142). Figure 3.1 shows a schematic of the ultrafiltration LC-MS process. Natural products display great potential as a reservoir of compounds with vast structural diversity, which may be used to hasten lead discovery and development of new therapeutic agents (143).

The van Breemen laboratory actively screens natural products against a variety of biological targets implicated in neoplastic diseases including the retinoid X receptor (RXR) (130, 135, 142, 144). Recently, the discovery of the novel RXR agonist, 3-amino-6-(3-aminopropyl)- 5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride designated as AM6-36, prompted the development of screening assays for the other members of the nuclear receptor subfamily 1 (NR1), which form heterodimers with RXR (126). Complementary to our current chemoprevention screening platform, an ultrafiltration assay was developed for the VDR, which was used as part of this dissertation to discovver structurally diverse binding partners for VDR that might resolve the aforementioned issues with calcitriol administration. The proof of principle assay was performed with the VDRs putative endogenous ligand calcitriol, and members of the indenoisoquinline class were subsequently screened with promising results. 60

Figure 3.1 Conceptual diagram of ultraﬁltration LC-MS

Ligand-receptor Unbound ligands complexes Wash unbound compounds to waste

H Botanical/Marine extracts or a Ultraﬁltration Dissociate macromolecular complex, P add internal standard, dry under N library of puriﬁed compounds separation 2 L C

LC-MS Elute desalted Identiﬁcation ligands into MS

m/z 61

3.2 Materials and methods

3.2.1 Chemicals and reagents

Cholecalciferol (vitamin D) and calcitriol (figure 3.2) were purchased from Sigma Aldrich (Saint Louis, MO). AM6-36 was synthesized and provided by Dr. Mark Cushman of the Department of Medicinal Chemistry and Molecular Pharmacology at Purdue University. 8-prenylnaringenin (8PN, figure 3.2) was obtained from the UIC/NIH Center for Botanical Dietary Supplements where it had been isolated from hops, humulus lupulus (124). The ligand binding domain (amino acids 118-427) of human recombinant VDR (determined to be greater than 95% homogeneous by SDS-PAGE) was purchased from ProteinOne (Bethesda, MD). Upon receipt, the VDR solution, which contained a storage buffer consisting of 20 mM Tris-Cl, 20% glycerol, 100 mM KCl, 1 mM DTT, and 0.2 mM EDTA, was partitioned into 20 µl aliquots, flash frozen in a dry ice acetone bath, and stored at -80 °C until use. All organic solvents were HPLC grade or better and were purchased from Thermo Fisher (Hanover Park, IL). High purity water was prepared using a Millipore (Bedford, MA) Milli-Q system.

3.2.2 Ultrafiltration Screening Conditions

A total volume of 50 µL consisting of 50 nM VDR, 100 nM calcitriol, and a binding buffer

(20 mM Tris-HCl, 100 mM NaCl pH 8.0) was incubated at 37 °C for 1 h. The entire reaction mixture was then transferred to a Millipore (Bedford, MA) YM-10 ultracentrafugation filter fitted with a regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 10, 000 kDa. The filter device was centrifuged twice at 4 °C at 12,000 x g for 10 min to remove unbound low molecular weight compounds and rinse the VDR ligand complex with ice cold buffer (50 mM ammonium acetate, 50 mM NaCl pH: 8.0.

Once the spin column has been washed thoroughly, the concentrated ultrafiltrate was collected by inversion of the spin column and subjected to another centrifugation step at 3,000 x g for 10 min. The protein/ligand complex was then disrupted with a 6x volume of methanol, 62

Figure 3.2 Structures of compounds utilized during the proof of princple stage of ultraﬁltration LC-MS screening for the VDR

H H

IS: Cholecalciferol 1,25-Dihydroxycholecalciferol Chemical Formula: C27H44O HO Chemical Formula: C27H46O3 Molecular Mass: 384.64 HO OH Molecular Mass: 416.64

O HO O

N NH2 H2N OH O

O AM6-36 IS: 8PN Chemical Formula: C H O Chemical Formula: C19H17N3O2 20 20 5 Molecular Mass: 319.36 Molecular Mass: 340.37 63 and vitamin D was added as the internal standard giving a final injection concentration of 200 nM. Each sample was dried under a steady stream of nitrogen and reconstituted with 50 µl of the initial HPLC solvent immediately before LC-MS analysis. As a control for non-specific binding, identical incubations were carried out with denatured VDR. Any inter-assay variations were controlled by normalization of chromatographic response to the internal standard. Comparison of the normalized LC-MS profiles of the experiment and control displaying an increased area of 2-fold indicates a binding event. The sample preparation for the screening of VDR with the indenoisoquinoline class of compounds was performed in an identical manner as above with the following exceptions; 8PN was utilized as an internal standard at a final concentration of 1 µM, and the incubation concentration of VDR was 1 µM.

3.2.3 LC-MS Conditions for the Proof of Principle Assay

A 10 µl injection volume of the reconstituted ultrafiltrate was chromatographically separated with a Shimadzu (Columbia, MA) LC20AD HPLC system and an Agilent (San Jose,

CA) Zorbax XBD C18 (5 μm, 2.1 X 50 mm) column. A flow rate of 400µL/min was used with a rapid linear gradient of 75-100% of methanol and 0.1%water/formic acid. An isocratic hold for 2 mins at 100% methanol was to ensure that vitamin D was completely eluted from the column. The column was equilibrated for 5 mins at 75% methanol before the next analysis. An Applied Biosystems (Foster City, CA) API 4000 triple quadrupole mass spectrometer was operated in selected ion monitoring (SIM) mode during this analysis. Analyst 1.5 was used for operational control of the LC-MS system the instrument parameters were optimized as follows; curtain gas 20, ion source gas 60, ion spray voltage 5 kV, temperature 500 °C, declustering potential 70, entrance potential 10, collision cell rod offset -20, collision cell exit potential 15, and a dwell time of 500 ms for each ion.

As a class, sterols characteristically lose water through charge mediated or thermal processes during atmospheric pressure chemical ionization (APCI). During SIM multiple ions were monitored corresponding to these sequential neutral losses. The protonated molecule (m/z 64

+ 416.3) of calcitriol was scarcely observed, but instead abundant ions of [MH-H2O] of m/z 399.3,

+ + [MH-2H2O] of m/z 381.3, and [MH-3H2O] of m/z 367.3 were observed as well as the ions for the

+ + internal standard vitamin D [M+H] m/z 381.3 and [MH-H2O] m/z 367.3). Confirmation of these species as valid fragments and not randomly interfering contaminants acquired through the ultrafiltraton process was conducted through high-resolution accurate mass measurements and tandem mass measurements of vitamin D and calcitriol standards acquired using a Shimadzu ion trap time-of-flight of hybrid mass spectrometer (figure 3.3). Operational parameters for the ion trap were as follows; argon was used as the bath gas, normalized collision energy for collision induced dissociation was set to 50%, ion accumulation time was set to auto sensitivity control, and an activation q value of 0.210 was employed. The ion source parameters are as follows; corona needle voltage of 4.0 kV, source block temperature 300 °C, curved desolvation line temperature 300 °C, and nebulizer gas flow of 2.5 L/min. An external calibration was performed usinga solution of sodium trifluoroacetate to obtain sub-5 ppm mass accuracy. A standard compound chemically similar to the target analyte was often included as a reference mass to compensate for room temperature fluctuations that might adversely affect mass accuracy. This practice provided additional confidence that the most accurate mass measurement for the IT-TOF was obtained.

3.2.4 LC-MS Conditions for Screening of AM6-36 for Binding to VDR

A 10 µl aliquot of the reconstituted ultrafiltrate was chromatographically separated using a Shimadzu LC20AD HPLC system and an Agilent (San Jose, CA) Zorbax XBD C18 (5 μm, 2.1 X 50 mm) column. However, a flow rate of 250 µL/min was used with a linear gradient from 20- 100% methanol in water/0.1% formic acid. After an isocratic hold at 20% methanol for 1 min, a linear gradient for 3 mins followed by an isocratic hold at 100% methanol for 2 mins. The column was equilibrated for 5 min before the next analysis. Instead of APCI, electrospray was used for ionization of AM6-36 and the internal standard 8PN. The protonated molecues of AM6-36 and 8PN at m/z 320.1 and m/z 340.1, respectively, were measured using SIM with an API 4000 triple quadrupole mass spectrometer. The optimized mass spectrometry conditions are as follows; 65 declustering potential 70, exit potential 10, curtain gas 15, ion source gas 20, nebulizing current 4, probe temperature of 450 °C, and a dwell time of 500 ms.

3.3.1 Results

Vitamin D metabolites have usually been derivatized prior to electrospray LC-MS analysis to enhance their ionization efficiency (145). A rapid mass spectrometry based screening assay was needed that could be used to process thousands of sample without undesirable derivatiation steps. Therefore, APCI without derivatiation was utilized instead of electrospray with derivatiation for the analysis of vitamin D analytes. Due to high temperatures during APCI an increased loss of neutral water molecules can be seen, resulting in a diminished response. However, instrumentation has become increasingly sensitive alleviating this issue with APCI.

Demonstration of the proof of principle ultrafitration LC-MS platform for the new chemoprevention target, VDR was carried out using the endogenous VDR ligand, calcitriol. Other endogenous compounds are known to bind VDR and could have been employed for this analysis such as the bile salt lithocholic acid. Calcitriol was used do it’s much higher affinity for VDR which facilitated assay development using very low concentrations of VDR to reduce costs. Note that lithocholic acids binding affinity for VDR is 1000-fold lower affinity than calcitriol. Likewise, a sensitive mass spectrometer operated in SIM mode was employed allowing for the detection of calcitriol at nanomolar levels. Again this ensured that only small quantities of VDR were needed for each assay.

Denatured VDR was used as a negative control experiment during ultrafiltration LC-MS screening to correct for non-specific binding to the protein and the ultrafiltration apparatus. Peak enhancement for compounds in the LC-MS analysis of experimental ultrafiltrates compared with controls indicates specific binding to the VDR. Figure 3.3 displays the resultant chromatogram of standard compounds with a retention time of 4.0 min for calcitriol and 5.4 min for vitamin D, also shown is the accurate mass spectra generate of the Shimadzu ITTOF for confirmatory purposes. Figure 3.4 shows overlaying LC-MS screening of calcitriol and vitamin D for binding to the VDR, 66

Figure 3.3. Positive ion APCI of standards with calcitriol eluting at 4.0 min and vitamin D eluting at 5.4 min. The accurate mass spectra for calcitriol is displayed (below). Note the in-source fragment ions corresponding to the losses of water from the protonated molecule.

m/z 399.3 [MH-H O]+ 2 m/z 385.3 [M+H]+ API 4000, SIM mode, Positive ion APCI

4.0e5

3.2e5

2.4e5

m/z 381.3 [MH-2H O]+ 5.40 2

LC-MS Response 1.6e5 m/z 385.3 [M+H]+

1.0e5

+ m/z 367.3 [M-H2O]

2.0e4

0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5

x1,000,000 8.0 + m/z 381.3148 [MH-H2O]

6.0 + m/z 399.3253 [MH-H2O]

+ m/z 363.3145 [MH-3H2O] 4.0 MS Response 2.0

0.0 225.0 275.0 325.0 375.0 425.0 475.0 525.0 m/z 67 the negative control is displayed as a dotted trace. While operating in SIM several unrelated interfering peaks between 4.5 and 5.0 mins were displayed, therefore it is a prudent operation to check a standards accurate mass as well as match the retention times of standards to experimental chromatograms.

Our longstanding search for a RXR agonist that elicits pronounced effects in cancer related pathways resulted in our discovery of AM6-36, which is a novel indenoisoquinoline originally synthesized as an analog to camptothecin (145). AM6-36 demonstrated significant antiproliferative responses within MCF7 cell lines, which were consistent with genomic actions normally controlled by RXR (126). However, the resulting experimental data concerning AM6-36 activity through the RXR pathways, suggested other complementary pathways might be involved. RXR and VDR roles in cancer regulation have many overlapping actions, such as the upregulation of the cyclin dependent kinase inhibitor p21 (106, 126), therefore we felt it would be prudent to develop an assay and check for AM6-36 cross reactivity. Structurally, AM6-36 is similar to calcitriol in that they are both lipophilic in nature, possess an extended aliphatic appendage, and contain several hydrogen bond donors, which are vital for the potent binding affinity of calcitriol. Therefore AM6-36 was tested for binding to the VDR using the new ultrafiltraion LC-MS assay.

Figure 3.5, the overlaid control (dotted trace) and experimental (solid trace) LC-MS responses of the protonated AM6-36, which eluted at 4.5 mins. 8PN eluted at 5.7 min during this analysis and was utilized for inter-assay ionization efficiency normalization by comparing the area of the curve. AM6-36 shows little non-specific binding to either the apparatus of the VDR, which allows for an unequivocal determination of a binding event. A VDR induced transactivation assay was developed for confirmation of our primary screening platform by Dr. John Pezzuto at the University of Hawaii. Breifly, HeLa cells were transiently transfected with a report vector containing the bioluminescent firefly luciferase with an upstream VDRE and Renilla reniformis luciferase vector. Samples were then treated for 48 h with AM6-36 and the subsequent dual measurement of luciferase activities was performed. As a positive control, vitamin D was 68

Figure 3.4. Positive ion APCI, ultraﬁltraion LC-MS screening of calcitriol (retention time 4.0 min) for binding to the VDR (ligand binding domain). Calcitriol peak enhancement can be seen relative to the normalized control indicating speciﬁc bindind to the VDR.

API 4000, SIM mode, Positive ion APCI m/z 385.3 [M+H]+

+ 4.6e5

Normalized respone - - - - / 3.8e5

3.0e5

2.2e5 5.40 m/z 385.3 [M+H]+ LC-MS response

1.4e5 Chemical interference

m/z 399.3 [MH-H O]+ 6.0e4 2 Negative control - - - -

0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 Retention time (min) 69 utilized. This secondary assay confirmed our discovery that AM6-36 can induce transcriptional activation of the VDRE in HeLa cells.

3.4 Conclusion

We have developed and demonstrated a highly selective and sensitive screening tool for the pleiotropic nuclear receptor, VDR. This assay complements our current portfolio of chemoprevention targets. We then used this assay to discover that the RXR ligand AM6-36 also binds to the VDR. Based on the recently developed proof of principle assay outlined above, we have suc- cessfully identified a nonsecosteroid ligand for the VDR. Also, as AM6-36 is a dual acting ligand by inducing a response for RXR and VDR the development of more potent ligands can further exploit these compounds overlapping pathways in neoplastic conditions. CHAPTER 4

HIGH THROUGHPUT ULTRAFILTRATION LC-MS SCREENING

4.1 Introduction The human vitamin D receptor (VDR) is one of the 48 members of the nuclear receptor superfamily, which is a group of ligand-dependent transcription factors with a distinct modular structural architecture. As a class, nuclear receptors mediate a diverse set of biological actions including metabolic homeostasis, development, and detoxification (35). Additionally, nuclear receptors have evolved into model candidates for targeted small molecule drug discovery; they selectively sequester their drug-like natural ligands within a large internal binding pocket with high affinity resulting in pronounced diverse biological responses dictated by the singular ligand activated complex.

The classically described endocrine actions of the VDR on bone metabolism, mediated through the potent endogenous secosteroid 1α, 25-dihydroxycholecalciferol (calcitriol), have recently been shown to be only a subset of its biological function. Furthermore, the regulatory factors that govern the systemic actions involved in bone maintenance are dissimilar to the paracrine/autocrine responses displayed within a variety of other tissue types (16, 146). In addition to

2+ the strict homeostatic regulation of Ca and Pi, VDR has been shown to attenuate the growth and formation of a variety of neoplastic diseases by inhibiting proliferation, promoting differentiation, inducing apoptotic pathways, and mitigating a variety of signal transduction pathways (106, 147). Ancillary to its emerging role in cancer, mounting epidemiological evidence and numerous in vitro studies and animal models implicate VDR-mediated responses within autoimmune regulated processes (148), cardiovascular pathology (149-151), fetal development and maternal health8, infectious diseases (72), skeletal muscle (152, 153) and skin disorders. However, a direct causal link between the activated VDR actions mediated through calcitriol binding has remained

70 71 elusive for many of these disease states.

Ideally, administration of calcitriol would improve health and attenuate many of these aberrant conditions, however, supraphysiological doses of calcitriol are generally needed to elicit a significant favorable response once a pathological condition has been diagnosed. Prolonged exposure to high doses of calcitriol can result in hypercalcemia and or hypercalciuria responses leading to a host of adverse biological consequences. Many attempts to abrogate the detrimen- tal calcemic effects as well as to improve its metabolic profile through the modification of the secosteroid structure have proved to be advantageous. However, targeted drug therapies based on these compounds have yet to demonstrate a significant improvement over calcitriol in many conditions.14 In an attempt to hasten the process of finding novel compounds which diminish the toxicity associated with high doses of calcitriol, we propose a high-throughput approach that effectively unites our existing ultrafiltration concept and our unique collection of extracts and synthetic compounds.

For more than a decade, ultrafiltration coupled with liquid chromatography-mass spectrometry (LC-MS) has served as our primary platform for screening structurally diverse botanical and marine extracts as well as libraries of natural products and synthetic compounds for ligands to pharmacologically significant targets.(141, 154, 155) We have developed an extensive portfolio of ultrafiltration LC-MS chemoprevention screening assays that include the estrogen receptors

(ERα/β) (156), quinone reductase-2 (QR2) (135), cyclooxygenase-2 (COX2) (142), and retinoid receptors RXR (130) and RAR. Here we report the development of a high-throughput ultrafiltration LC-MS VDR assay to complement this suite of chemoprevention screening assays. Since our traditional ultrafiltration LC-MS assays were comparatively slow, we report here innovations designed to enhance the throughput of the new target VDR as well as all ultrafiltration LC-MS screening assays by at least 100 fold. Our high throughput objective came to fruition with the utilization of a 96 well-plate membrane system and the strategic placement of a newly acquired extended pressure LC system with our existing high resolution accurate mass instrument (IT- 72

TOF). This system not only affords a more rapid screening tool but also provides a significant increase in sensitivity and selectivity compared with the previous ultrafiltration platforms. The use of UHPLC columns and the high resolution capabilities of the ITTOF are the primary sources for the aforementioned advantages.

4.2 Experimental section

4.2.1 Chemicals and reagents

Lithocholic acid, deoxycholic acid and genistein were purchased from Sigma Aldrich (St. Louis, MO), and 6-prenylnaringenin and isoxanthohumol were isolated as described previously, see structures in figure 4.1 (124). Acetonitrile and formic acid (HPLC grade) were purchased from Thermo Fisher (Hanover Park, IL). Deionized water was purified using a Siemens (Warrendale, PA) PURELAB Ultra system. The ligand binding domain (amino acids 118-427) of the human recombinant VDR (determined to be greater than 95% homogeneous by SDS-PAGE) was purchased from ProteinOne (Bethesda, MD). Upon receipt, the VDR solution, which contained a storage buffer consisting of 20 mM Tris-Cl, 20% glycerol, 100 mM KCl, 1 mM DTT, and 0.2 mM EDTA, was partitioned into 20 µl aliquots, flash frozen in a dry ice acetone bath, and stored at -80 °C until use.

4.2.2 Sample preparation

Ultrafiltration separations were carried out using centrifugation with either a Millipore (Billerica, MA) vial system (MWCO 30 kDa) for screening pooled compound libraries or a 96-well plate membrane system (MWCO 10 kDa) for screening extracts of marine bacteria and botanical extracts. A library of purified natural products and synthetic compounds was prepared from compounds isolated in-house at the UIC/NIH Center for Botanical Dietary Supplements Research or purchased from multiple vendors. The purity and identity of each compound was verified using LC-MS. Stock solutions were prepared by the dissolution of powdered material in dry DMSO and then stored at -20 °C. Marine and botanical extracts were obtained as organic solutions and were also stored at -20 °C until use. A standard stock solution of pooled samples was prepared containing ~100 compounds in DMSO at an equimolar concentration of 150 µM. Working solu- 73

Figure 4.1. Structures of compounds used a internal standards (IS), positive controls, and novel analytes that display binding to the VDR

HO O OH

OH O H OH IS: Genistein Calcitriol Chemical Formula: C H O 15 10 5 Chemical Formula: C27H44O3 Molecular Mass: 270.24 Molecular Mass: 416.64

HO OH

HO OH OH

O O H O OH

H H Lithocholic acid Curcumin Chemical Formula: C24H40O3 Chemical Formula: C21H20O6 HO Molecular Mass: 376.57 Molecular Mass: 368.38 H

O O

OH OH OH OH

OH OH

HO O H H HO O

H H H H

HO HO OH OH O H O O H Cholic acid 8-Prenylnaringenin Isoxanthohumol Deoxycholic acid Chemical Formula: C H O Chemical Formula: C24H40O5 20 20 5 Chemical Formula: C H O Chemical Formula: C24H40O4 21 22 5 Molecular Mass: 408.57 Molecular Mass: 340.37 Molecular Mass: 354.40 Molecular Mass: 392.57 74 tions of the pooled ligands were prepared by diluting the DMSO standard stock solution with 50 mM ammonium acetate buffer (pH 8.0) containing 100 mM NaCl to produce final incubation concentrations between 2.5-5 µM. The volume of DMSO in the final incubations was less than 3%.

The assay protein concentration of 1 µM was selected for the detection of ligands with dissociation constants of 50 µM or lower, which included the natural ligand calcitriol (Kd ~ 0.1 nM) and a wide range of potential ligands with lower affinity. The concentration of VDR within each incubated solution was calculated using the molecular mass of 35,000. Genistein was used as an internal standard for all incubations, and a stock solution of 100 µM genistein in DMSO was prepared from powdered material.

4.2.3 Screening protocol

The VDR (4.7 µl) in storage buffer was was mixed with 15.3 µl of a working ligand solution and incubated in the absence of light for 30 min at 37 °C with shaking at 300 rpm. The entire 20 µl volume was transferred to a Millipore membrane apparatus with a molecular weight cut off (MWCO) of either 30 kDa (single vial) or 10 kDa (96 well plate), and subjected to 5 min of ultracentrifugation at either 12,000 X g (single vial) and 3,000 X g (96-well plate). A temperature controlled centrifuge allowed for a constant temperature of 4 °C throughout the ultrafiltration process. Each membrane was rinsed with 200 µl of ice cold buffer (50 mM ammonium acetate, 0.1 mM NaCl at ph 8.0) with centrifugation at 12,000 X g (single vial) for 15 min or 3000 X g (96-well plate) for 1.5 hrs. Inversion of the apparatus and subsequent centrifugation at 3000 X g allowed for the collection of the ultrafiltrate. The ligand bound protein complex was dissociated with the addition of 300 µl methanol containing 0.5 µl of 100 µM genistein. A stream of nitrogen was used to evaporate each ultrafiltrate to dryness, and 20 µl of the initial LC mobile phase was added to reconstitute each sample. A conceptual representation of the evolution of the ultrafiltration process is displayed in figure 4.2.

LC-MS profiles were obtained of each unprocesses samples, each ultrafiltrate after incubation with VDR, each control ultrafiltrate were incubated with denatured VDR, and each control 75

Figure 4.2. Evolution of ultraﬁltration LC-MS

First generation ﬂow cell designed for binding parameter determination

A screening based ultraﬁltration design

High throughput ultraﬁltration platform 76 ultrafiltrate afterwithout VDR. An increase in peak area displayed within the LC-MS chromatogram on the order of 2-fold after normalization with the internal standard was used a the criteria for the determination of a specific binding event, which compares the ultrafiltrate of the VDR incubation with the denatured VDR or void incubations. Positive control experiments for the HTS approach were performed with the bile salt lithocholic acid (LCA) and curcumin (CUR). Control analyses were carried out using either denatured VDR, which was prepared by boiling in water for 10 min, or in the absence of protein. Multiple factors were considered for the identification of VDR binding partners including chromatographic retention times, accurate mass measurement, and comparison with standards.

4.2.4 LC-MS Conditions

LC-MS analysis of the ligands isolated during the ultrafiltration process utilized a Shi- madzu (Columbia, MA) LC20ADXR enhanced pressure tolerance HPLC system (max. 9,600 psi) coupled with an ion-trap time of flight (ITTOF) mass analyzer. A universal LC-MS method for all sample types was developed allowing for an uncomplicated batch setup and data processing workflow of large sample sets. A Shimadzu 2.0 X 50 mm, UHPLC 1.6 µm column was utilized with the solvent system consisted of water/0.1% formic acid channel A and acetonitrile channel B at 500 µl/min employing a linear gradient, conditions are as follows: isocratic hold at 25% B for 0.2 min, 25-100% B in 1 min, isocratic 100% B for 0.8 min, re-equilibrate at initial condition for 1 min. A total run time of 3 min proved to be sufficient to elute all compounds and eliminate run to run carry over. Electrospray mass spectra were acquired in both positive and negative polarities. Operating parameters were defined as follows: Vcap = +4500 V/-3500 V, heating block and curved desolvation line set to 200 °C, nitrogen drying gas flow 1.5 L/min. A total of four events with two assigned for each polarity were employed to cover the desired mass range m/z 100-1500 with a total cycle time of 497 ms.

4.4 Results and discussion

Within the functional complexity of the nuclear receptor superfamily, a more narrowly de- 77 fined grouping has been established based on sequence similarity and function. The constituents of the NR1I subfamily include the vitamin D receptor (NR1I1), pregnane X receptor (PXR; NR1I2), and the constitutive androstane receptor (CAR; NR1I3). Each member of this class has developed the ability to promote the metabolic clearance of potentially harmful endogenous compounds through low affinity binding events, while a few members have expanded to recognize a range of xenobotics and potentiate the alteration of their biological outcome. VDR displays relatively defined ligand selectivity in contrast to the other group members, primarily restricted to the high affinity endocrine modulator calcitriol and its continual role in the detection and detoxification of several bile salts, such as lithocholic acid (LCA). Calcitriol was used during the initial proof of principal stage of the ultrafiltration binding process in order to exploit its high affinity character, which resulted in a 20-fold reduction of protein consumption and ultimately many more itera- tions of sample preparation conditions. Since, atmospheric pressure chemical ionization (APCI) must be employed for the measurement of calcitriol, LCA was utilized as an electrospray (ESI) positive control as a means of producing a consistent screening platform since most drug like compounds are amenable with type of ionization.

A pigmented class of compounds known as Curcuminoids is found in the Indian spice turmeric, which is a member of the ginger family (Zingiberaceae). Due to the medicinal qualities of this rhizomatous plant, this class of compounds has been implicated in a wide range of medical alignments. Curcumin is the most abundant curcuminoid in tumeric, has been extensively characterized and is under investigation as a nutrient with potential medicinal properties (157). Recently, curcumin has been shown to display affinity towards VDR and was therefore adopted as an additional positive control for this investigation.

The selection of genistein as the internal standard was based on several qualities; it readily ionizes in both positive and negative ion electrospray, it displays a similar hydrophobic character as the anticipated binding partners for VDR so that chromatographic elution should be similar, and as a small molecule with a molecular mass of 270 g/mole it’s mass is approximately a median 78

Figure 4.3. Positive ion electrospray UHPLC-MS analysis of ultrafiltraes for the ultrafiltration-MS screening of a library of 100 compounds for ligands to VDR. A) resolution of standard compounds, B) resultant overlayed ultrafiltration experiment, C) adverse feature of ultrafiltration A (x10,000,000) + CA CUR 8PN 4.00 [M+H] 355.1561 DCA [M+H]+ 369.1364 IX - [M-H] 339.1255 Extracted ion chromatograms generated 3.50 [M-H]- 375.2922 [M-H]- 391.2878 on the ITTOF 3.00 [M-H]- 407.2801 2.50

2.00

1.50 LCA 1.00

0.50

0.00 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 B (x10,000,000) [M+H]+ 271.0630 CUR [M+H]+ 355.1544 1.00 [M+H]+ 369.1320 [M-H]- 375.2923 IS: Genistein [M-H]- 269.0474 0.80 [M-H]- 391.2865 [M-H]- 339.1225 IX 0.60 8PN DCA 0.40

0.20 UHPLC MS response LCA

0.00 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 C (x1,000,000) IS: Genistein [M+H]+ 271.0624 7.0 [M+H]+ 355.2716 [M-H]- 391.2815 [M-H]- 269.0469 6.0

5.0

4.0

3.0 DCA

2.0 Isobaric interference

1.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

Retention time (min) 79 value for the majority of compounds screened.

Weekly tuning with an external calibrant (sodium TFA) routinely affords an accurate mass measurement of 5 ppm or better between the m/z values of 100-2000 in both ion polarities. How- ever, significant deviations in laboratory temperature are not uncommon and can adversely affect the instruments mass accuracy. When mass accuracy exceeded the 5 ppm threshold, a mass com- pensation function was used which adjusted the measured mass defect of all compounds based on the measured and predicted values of the internal standard. The scan rate of the instrument contributed to the quality of not only peak shape for the qualitative inspection of differences between control and sample, but also the reliability of the mass accuracy measurements. Defining a maximum flow rate of 500 µl/min was not solely based on the pressure limitations of the mobile phase pump, but with the number and type of scanned events present in the method. Peak shape began to falter at higher flow rates due to a diminishing number of data points allotted for each peak.

As a representative example of the ultrafiltration workflow, figure 4.3 displays a series of chromatograms which resulted from a single incubation of approximately 100 compounds in addition to the two positive control standards previously described. The extracted ion chromatograms representing the unprocessed mixture 4.3a is show to highlight those species showing binding to VDR. Once a compound was extracted from the total ion current, an composition was performed with the provided formula prediction software. Several compounds demonstrated binding as shown in Figure 4.3b and included 8-prenyl naringenin (8PN) and isoxanthohumol (IX) which are constituents of the Humulus lupulus female flower, otherwise known as hops (158), and the secondary bile acid deoxycholic acid (DCA). The structures of these compounds are shown in figure 4.1.

Confirmation of a binding event can only be made with an examination of the negative control sample. Figure 4c displays the chromatographic response for a negative control experiment. While most compounds are completely eliminated from the regenerated cellulose membrane 80 nonspecific non-specific signals for some compound persist due to adsorption to the ultrafiltration membrane. DCA is one such compound that remains on the column after significant washing steps were performed. DCA also exhibited specific binding to VDR as indicated by our minimum criteria of a 2-fold increase in AUC between control and sample response (see table 4.1). Another common adverse feature of the ultrafiltration process results from the type of mass analyzer used; with a large scan window, isobaric contaminants are a nuisance especially when the species co- elute with the analyte of interest. Fortunately, the high resolution accurate mass capabilities of the ITTOF MS provide a means to distinguish isobaric contaminants from potential binding ligands. Manual inspection of the chromatographic response is used in conjunction with peak identification software that displays the difference in response of complementary data sets. A completely automated workup has proven to be unreliable for species with low ion abundances; however the simplicity of the ion listing format significantly improves the likelihood for the identification of moderate to high affinity analytes.

A convenient 96-well plate membrane system significantly expands the volume of botanical and marine extracts can be screened per day. However, the single chamber membrane system is still appropriate for the analysis of simple mixtures of known compounds. This is due in part to the maximum centrifugal force that our rotor systems can produce for each membrane type. The rotor for the single chamber membrane can hold up to 24 vials and can withstand a centrifugal force up to 13,000 x g, while the 96-well plate format accommodates the simultaneous sampling of 192 ultrafiltration vials. However the plate system displays a diminished capacity for high speed centrifugation resulting in longer filtration time. Also, the 96 well plate system utilizes a 10 kDa MWCO membrane which significantly extends the time required for a complete centrifugation cycle compared to the 30 kDa counterpart. Even with the aforementioned disadvantages of the plate system, a 10 fold increase in sampling rate can be seen at full capacity.

4.5 Conclusions

Ultrahigh pressure HPLC (UHPLC) systems are becoming commonplace allowing for 81

TABLE 4.1

COMPARISON OF NORMALIZED RESPONSES WITH RESPECT TO THE MOST POTENT LIGAND 8PN

With hVDR w/o hVDR Normalized ppm Retention % of 8PN Measured Mass AUC AUC Ratio diﬀ time Response

Genistein 27602611 24234502 1.14 [M+H]+ 271.0630 10.7 1.43 N/A

Genistein 14685635 19609143 0.75 [M-H]- 269.0774 7.06 1.42 N/A

LCA 13043 0 - [M-H]- 375.2897 -2.14 1.93 1

DCA 175439 33301 5.3 [M-H]- 391.2865 2.81 1.68 10.3

CUR 316279 0 - [M+H]+ 369.1320 -3.52 1.57 19

IX 85180 0 - [M+H]+ 355.1541 0.28 1.47 5.11

8PN 1665390 0 - [M-H]- 339.1225 -3.83 1.61 100

82 fast analysis of complex mixtures with increased chromatographic resolution. Hybrid mass spectrometers with capabilities of fast scanning, polarity switching, and high resolution accurate mass measurements allow for the coupling to UHPLC systems without sacrificing data quality. In addition, sophisticated feature peaking software allows for the automation of data processing which leads to the generation of more reliable results in a much shorter amount of time.

Our ultrafiltration process has historically displayed a moderate success rate for the discovery of ligands within complex extracts. Similarly, the screening of mixtures of purified compounds has been predominately employed with relatively few compounds, traditionally 10 or less, due to the practical limitations associated with the analytical instrumentation and the deconvolution of data sets. Through the unification of technological advancements within all facets of our screening workflow, a streamlined screening platform which culminates in a significantly enhanced sample throughput and data processing capability has been developed. Based on this strategy our current sampling rate is 100 times faster than our previous platform, enabling the processing of 2,400 compounds or 96 extracts per day. These numbers reflect our current screening needs and by no means suggest that this is the limit to the sample volume that can theoreti- cally be screened. With continuous sample preparation and instrument usage, these values can be easily scaled up 10 fold. As demonstrated through the example of VDR, HTS-PUF can be uni- versally adopted as the standard operating procedure for all previously developed ultrafiltration screening targets. CHAPTER 5

METABOLOMICS GUIDED PROCESSING OF COMPLEX ULTRAFILTRATION DATA

SETS FOR LIGANDS WITH THERAPEUTIC VALUE

5.1 Introduction

Metabolomics is the process of metabolite identification within samples, which can be based on a targeted or untargeted approach (91). LC-MS has recently emerged as a dominant platform for un-targeted metabolomics studies, which are designed to visualize changes due to disease, therapy, exposure to toxins, etc., within complex samples consisting of thousands of small molecules. Utilization of UHPLC in combination with mass spectrometry improves column efficiencies and can significantly improve metabolomics coverage for complex samples by facilitating detection of additional compounds for characterization and identification (159). High resolution accurate mass instruments using TOF, orbitrap (93), and FT-ICR (92) mass spectrometers are frequently utilized for metabolomics workflows given their capability to distinguish isobaric species within the mass spectra thereby allowing for prediction of elemental composition (91).

A variety of software platforms designed for processing and comparing raw data files have been created as open source tools as well as proprietary commercial packages. Common vendor metabolomic software suites include Sieve (Thermo, San Jose, CA) MarkerLynx (Waters, Milford, MA), MarkerView (Applied Biosystems, Foster City, CA), MassHunter Profiling (Agi- lent Technologies, Santa Clara, CA), and Profiling solutions (Shimadzu, Columbia, MA), which provide a variety of functions including filtering, feature detection, alignment, de-isotopeing, and normalization (95). These tools feature easy to use graphical interfaces, but no insight into the processing functions is provided and access to the full complement of algorithms is prohibited. Many open source tools are available such as XCMS (160), MZmine (161), and OpenMS (94), which allow for a more in depth treatment of datasets, but steep programing learning curve asso-

83 84 ciated with these programs can be a deterrent for some users. In collaboration with Shimadzu, the van Breemen laboratory has acquired greater insight into Profiling solutions software. Therefore, this program was utilized for ultrafiltration analysis. Since there are similarities between XCMS and Profiling solutions the following section concerning processing features will be drawn from the open source program XCMS.

5.1.1 Metabolomics Software

XCMS is an acronym for “X” forms of Chromatography Mass Spectrometry and imple- ments the R statistical language for algorithm parameter selection (160). The processing of LC- MS data sets beings with “binning” of the mass domain into 0.1 m/z wide slices and operated on in the chromatographic time domain. Binning works well with unit resolution mass spectrometers, but the employment of instruments capable of accurate mass measurements necessitated the creation of another approach due to the large binning size. Therefore, centWave was recently added to XCMS which facilitates the processing of high resolution accurate mass data set by utilizing a density based feature detection that identifies regions of interest and applies a wavelet transformation or Gauss fitting (162). Within each mass domain slice a signal representing the maximum response for each time point is determined, resulting in an extracted base peak chromatogram (EIBPC). A second-derivative Gaussian function serving a model peak is applied to each slice operating as a matched filter (163). A signal to noise cutoff is then applied finishing the peak detection process.

Peak matching algorithms were designed to evaluate LC-MS chromatograms (in this dissertation the sample groups were ultrafiltration controls and experimental samples) by calcu- lating retention time deviations and ion intensities for comparison. Alignment is accomplished with a mass domain fixed overlapping interval bin and then employs a kernel density estimation providing an approximate distribution of retention times associated with each bin. De-isotoping functions such as SIRIUS (80) incorporate the natural isotopic distribution of elements generating a sum formula, which provides essential molecular information for identification purposes. Data 85 processing algorithms were explored for deconvolution of ultrafiltration data sets, which could greatly hastens the identification of bound ligands.

5.1.2 Hypercalcemic Effects of Lead Structures

As discussed in chapter 1, vitamin D plays a crucial role in mediating a wide variety of actions within normal and diseased cellular processes. Maintaining an adequate circulating level of vitamin D metabolites provides the best preventative option for overall health, however adverse effects administration of vitamin D or its structural analogs. In healthy adults a sustained daily intake (several months) of 40,000 IU vitamin D leads to a host of aliments due to hypercalcemica. Potential symptoms of toxicity include polyuria, polydipsia, weakness, and pruritus. If immedi- ate action is not taken renal failure, abnormal heart rhythms, and ectopic calcification of blood vessels will result (74). A variety of vitamin D structural analogs have designed, synthesized and tested in order to address the toxicity issue while maintaining clinical effectiveness. Calcipotriol

(76) and 1,25-hydroxy-2-16-ene-23-yne-vitamin D3 (78) are two such analogs that possess low calcemic activity while maintaining their intended actions against psoriasis and retinoblastoma, respectively. The mechanisms behind the diminished calcemic responses of such vitamin D derivatives have ranged from altered binding to the VDR (alternative binding pocket) (84), increased catabolism through 24-hydroxylase activity, and the involvement of the rapid response receptor associated with the plasma membrane (73, 164).

Recently, the protein-disulfide isomerase-associated protein (Pdia3) has been shown to mediate the signal transduction response in osteoblasts, modifying gene transcription of bone related products and mineralization in osteoblast-like MC3T3-E1 cells (77). The thiol-disulfide oxidoreductase known collectively as ERp57/GRP58/Pdia3/MARRS is a soluble multifunctional protein typically associated with endoplasmic reticulum (ER), however this protein has been localized to the nucleus, plasma membrane and extracellular matrix (87). Through the interaction with two lectin chaperones (calnexin and calreticulin) ERp57 promotes the oxidative folding of newly synthesized glycoproteins (89). Additionally, ERp57 elicits a variety of catalytic functions 86 include the reduction, isomerization, and oxidation of proteins. This protein demonstrates the ability to reduce partially folded MHC class I molecules facilitating the loading of short peptides for presentation to cytotoxic T lymphocytes (85).

The first development giving rise to a membrane receptor for calcitriol was the purification of a protein from the basal lateral membranes of chick intestinal epithelium displayed satu- rable binding to calcitriol inducing transcaltachia (79).Additional insights indicate that the intestinal epithelium and chondrocytes stimulated by calcitriol produce rapid membrane responses that are distinct from genomic effects involving the VDR (165). ERp57 has been postulated as an alternate candidate for the non-genomic rapid response membrane-associated receptor for calcitriol (63). VDR receptors localized to the caveoli of the plasma member demonstrating binding to the s-cis form of calcitriol remain the other contending species for the non-genomic processes (84). Recently, a report demonstrated the photo-protective effects of calcitriol may act through combined mechanisms involving both the VDR and ERp57 (61). As part of this dissertation the lead structures that display binding toward VDR were subsequently screened against ERp57 in an attempt to characterize potential hypercalcemic responses associated with these ligands.

5.2 Materials and Methods

5.2.1 Chemicals and Reagents

Calcitriol and reserpine were purchased from Sigma Aldrich (StLouis, MO). His-tagged human recombinant ERp57 protein (amino acids 25-505), determined to be greater than 95% pure by SDS-PAGE was purchased from Novus biological (Littleton, CO). Upon receipt, the ERp57 solution, which contained a storage buffer consisting of 20 mM Tris-HCl buffer (pH 8.0), 1mM DTT, 0.1 M NaCl, and 10% glycerol, was partitioned into 10 µl aliquots, flash frozen in a dry ice acetone bath, and stored at -80 °C until use. All organic solvents utilized were designated as Optima for LC-MS was purchased from Thermo Fisher (Hanover Park, IL). Deionized water was purified using a Siemens (Warrendale, PA) PURELAB Ultra system. 87

5.2.2 Extracts and Pure Compounds

A library of natural products and of synthetic compounds has been assembled in the van Breemen research group through screening for chemoprevention agents as part of a program project grant funded by the NIH for 17 years. Approximately 700 unrelated compounds contained in the library were screened that were dissolved in DMSO at 10 mM and stored at -20 °C. Two directed libraries were screened that are based on structural motifs (indenoisoquinolines and stilbenes) synthesized in Dr. Mark Cushman’s laboratory at Purdue University. The indenoisoquinoline structure is derived from a well described scheme presented within a number of articles. Briefly, homophthalic anhydrides are reacted with Schiff bases affording cis-carboxcylic acids, with subsequent acyl chloride formation. Ring closure is accomplished through an oxidative Freidel-Craft mechanism (86, 88). The stilbene derivatives are modeled after resveratrol with a concise synthetic protocol. Trans-stilbene analogs are derived from an initial Wittig reaction of substituted aryl aldehydes with a subsequent Heck coupling of the vinyl aromatic compound with iodo-benzene groups (119). The compounds in the directed libraries were either received as powdered material or as 10 mM DMSO solutions. Each compound stored in 96-well plate screening library at -20 °C.

Numerous botanical extracts were obtained for screening from the UIC/NIH Botanical Center for Dietary Supplements Research, and marine extracts were provided by Dr. Fenical of the University of California San Diego and Dr. Brian Murphy laboratory of UIC. Random selec- tions of 5000 structurally similar compounds were screened that were provided by Dr. Steven Kron of the University of Chicago. In an effort to be concise, a list of structurally related groups will be provided instead of individual chemical names for these libraries. A myriad of flavonoids, steroids, alkaloids, pharmaceutical drugs, and pure compounds derived from botanical and marine extracts were screened. 88

5.2.3 High Throughput Screening

The detailed procedure for screening extracts and compounds against VDR utilizing the new high throughput workflow was presented in chapter 4. This protocol was performed in an iterative manner with individual extracts or mixtures of purified compounds totaling 100 per well in equal molar concentrations. Ultrafiltration-UHPLC screening of libraries and extracts for ligands to ERp57 was carried out in an analogous fashion with a 1 µM protein concentration, 20 µl incubation volume, less than 2% DMSO content, as well as a binding buffer (20 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl) and washing buffer (50 mM ammonium acetate pH (8.0), 50 mM NaCl). The proof of principle assay for ERp57 binding was demonstrated using calcitriol, and the internal standard reserpine was used to normalize signals between analyses.

All binding events were defined as at least a 3-fold enhancement of signal for the experimental signal relative to control. Because the extracted ion chromatograms for compounds that bind to VDR were complex, the negative control signals will be shown. However, it should be noted that the washing step typically removes all unbound material.

5.2.4 LC-MS Conditions

The LC-MS conditions employed for screening against VDR were described in chapter 4. Several procedural changes were implemented for the LC-MS analysis of the ultrafiltrates containing ligands to ERp57. A Shimadzu LC20ADXR HPLC system coupled to the ITTOF was again utilized. A 10 µl injection of reconstituted ultrafiltrate was injected onto a Shimadzu 2.0 X 50 mm, 1.6 µm column. The UHPLC mobile phase consisted of a 1.5 min linear gradient from 85% to 100% acetonitrile containing 0.1% aqueous formic acid. APCI mass spectra were acquired in positive mode. Operating parameters were defined as follows: corona needle = +4000 V, heating block and CDL, 300 °C; nitrogen drying gas flow 2.5 L/min. The mass range m/z 100-500 was recorded every 200 ms. 89

5.2.5 Profiling Solutions

The profiling solutions software contains many user defined setting, which are dependent the chromatographic quality of each sample. Careful inspection of the resultant chromatogram for each pooled standard or extract must be performed in order to adjust integration and spectral processing parameters. Optimization of these parameters is crucial in order to achieve a selective tool that discerns significant peak enhancements between experimental and control samples and the chemical and detector noise present in the total ion chromatogram.

Data file parameters were selected to process both ion polarities simultaneously. Spectra processing parameter were defined as follows; m/z ion tolerance 10 mDa, ion retention tolerance 0.2 min, ion intensity threshold 1000 counts, detect isomer valley 20%, allow for low abundance peaks with no isotope peaks, de-isotoping the ion matrix was enabled. Integration parameters included a peak; width of 3 s, slope 500/min, base line drift 0/min, min peak area 100,000 counts, and Savitzky-Golay smoothing with a 25 point width .

5.3 Results and Discussion

Profiling solutions software was utilized to sift through large data sets generated by the high-throughput ultrafiltration process UHPLC-MS screening process. This process facilitated rapid and accurate assessment of complex samples such as botanical and marine extracts and proved to be a reliable tool for screening large numbers of compounds within a single mixture. The alternative, manual selection of ultrafiltration peaks can be a tedious process even for known compounds and proved to be exceedingly difficult for unknown constituents in complex extracts. Although valuable, Profiling solutions software did not always find all meaningful constituents and produced some unwanted artifacts. In a typical workflow, Profiling solutions software would filter tens of thousands of peaks resulting in a list of several hundred accurate masses, and then with manual inspection an accurate selection of binding partners was realized. This software is under continual refinement by Shimadzu, and with each successive generation of software, an 90 enhanced product tailored to our ultrafiltration screening application should eventually emerge as a standard screening tool.

Although AM-6-36 was the first indenoisoquinoline compound identified as a ligand for VDR, subsequent screening of numerous analogs has revealed more potent compounds within this class. These compounds are relatively similar in structure which allows for an assessment of their relative binding affinities when these compounds are screened together as equal molar solutions. The relative affinity of each compound for VDR in this competitive screening can be extrapolated based on the peak areas observed during ultrafiltration UHPLC-MS analysis of the ultrafiltrates. Since these compounds were similar in structure individual compound variations in ionization efficiency were considered negligible.

Examples of ultrafiltration UHPLC-MS screening data showing indenoisoquinolines that bound to VDR are shown in figure 5.1, and the chemical structures of these ligands are shown in figure 5.2. The indenoquinoline structures were found to bind toVDR are as follows; N-(6-(3-aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetamide (MAR-V-1), 6-(3-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)- dione (MAR-VI-46), 6-(3-aminopropyl)-8,9-dimethoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (AM-IX-70), 3-amino-6-(3-(dimethylamino)propyl)-5H-indeno[1,2-c] isoquinoline-5,11(6H)-dione (PVN-6-21), 6-(3-aminopropyl)-9-methoxy-5H-indeno[1,2-c] isoquinoline-5,11(6H)-dione (AM-XII-70), 3-amino-6-(3-aminopropyl)-6,11-dihydro-5H- indeno[1,2-c]isoquinolin-5-one (PVN-6-16), 3-amino-11-hydroxy-6-(3-hydroxypropyl)-6,11- dihydro-5H-indeno[1,2-c]isoquinolin-5-one (MAR-VI-24, N-(6-(3-hydroxypropyl)-

5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetamide (MAR-VI-1), 3-amino-6-(3-aminopropyl)-11-hydroxy-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-5-one (MAR- VI-30), N-(6-(3-aminopropyl)-11-hydroxy-5-oxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl) acetamide (MAR-VI-38), N-(3-(3-amino-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)acetamide (MAR-V-80), 3-amino-6-(3-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)- 91 dione (AM6-36), 6-(4-aminobutyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (TN-1-65), 6-(3-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (TN-1-62), 6-(6-aminohexyl)-5H- indeno[1,2-c]isoquinoline-5,11(6H)-dione (TN1-75), 6-(5-aminopentyl)-5H-indeno[1,2-c] isoquinoline-5,11(6H)-dione (TN-2-37), 6-(3-(dimethylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione and 3-amino-6-(3-(methylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (two structures with the same designation PV-6-43), and 3-amino-6-(3- aminopropyl)-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-5-one (PVN-6-15).

More than one hundred indenoisoquinoline compounds were screened, and several structural features were identified that were essential for binding to the VDR. An extended aliphatic chain with a terminal hydrogen donor as well as a hydroxyl or carbonyl placed on the indene portion of the indenoisoquinoline molecule was essential for binding to the VDR. The 10 indenoisoquinolines showing the highest affinity for VDR based on the initial ultrafiltration UHPLC-MS screening were retested as an equimolar mixture to determine their relative affinities for VDR. This experiment is based on the work of Sun, et. al., who showed that ultrafiltration UHPLC-MS screening may be used to rand order ligands during competitive binding (144). The chromatograms for this analysis are shown in figure 5.3. The most potent VDR ligand was TN-1- 75, followed by TN-2-37 and then MAR-5-80, AM6-36, which was the first compound in this series found to bind to VDR, was ranked 7th in affinity.

The indenoisoquinoline structure has been of great interest to our chemoprevention studies based on the multiple functionality of these compounds. An analog of nitidine and originally developed by Cushman et. al. (166) as a possible topoisomerase inhibitor, AM6-36 was later discovered to be an agonist of RXR. In this dissertation, AM6-36 and related indenoisoquinolines were also shown to bind to VDR. By displaying affinity toward both RXR and VDR and in some cased to Topoisomerase-1, these compounds might be more potent inhibitors of carcinogensis than mono-functional compounds. The chemical structures of the indenoisoquinolines that were found to bind to both VDR and RXR are shown in figure 5.4. 92

Figure 5.1. Initial screening of 50 indenoisoquinoline analogs against VDR using positive ion ultraﬁltration UHPLC-MS. The concentration of VDR was 1 µM while the concentration of each indenoisoquinoline was 5 µM

(x10,000,000) [M+H]+ 321.1279 4.00 IT-TOF; (+) ion ESI Extracted ion chromatogram

IS:[M+H]+ 271.1022 3.00

2.00

+ + LC-MS response [M+H] 362.1538 [M+H] 363.1379 [M+H]+ 410.1401

1.00 [M+H]+ 305.1305 [M+H]+ 348.1728 [M+H]+ 322.1296 [M+H]+ 362.1538 [M+H]+ 306.1650 [M+H]+ 335.1418 0.00 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 Retention time (min) 93

Figure 5.2. Structures of the indenoisoquinolines that displayed affinity to VDR during ultrafiltration UHPLC-MS screening in figure 5.1

O HO O

N OH N OH HN N OH

H2 N H 2N O O O O MAR-VI-24 MAR-VI-1 MAR-V-49 Molecular mass: 322.36 Molecular mass: 362.38 Molecular mass: 320.34

O O O O O O

N NH2 N NH2 N N N NH2

H 2N O 2N O O O O MAR-VI-46 AM-XII-70 PVN-6-21 AM-IX-70 Molecular mass: 304.34 Molecular mass: 334.37 Molecular mass: 347.41 Molecular mass: 409.39

HO O O

O O

N NH2 N NH 2 N NH N NH2 H2N H2N H2N N H O O O O MAR-VI-38 PVN-6-15 MAR-V-80 MAR-V-80 Molecular mass: 321.37 Molecular mass: 305.37 Molecular mass: 361.39 Molecular mass: 361.39 94

Figure 5.3. Competitive ultrafiltration UHPLC-MS profiling of a mixture of 10 indenoisoquinoline ligands for relative affinities to VDR

(x10,000,000) 4.00 IT-TOF; (+) ion ESI + [M+H]+ 347.1669 [M+H} 333.1547 Extracted ion chromatogram 3.50

3.00 [M+H]+ 362.1406 2.50 Isobaric interference

2.00 [M+H]+ 319.1376

1.50 + LC-MS response [M+H] 320.1387

1.00 [M+H]+ 334.1550 [M+H]+ 305.1220 [M+H]+ 306.1594 0.50 [M+H]+ 348.1689 0.00 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 Retention time (min)

TABLE 5.1 RANKING OF INDENOISOQUINOLINE ANALOGS RELATIVE TO TN-1-75

Compound Positive VDR signal Ratio (X/TN -1-75) Percent of TN -1 -75 AM6 -36 1903415 0.173 17.3 TN -1-75 9397620 1 100 TN -2-37 7877048 0.838 83.8 MAR -5-80 4875144 0.506 50.6 TN -1-65 3320824 0.353 35.3 PVN -6-43 2011978 0.214 21.4 TN -1-62 4013179 0.427 42.7 PVN -6-15 1422175 0.151 15.1 PVN -6-80 152467 0.016 1.6 PVN -6-43 2155086 0.222 22.2

Figure 5.4. Structures of indenoisoquinoline analogs with aﬃnities for both RXR and VDR

O O O

N NH2 N NH N 2 N H2N H NH2 O O O AM6-36 MAR-5-80 TN-1-65 Molecular mass: 319.26 Molecular mass: 361.39 Molecular mass: 318.37

N N NH2 NH2 N NH2 H2N O TN-1-75 PVN-6-15 TN-1-62 O O Molecular mass: 346.42 Molecular mass: 346.42 Molecular mass: 304.34

O O O

N N H N NH2 N N H2N

H2N O PVN-6-43 O TN-2-37 O PVN-6-43 Molecular mass: 347.41 Molecular mass: 332.40 Molecular mass: 333.38 96

Figure 5.5 shows extracted ion chromatograms for ultrafiltration UHPLC-MS screening against VDR, of structurally diverse compounds that were determined to be ligands. Note that these ultrafiltration UHPLC-MS data were obtained during screening of equimolar mixture of 100 compounds that included all of these hits. The chemical structures for these compounds are shown in figure 5.6, and the chemical names for these ligands are as follows, (2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a ,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid (botanical antioxidant, celastrol), N-(2-(3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)quinoxaline-2-carboxamide (a proposed SIRT1 activator, SRT1720), 2-(isopropylamino)-1-(naphthalen-1-ylmethoxy)ethanol (non-selective beta blocker, propranolol), 2-(4-(dimethylamino)phenyl)-3,6-dimethylbenzo[d] thiazol-3-ium (basic yellow 1 dye, thioflavin T), (8S,10S)-8-acetyl-10-((1R,3S,4R,5R)-3-amino-4-hydroxy-5-methylcyclohexyloxy)-6,8,11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotet- racene-5,12(5aH,11aH)-dione (chemotherapeutic, daunorubicin), (E)-4-styrylaniline (resveratrol analog), the antitussive (dextromethorphan), 2-(2-fluorobiphenyl-4-yl)propanoic acid (NSAID, flurbiprofen), matridin-15-one (kappa opioid receptor agonist, matrine), bis-benzylisoquinoline alkaloid (calcium channel blocker, tetrandrine), methoxy-5-methyl-;benzo(e)-1,3-dioxolo(4,5- l)(2)benzazecin-12(5h)-one,4,6,7,13-tetrahydro-9,10-d;4,6,7,13-Tetrahydro-9,10-dimethoxy- 5-methylbenzo(e)-1,3-dioxolo(4,5-l)(2)benzazecin-12(5H)-one (alkaloid, crytopine), (Z)-2-(4-(1,2- diphenylbut-1-enyl)phenoxy)-N,N-dimethylethanamine (ER antagonist, tamoxifen), [2aR-[2aα, 4β,4aβ,6α,9α(αR*,βS*),11α,12α,12aα,12bα]]-β-(Benzoylamino)-α-hydroxy-6,12b-bis(acetyloxy)- 12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-tetra- methyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxe t-9-yl ester benzenepropanoic acid (mitotic inhibitor, paclitaxel), and 7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (isofla- vone, daidzein). 97

Figure 5.5. Computer reconstructed mass chromatograms of VDR ligands discovered during ultraﬁltration UHPLC-MS screening of 100 compounds

[M+H]+ 260.1678 (x10,000,000) IT-TOF; (+/-) ion ESI 3.00 [M+H]+ 272.2033 Extracted ion chromatogram [M+H]+ 245.1005 [M+H]+ 372.2348 + [M+H] 370.1690 M+H]+ 854.5880 [M+H]+ 470.1794 2.50 [M+H]+ 623.3159 [M+H]+ 196.1125

[M+H]+ 451.2862 2.00 [M+H]+ 255.0679

[M+H]+ 249.1498 1.50 [M+H]+ 304.2948 LC-MS response 1.00 [M+H]+ 528.1932

0.50

0.00

0.50 1.00 1.50 2.00 2.50 3.00 Retention time (min) 98

Figure 5.6. Structures of compounds that displayed binding towards VDR during ultraﬁltration UHPLC-MS as shown in ﬁgure 5.5 O COOH O O S

N N N N N O H H H O O N HN Thioﬂavin T Molecular mass: 283.41 S N HO NH2

N O Celastrol Tetraudrine Molecular mass: 450.61 SRTT1720 N Molecular mass: 622.75 O HN Molecular mass: 469.56 (E)-4-styrylaniline Molecular mass: 195.26 O N O

H H O N O O H N H N O N O H O Tamoxifen Cryptopine Molecular mass: 371.5 Matrine Dextromethorphan HO O Molecular mass: 369.41 Molecular mass: 248.36 Molecular mass: 283.41

O N H O Diadzein OH O OH OH Molecular mass: 254.2 O O OH O Propranolol OH O Molecular mass: 259.34 H O NH O

O OH O O O OH O O OH O OH O O O Paclitaxel Daunorubicin Molecular mass: 853.91 F Flurbiprofen Molecular mass: 527.25 OH Molecular mass: 244.26 NH2 99

In addition to the endogenous and synthetic secosteroid as well as the bile acids a few other compounds have displayed affinity towards VDR. Currently the ω3- andω 6-essential polyunsaturated fatty acids, docosahexaenoic acid, and arachidonic acid, γ-tocotrienol a vitamin E derivative, and curcumin have all been documented as substrates for VDR (62, 167). It is not surprising that a few of the binding partners discovered above are chemotherapeutic agents. Cal- citriol and its analogues have demonstrated synergistically or at least additive responses when co-administered with several known antineoplastic agents. Also, some chemotherapeutic drugs display inhibitory actions towards CYP24 the major degradation enzyme of vitamin metabolites (168). Some of these structures may elicit a similar response in neoplastic diseases or other hyper- proliferative disorders and may even produce less of the adverse calcemic response compared to calcitriol.

Figure 5.7 shows the extracted ion chromatograms for ligands of VDR detected during ultrafiltration UHPLC-MS screening of another 100-compound library. The corresponding structures of these hits are shown in figure 5.8, and the chemical names and common names of these compounds are as follows: 10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide (anticonvul- sant, carbamazepine), 1-(1H-indol-4-yloxy)-3-(isopropylamino)propan-2-ol (non-selective beta blocker, pindolol), N-(3-acetyl-4-(2-hydroxy-3-(isopropylamino)propoxy)phenyl)butyramide (cardio-selective beta blocker, acebutolol), alpha-methylornithine), 6-methyl-1,3,8-trihydroxyan- thraquinone (inhibitor of 11β-SHD1 enzyme, emodin), 8-chloro-11-(4-methylpiperazin-1-yl)-5H- dibenzo[b,e][1,4]diazepine (antipsychotic, clozapine), 5,7-dihydroxy-2-phenyl-4H-chromen- 4-one (flavones, chrysin), (1S)-(6-methoxyquinolin-4-yl)(8-vinylquinuclidin-2-yl)methanol (class I antiarrhythmic agent, quinidine), and (2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2-isoprope- nyl-8,9-dimethoxychromeno[3,4-b]furo(2,3-h)chromen-6-one (broad spectrum insecticide, rotenone). 100

Figure 5.7. Screening of a 100-compound library for ligand binding to VDR

(x10,000,000) IT-TOF; (+/-) ion ESI [M+H]+ 237.1051 Extracted ion chromatogram

4.00 [M+H]+ 327.1382

3.00 [M+H]+ 249.1627

[M+H]+ 325.1950 Isobaric interference [M+H]+ 337.2120 2.00 LC-MS response [M+H]+ 255.0694

[M+H]+ 395.1498

1.00 [M-H]- 269.0471

0.00 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1

Retention time (min) 101

Figure 5.8. Structures of compounds that displayed binding towards VDR during ultraﬁltra- tion UHPLC-MS screening shown in ﬁgure 5.7

H O N O

N H O N O H O H2N O O Acebutolol Carbamazepine Molecular mass: 336.42 Molecular mass: 236.27 H Rotenone O Molecular mass: 394.42 O

OH OH O OH O H O N

HO O

OH Pindolol Emodin Chrysin HN O Molecular mass: 248.3 Molecular mass: 270.2 Molecular mass: 254.2 OH O

N O

N OH

Cl N

N N N H Clozapine Quinidine Molecular mass: 326.8 Molecular mass: 324.4 102

Another structural motif that has been well characterized by our chemoprevention drug discovery program is the stilbene (modeled after resveratrol). A series of resveratrol analogs was synthesized as part of our natural products-based drug discovery program in an effort to obtain chemoprevention agents with enhanced selectivity and potency. This directed library of synthetic resveratrol analogs was screened for binding to the VDR as part of this dissertation, and many hits were obtained as shown in the ultrafiltration UHPLC-MS chromatogram for these assays in figure 5.9 and 5.10.

Structures for these resveratrol-related VDR ligands are shown in figure 5.11 and 5.12. The chemical names of these hits shown in figure 5.11 are as follows: (E)-4-styrylaniline, (E)-3-styrylaniline, (E)-4-styrylbenzamide, (E)-4-(2,3,5-trimethoxystyryl)aniline, N-(4-aminophenyl)benzamide, 4-amino-N-phenylbenzamide, (E)-4-(2,4-dimethoxystyryl)aniline, (E)-3-(3,5-dimethoxystyryl) aniline, (E)-4-(3,5-dimethoxystyryl)phenol, (E)-4-(3,5-dimethoxystyryl)aniline, (E)-4-(2,3,4-trimethoxystyryl)aniline, (E)-4-(3,5-dimethoxystyryl)-N-methylaniline, (E)-4-(3,4,5-trimethoxystyryl) aniline, and (E)-3-(2,4-dimethoxystyryl)aniline. The chemical names for the resveratrol analogs that are VDR ligands and shown in figure 5.12. (E)-4-(3,4-dimethoxystyryl)aniline, (E)-5-(4-nitrostyryl)benzene-1,3-diol, 4-(3,5-dimethoxyphenethyl)aniline, 2-(3,4-dimethoxyphenethyl)aniline, (Z)-1-(3-(3,5-dimethoxyphenyl)-2-(4-nitrophenyl)allyl)-1H-imidazole, (E)-3-(2-(benzo[d][1,3] dioxol-5-yl)vinyl)aniline, 4-(3,4,5-trimethoxyphenethyl)aniline, (Z)-4-(1-(3,5-dimethoxyphenyl)- 3-(1H-imidazol-1-yl)prop-1-en-2-yl)aniline, and (E)-3-(3,4,5-trimethoxystyryl)aniline.

Surprisingly, resveratrol did not display binding to the VDR as a mixture or as a single ligand screened against this receptor. It can be noted that the VDR is receptive to larger structures with hydrogen bond donor groups positioned in the right place. Many researchers have noted that slight changes to the secosteroid structure of calcitriol can markedly decrease the binding affinity case in point 3-epi-calcitriol (169). Therefore, based on these structures is it is hard to de- finitively define essential qualities of these stilbene analogs. 103

Figure 5.9. Ultraﬁltration UHPLC-MS screening of a directed library of reseveratrol analogs for ligands to the VDR. These computer reconstructed chromatograms show hits for approximately half of the directed library. See ﬁgure 5.10 for hits from the rest of the library

IT-TOF; (+/-) ion ESI (x10,000,000) [M+H]+ 286.1442 3.50 Extracted ion chromatogram [M+H]+ 256.1338

3.00 [M+H]+ 270.1499

2.50

2.00 [M+H]+ 224.1071 [M+H]+ 258.1514 [M+H]+ 196.1131

LC-MS response 1.50 [M+H]+ 213.1022

[M+H]+ 257.1207 [M+H]+ 196.1131 1.00

[M+H]+ 213.1022 0.50

0.00 0.50 1.00 1.50 2.00 2.50 3.00 Retention time (min) 104

Figure 5.10. Ultraﬁltration UHPLC-MS screening of a library of resveratrol analogs for ligands to the VDR. Part 2 of 2 (see ﬁgure 5.9 for part 1)

3.0 [M+H]+ 336.1745 [M+H}+ 366.1466 IT-TOF; (+/-) ion ESI Extracted ion chromatogram

[M+H]+ 256.1354 2.5

[M+H]+ 286.1461 2.0

[M+H]+ 258.1485

[M+H]+ 258.1485 1.5 [M+H]+ 256.2291 [M+H}+ 288.1611 LC-MS response 1.0

0.5

[M-H]- 256.0602 0.0 0.50 1.00 1.50 2.00 2.50 3.00 Retention time (min) 105

Figure 5.11. Chemical structures of resveratrol analogs that displayed binding towards VDR during ultraﬁltration UHPLC-MS screening in ﬁgure 5.9

NH NH 2 O 2

NH2 O NH2

O (E)-4-styrylaniline (E)-3-styrylaniline (E)-4-styrylbenzamide Molecular mass:195.26 Molecular mass:195.26 Molecular mass: 223.27 (E)-4-(2,3,5- NH2 NH2 trimethoxystyryl)aniline O O

N N H H

O O H2N N-(4-aminophenyl)benzamide 4-amino-N-phenylbenzamide (E)-4-(2,4-dimethoxystyryl)aniline Molecular mass: 212.25 Molecular mass: 212.25 Molecular mass: 255.31 NH OH 2

O O

H N (E)-4-(3,5-dimethoxystyryl)aniline Molecular mass: 255.31 O (E)-4-(3,5-dimethoxystyryl)phenol O O Molecular mass: 256.30

NH2

O (E)-4-(3,5-dimethoxystyryl)-N- NH2 O methylaniline

Molecular mass: 269.34 O O O (E)-4-(3,4,5- (E)-3-(2,4-dimethoxystyryl)aniline trimethoxystyryl)aniline Molecular mass: 255.31 O

NH2

O O (E)-4-(2,3,4-trimethoxystyryl)aniline (E)-3-(3,5-dimethoxystyryl)aniline O Molecular mass: 285.34 O Molecular mass: 255.31 106

Figure 5.12. Chemical structures of resveratrol analogs that showed binding to VDR (Part 2 of 2. See part 1 in ﬁgure 5.11)

NO2

NH2

NH2 (E)-5-(4-nitrostyryl)benzene-1,3-diol O Molecular mass: 257.24 (E)-4-(3,4-dimethoxystyryl)aniline OH O O Molecular mass: 255.31 O

NH2 (E)-3-(3,4,5-trimethoxystyryl)aniline Molecular mass: 285.34 O NH2

O NH2 2-(3,4-dimethoxyphenethyl)aniline O 4-(3,5-dimethoxyphenethyl)aniline Molecular mass: 257.33 O

O Molecular mass: 257.331

NH 2 O 4-(3,4,5-trimethoxyphenethyl)aniline O (E)-3-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)aniline Molecular mass: 287.35 O O Molecular mass: 239.27 O

O O

O2N N

H2N N (Z)-1-(3-(3,5-dimethoxyphenyl)-2-(4-nitrophenyl)allyl)- N 1H-imidazole (Z)-4-(1-(3,5-dimethoxyphenyl)-3-(1H-imidazol-1- Molecular mass: 365.38 yl)prop-1-en-2-yl)aniline Molecular mass: 335.16 107

Additional compounds, screened in 100-compound groups that were determined to bind to VDR included a series of flavonoid natural products. As shown in chapter 4 8PN displayed much more efficient binding to VDR than curcumin. Curcumin has been shown to bind to VDR as well as LCA, which correlates to low micromolar affinity (167). Therefore, structures of this class show great promise as potent VDR ligands. Ultrafiltration UHPLC-MS chromatograms for these ligands are shown in figure 5.13. Chemical structures for these compounds are shown in figure 5.14, and the chemical names of these compounds are as follows: 3-(1-(furan-2-yl)-3-oxobutyl)- 2-hydroxy-4H-chromen-4-one (fumarin), (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl) prop-2-en-1-one (butein), 5,7-dihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (biochanin A), (E)-4-(5-hydroxy-3,7-dimethylocta-2,6-dienyloxy)-7H-furo[3,2-g]chromen-7-one (MA4 121-6),

(S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)chroman-4-one (8PN), and (S)-5,7- dihydroxy-2-(4-hydroxyphenyl)-6-(3-methylbut-2-enyl)chroman-4-one (6PN).

The compounds that displayed affinity for VDR based on the ultrafiltration LC-MS screening were subsequently tested for binding to the ERp57 protein. This assay was designed to predict potential hypercalcemic responses for each hit in the VDR binding assay. A proof-of- principle ultrafiltration UHPLC-MS assay for ERp57 was carried out using the known ligand, calcitriol. As indicated in the ultrafiltration UHPLC-MS chromatograms shown in figure 5.15, the calcitriol peak eluting at 0.5 min was enhanced in the experiment containing active ERp57 relative to the negative control experiment containing denatured ERp57. Therefore, calcitriol showed specific binding to ERp57. Since the protonated molecule of calcitriol was not observed during positive ion APCI mass spectrometry (see mass spectrum shown as an inset to figure 5.15), the abundant fragment ions of m/z 381 and 399 were used to monitor the elution of calcitriol during ultrafiltration UHPLC-MS.

All of the compounds that demonstrated binding to VDR in the initial screening were assayed for binding to ERp57 using this new assay. None showed any affinity for ERp57, which suggests that none of the hits for the VDR assay would be expected to produce hypercalcemia,. 108

Figure 5.13. Ultraﬁltration UHPLC-MS Screening computer reconstructed chromatograms showing hits from a 100 compound library IT-TOF; (+/-) ion ESI (x10,000,000) Extracted ion chromatogram + 4.00 [M+H] 285.0781

[M-H]- 339.1230 3.50

3.00

[M+H]+ 299.0937 2.50 [M+H]+ 355.1479 [M+H]+ 273.0704 2.00 LC-MS response 1.50

1.00

[M-H]- 339.1244 0.50

0.00 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 Retention time (min) 109

Figure 5.14. Structures of compounds that displayed binding towards VDR as displayed in the chromatogram in ﬁgure 5.13

O OH HO O O O

OH O HO O O OH O Fumarin Biochanin A Butein Molecular mass: 298.2 Molecular mass: 284.3 Molecular mass: 372.25

OH OH HO O

HO O

OH O

O O O 8PN 6PN MA4 121-6 Retention time: 1.6 min Retention time: 1.9 min Molecular mass: 354.2 Molecular mass: 340.37 Molecular mass: 340.37 110

Figure 15.5. Proof-of-principle binding of calcitriol to ERp57 obtained using a new ultrafiltration UHPLC-MS assay. Chromatograms for m/z 399 and m/z 381, which correspond to the sequential losses of water form calcitrol are shown. The dotted lines represent chromatograms for the negative control (nonspecific binding). Specific binding of calcitriol to ERp57 is indicated by enhancement of the peakat 0.5 min in the experimental ultrafiltrates comparedwith the negative control

(x1,000,000) IT-TOF; (+) ion APCI Extracted ion chromatogram [MH-2H O]+ 381.3128 3.00 2 Positive ion APCI accurate mass spectrum showing the multiple losses of water from calcitriol. The protonated molecule of m/z ws not detected

(x1,000,000) 2.50 1.50

381.3134 1.00 2.00

363.3032 + 399.3251 [MH-H20] 399.3253 0.50

1.50

LC-MS response 0.00 310 330 350 370 390 410 430 450 470 m/z

1.00

0.50

0.00 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Retention time (min) 111

However, this prediction needs to be verified using secondary assays. Lack of binding of these compounds to ERp57 might be the result of improper binding modes, since many are cyclic structures locked into a defined position while calcitriol possess many freely rotating bonds. A complex assessment of the metabolic clearance of each compound in vivo might also be necessary, since many secosteroid analogs associated with diminished hypercalcemia show rapid clearance compared with calcitriol as the main determinate of their low calcemic response (73).

5.4 Conclusions

A high throughput assay for screening compound libraries for ligands to the VDR was applied that utilized a metabolomics-based post data acquisition platform. Binding partners for VDR were identified in a short period of time. As a result of ultrafiltration UHPLC-MS screening, a sub-set of a library of indenoisoquinolines, a sub-set of a library of resveratrol analogs, several natural product flavonoids, and several unrelated synthetic compounds were found to be ligands to the VDR. A long aliphatic extension such as a prenyl group was found to be a major factor for enhancing binding affinity as well as the presence of hydrogen bond donors.

An alternative assay that is routinely utilized to demonstrate binding of ligands to the VDR is based on the biolumenescene properties of luciferase systems. Specifically a direct mea- sure of promoter response in cells is measured by a chemical reaction generated through an oxidative process where D-luciferin is adenylated, and then molecular oxygen produces the oxidative product dioxetanone. Subsequent decarboxylation forms oxyluciferin, which tautomerizes between keto-enol states generating an emission of light during its return from an excited state back to ground. Luciferase can generate a variety of bioluminescene colors ranging between yellow-green to red (λmax 550-620). Dual reporter assay kits have become available for many cellular targets including the VDR. This dual acting system has been shown to improve experimental accuracy due to the simultaneous expression and measurement of two separate reporter enzymes within a single system. While one bioluminescence event (e.g. fire fly luciferase) provides the analytical measurement an internal standard such as (e.g. Renilla luciferse) normalizes for cellular 112 variability and transfection efficiencies (126, 167).

These bioluminescence-based assays provide an effect alternative to the described high throughput ultrafiltration screening approach for VDR ligands. However, no structural information is provided with the luciferase reporter assay, which dramatically reduces this assays effectiveness when screening complex chemical libraries or extracts. Therefore, the major advantage of ultrafiltration LC-MS over the dual report approach is the structural information provided by high resolution accurate mass measurements.

As a potential test of hypercalcemic activity, an ultrafiltration UHPLC-MS assay for binding to the protein receptor ERp57 was developed and validated using calcitriol. Since none of the ligands discovered using the VDR binding assay were also ligands for ERp57, this implies a diminished rapid response mechanism for these compounds in addition to a reduced calcemic effect. Potentially, these ligands would show beneficial effects resulting from genomic actions. However, additional secondary screens are necessary to verify that the hits lack undesirable hypercalcemic activity. CHAPTER 6

CONCLUSION AND FUTURE DIRECTIONS

Vitamin D, a pleiotropic acting member of the nuclear receptor superfamily has demonstrated the ability to affect many auxiliary biological sites beyond the classically described homeostasis of calcium and phosphorous. Apart for the genomic actions of vitamin D, a myriad of rapid response mechanisms have been elucidated including signal transduction pathways acting in an autocrine/paracrine manner. Drug discovery efforts have been increasingly promising for many diseases states through the structural modification of the secosteroid structure. However, toxicity of high doses of vitamin D and analogs has spurred an investigation into potent nonsecosteroid binding partners for the VDR with the promise of alleviating these side effects.

A rapid preclinical quantitative analysis of 6 chemopreventative candidates was performed in chapter 2, which has giving rise to promising new class of ligands that display binding towards vitamin D receptor as demonstrated in chapter 3. A high throughput approach to our ultrafiltration LC-MS assay was developed to process the large libraries of purified compounds and extracts, described in chapter 4. Then, once the high throughput assay was optimized the vitamin D receptor assay served as a model for the processing capabilities of this approach. Chapter 5 uses a variety of LC-MS and metabolomics software techniques, which when combined allows for a 100-fold increase in sample throughout. Within this chapter a variety of compounds displayed binding to the VDR. We have a special interest in the indenoisoquinoline class as a dual acting RXR and VDR binding partner, which will increase the potency within neoplastic and potentially other diseases. Also, the prenylated hops constituents have been shown to out compete curcumin a known VDR binding partner. Ultrafilatration was also utilized to screening against the proposed plasma membrane bound receptor ERp57 for binding to vitamin D metabolites. Although calcitriol displayed binding to this receptor no other compounds presented within this dissertation showed the same affinity toward this receptor. This is circumstantial evidence that

113 114 these compounds will elicit a diminished hypercalcemic effect, will have to be tested further inin vivo systems to confirm this response.

Continual refinement of the ultrafiltration platform including new plate and rotor systems, which could withstand faster ultracentrifugation speeds would allow for increases in throughput. A major limitation for the vitamin D ultracentrifugation assay was that the vitamin D receptor must be purchased. In order to realize the full potential of the high throughput screening approach with respect to the vitamin D receptor an in-house purification of this receptor should be accomplished. 115

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VITA

NAME Jerry J. White

EDUCATION PhD., Medicinal Chemistry, University of Illinois at Chacago, Chicago, Illinois

B.S., Biochemistry, University of California at California at Riverside (magna cum laude), Riverside California (2006)

RESEARCH Department of Medicinal Chemistry and Pharmacognosy, EXPERICENCE University of Illinois at Chicago, Chicago, IL: Research Assistant, 2007-Present

Research Resource Center (RRC), University of Illinois at Chicago, Chicago, IL: Research Assistant, 2009-2011

TEACHING Department of Medicinal Chemisty and Pharmacognosy, EXPERIENCE University of Illinois at Chicago, Illinois: Teaching Assistant, 2007-2008

AWARDS AND Oscar Robert Olberg Prize in Medicinal Chemistry, University HONORS of Illinois at Chicago, 2009

Rho Chi Honor Society

PROFESSIONAL American Society for Mass Spectrometry MEMBERSHIPS

PUBLICATIONS Park, E.-J.; Kondratyuk, T. P.; Morrell, A.; Kiselev, E.; Conda Sheridan, M.; Cushman, M.; Ahn, S.; Choi, Y.; White, J. J.; van Breemen, R. B.; Pezzuto, J. M., Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21 WAF1/CIP1 by Indenoisoquinolines in MCF7 Cells. Cancer Prevention Research 2011, 4 (4), 592-607.

Nikolić, D.; Gödecke, T.; Chen, S.-N.; White, J.; Lankin, D. C.; Pauli, G. F.; van Breemen, R. B., Mass spectrometric dereplication of nitrogen-containing constituents of black cohosh (Cimicifuga racemosa L.). Fitoterapia