A High Throughput Ultrafiltration LC-MS Platform for the Discovery of Vitamin D Receptor Ligands
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A High Throughput Ultrafiltration LC-MS Platform for the Discovery of Vitamin D Receptor Ligands BY 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 pa- tient when life looked bleak at times. JW 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 .