Screening, hit expansion and mechanistic studies for natural product analogs derived from traditional herbal medicine: curcumin and triptolide. Open Access

Brown, Andrew Patrick (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/4m90dv96c?locale=en
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Abstract

The use of naturally-derived compounds in traditional medicine dates back thousands of years. Many of these compounds have served as predecessors for modern drug therapies. Despite this fact, obstacles such as variation in preparation and inconsistencies in chemical makeup have prevented mainstream acceptance of herbal medicine. Advances in medicinal chemistry have begun to allow for the purification and characterization of active ingredients in a number of traditional herbal preparations. Armed with this information, it has become possible to synthesize compounds inspired by the active ingredients in these preparations that possess better drug properties, such as a lower toxicity, better pharmacokinetics or increased potency. The work discussed herein details the characterization of analogs of two such compounds; curcumin, from the root of the curcuma longa plant, and triptolide, from the Chinese herb Tripterygiumwilfordii Hook F (TWHF).

Table of Contents

Table of Contents

CHAPTER I: Introduction..................................................................................................1

1.1 1Traditional herbal medicine...................................................................................2

1.2 Identifying biologically active compounds in traditional medicine and natural products......3

1.3 Triptolide, a biologically active compound in Lei Gong Teng...........................................5

1.4 Curcumin, a biologically active compound from the tumeric spice...................................10

1.5 Curcumin analogs.........................................................................................................13

1.6 Initial screening of curcumin analogs..............................................................................17

1.7 NF-κB pathway...........................................................................................................17

1.8 NF-κB, kinases, and cancer.........................................................................................18

1.9 NF-κB and autoimmune disease...................................................................................19

1.10 Hypothesis.................................................................................................................19

CHAPTER II: Triptolide....................................................................................................20

2.1 Introduction.................................................................................................................21

2.2 Experimental Procedures.............................................................................................21

2.3 Results........................................................................................................................29

2.3.1The structure of the natural product triptolide inspires a series of analogs....................29

2.3.2 Screening of analogs for anti-NF-κB activity............................................................35

2.3.3 Further testing in cell based assays...........................................................................38

2.3.4 Inhibition of NF-κB target genes..............................................................................43

2.3.5 Pharmacokinetics....................................................................................................43

2.3.6 Toxicity...................................................................................................................46

2.3.7 In vivo studies........................................................................................................51

2.3.8 Dextran sulfate sodium (DSS) induced experimental colitis.......................................54

2.3.9 Triptolide mechanism of action................................................................................59

Chapter III: Curcumin.....................................................................................................67

3.1 Introduction...............................................................................................................68

3.2 Experimental Procedures...........................................................................................69

3.3 Results......................................................................................................................77

3.3.1 Curcumin analog's inhibitory effects on various kinases...........................................77

3.3.2 EF31 (2.4) is a pleiotrophic kinase inhibitor............................................................82

3.3.3 Rationale for analogs selected in focused screen.....................................................92

3.3.4 Pleiotrophic kinase inhibition..................................................................................92

3.3.5 RAF1-MEK1-ERK2 kinase cascade....................................................................93

3.3.6 MAPK14 (p38α)-MAPKAPK2 kinase cascade..................................................96

3.3.7 Pan kinase inhibition and interrelated signaling pathways.........................................96

3.3.8 Enzyme kinetics of curcumin analog inhibition.........................................................99

3.3.9 Mixed model inhibition..........................................................................................102

3.3.10 Possible mixed model inhibition mechanism of action............................................102

3.3.11 Rationalizing the IC50 data by molecular modeling................................................103

3.3.12 Additional notes on kinase inhibition mechanisms.................................................112

3.3.13 In vivo studies, inflammation and anti-tumor activity.............................................122

3.4 Discussion...............................................................................................................128

Chapter IV: Conclusions and Future Studies..................................................................129

4.1 Triptolide................................................................................................................130

4.2 Curcumin................................................................................................................131

References...................................................................................................................134

List of Figures

Figure I-1. Lei gong teng is the source of natural compound triptolide..........................................................................7

Figure I-2. Curcuma longa is the source of the spice tumeric and the biologically active compound curcumin................11

Figure I-3. Structurally similar series of compounds synthesized, based on the structure of curcumin............................15

Figure II-1. Chemical synthesis of triptolide analogs...................................................................................................30

Figure II-2. Structures of triptolide analogs................................................................................................................32

Figure II-3. Triptolide analogs exhibit better potency the triptolide itself......................................................................36

Figure II-4. Inhibition of COX-2 mRNA by triptolide analogs...................................................................................39

Figure II-5. Inhibition of TNF-a and IFN-a mRNA by triptolide analogs.................................................................41

Figure II-6. In vivo plasma stability study in mice.....................................................................................................44

Figure II-7. Toxicity toward MDA-MB-231 cells....................................................................................................47

Figure II-8. Five day toxicology study in mice..........................................................................................................49

Figure II-9. Carrageenan paw edema model............................................................................................................51

Figure II-10. Mouse liver RNase protection assay...................................................................................................53

Figure II-11. Histological evaluation of colonic tissue...............................................................................................56

Figure II-12. Intestinal tissue mRNA levels..............................................................................................................58

Figure II-13. IKKβ kinase inhibition........................................................................................................................61

Figure II-14. NF-κβ Translocation.........................................................................................................................63

Figure II-15. Protein DNA binding specificity.........................................................................................................65

Figure III-1. Z' Lyte In vitro kinase assay (Invitrogen)...........................................................................................69

Figure III-2. Z' Lyte kinase cascade assay.............................................................................................................71

Figure III-3. Curcumin analogs..............................................................................................................................78

Figure III-4. Preliminary kinase screen...................................................................................................................80

Figure III-5. Screening of a 50-kinase panel by curcumin analog EF31 (2.4)..........................................................84

Figure III-6. IC50 curves: selection and testing of kinases and analogs.....................................................................87

Figure III-7. Blockade of Signal Transduction Pathways.........................................................................................94

Figure III-8. Inhibition of multiple signal transduction pathways................................................................................97

Figure III-9. Enzyme kinetics of AKT-2 kinase inhibition........................................................................................100

Figure III-10. Homology of AKT-1 and AKT-2....................................................................................................104

Figure III-11. Proposed analog binding in ATP binding pocket of AKT-2...............................................................106

Figure III-12. IC50 data rationalized by conformational changes due to protonation of compound UBS109 (2.12)...108

Figure III-13. Hypothesis for mixed model inhibition...............................................................................................110

Figure III-14. AM series curcumin analogs.............................................................................................................113

Figure III-15. IKKβ inhibition by AM series analogs..............................................................................................116

Figure III-16. Inhibition of NF-κB Translocation by curcumin analogs....................................................................118

Figure III-17. Cell toxicity studies..........................................................................................................................120

Figure III-18. Paw edema model.......................................................................................................................... 122

Figure III-19. MDA-MB-231 xenograft tumor model............................................................................................124

Figure III-20. In vivo radiosensitization by EF31 (2.4) in a subcutaneous xenograft tumor model............................126

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