CITY UNIVERSITY OF HONG KONG

Syntheses and Studies of Multifunctional Inhibitors

for Enzymes in Mevalonate Pathway

Submitted to

Department of Biology and Chemistry

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

by

Gao Jinbo

July 2009

Syntheses and Studies of Multifunctional Inhibitors

for Enzymes in Mevalonate Pathway

Abstract

The mevalonate pathway is an important biosynthetic pathway, which plays a key

role in multiple cellular processes for synthesizing cholesterol, prenylated proteins,

isopentenyl tRNA, dolichol, hormones, ubiquinone, heme A, and N-glycosylation.

These end-products are vital for diverse cellular processes including cell growth and

differentiation, signal transduction pathways, and mitochondrial electron transport.

Manipulation of this pathway results in alteration of malignant cell growth and survival

in cell culture and animal models, with promising potential interesting therapeutic

targets for many other areas of ongoing research, including oncology, autoimmune

disorders, atherosclerosis, Alzheimer disease, and cardiovascular disease.

The mevalonate pathway has been exploited in the design of cholesterol-lowering

drugs by inhibiting the enzyme HMG-CoA reductase with statin drugs as a means of

reducing the risk of cardiovascular disease. Statin is not satisfactory in treatment of

cardiovascular disease due to significant side effects, such as headaches, nausea, fever,

extreme muscle pain, serious liver problems, and even deaths. Therefore, it is important

to design and develop other cholesterol-lowering inhibitors targeting other enzymes

instead of HMG-CoA reductase for the inhibition of cholesterol biosynthesis. Several

enzymes in mevalonate pathway, such as mevalonate kinase, phosphomevalonate kinase,

mevalonate 5-diphosphate decarboxylase, farnesyl pyrophosphate synthase, and

squalene synthase, have also been suggested as important regulatory enzymes in the

biosynthesis of cholesterol and cell growth and differentiation.

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The objective of the present study is to design, synthesize, and study multifunctional

inhibitors for simultaneous inactivation of more than one enzyme in the mevalonate

pathway, which may block effectively the formation of cholesterol and downstream

products and regulate cell growth and differentiation for the treatment of heart disease

and cancer. In the present study, various types of potential multifunctional inhibitors

based on bisphosphonates were designed and synthesized to inhibit mevalonate kinase,

phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase, and farnesyl

pyrophosphate synthase.

The results showed that these compounds exhibited potent inhibition on the four

enzymes simultaneously and the IC50 values are at several nanomole to micromole

range. One bisphosphonate compound with geranyl and mevalonate group exhibits

potent inhibition for rat mevalonate kinase, phosphomevalonate kinase, mevalonate 5-

diphosphate decarboxylase, and farnesyl pyrophosphate synthase with IC50 values of

2.7 M, 4.2 M, 0.8 M, and 0.029 M, respectively. This inhibitor also strongly

inactivate the corresponding enzymes from S. pneumoniae with low IC50 values of 0.9

M, 6.5M, 1.1 M, and 0.015 M, respectively. The results show that the

bisphosphonate moiety and the mevalonate analogs all make important contribution for

the inhibition of the four enzymes. It is usually difficult to inhibit efficiently one enzyme

completely with a single inhibitory drug, which potentially result in toxic side effects.

The inhibition of more than one enzyme in a metabolic pathway using a combined drug

treatment might be more effective than inhibition of only one individual enzyme.

To predict the structure and orientation of the multifunctional inhibitors in the binding

cavity of these four enzymes, the binding interactions between small molecule inhibitor

and enzyme were also investigated by using the computer molecular docking simulation.

Molecular docking analysis illustrated that these compounds bind to the active site of

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the enzyme in a favorable position via hydrogen bonds, metal ions, hydrophobic and

van der Waals interactions with the amino acid residues. These results indicated that the

active compounds are likely to be competitive inhibitors. Characterization of enzyme

kinetics is in good agreement with the predicted binding mode based on simulation

study.

Meanwhile, a convenient colorimetric high throughput assay method was developed

for the measurement of FPPS activity and inhibitor screening instead of traditional

radioactive assay that is inconvenient, expensive, time-consuming, and inaccurate due

to product inhibition. This novel sensitive high-throughput screening assay is based on

the amount of free phosphate generated in a reaction. The resulting Pi reacted with

molybdate to form a colorless Keggin type phosphomolybdate (PMo12MoO403)

complex, which was then reduced to give a phosphomolybdate-blue (PMo12MoO407)

complex with absorbance at 830 nm. The sensitivity of this assay method was found to

be much higher than that of the traditional radioactive assay method. This FPPS assay

method can be used in high-throughput screening procedures for rapid screening of

large numbers of compounds under similar test conditions.

The bisphosphonates can induce cancer cell apoptosis by inhibiting enzymes in

mevalonate pathway, preventing the generation of isoprenoid moieties and thereby

impairing the isoprenylation of small GTP proteins. So the bisphosphonate compounds

were also evaluated against cancer cell lines using MTT assay methods with high-

throughput screening. Some multifunctional inhibitors also inhibited effectively cancer

cell growth and induced apoptosis. One bisphosphonate compound with geranyl and

mevalonate group showed a significant anti-proliferative activity on Hela cells with IC50

of about 20 M. In addition, Hela cancer cells treated with our fluorescent inhibitors

were examined by confocal fluorescence microscopy. Many fluorescent particles appear

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in both cytoplasm and nucleus. The particle density is higher in the cytoplasm than that

in the nucleus, indicating that the fluorescent inhibitors can get into cell although they

are highly charged compounds.

In conclusion, a series of multifunctional inhibitors based on bisphosphonate analogs

were designed and synthesized, which show potent inhibition for mevalonate kinase,

phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase, and farnesyl

pyrophosphate synthase from both rat and S. pneumoniae. An efficient colorimetric

high-throughput enzyme assay method for FPPS was successfully established, which

provided a convenient screening method for inhibitors of FPPS. The confirmation of

multi-targeted inhibition and establishment of SAR analysis with concurrent aid of

computational simulation helped to unravel the identified multifunctional compounds as

a new class of multifunctional enzyme inhibitors. Cell survival assay with new

bisphosphonate analogs demonstrated their potential usage for cancer treatment.

Therefore, inactivation of these enzymes involved in mevalonate pathway provides an

alternative potentially useful method for treating cardiovascular disease, cancer, and

bacteria infection.

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LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

AD Alzheimers disease

ADP Adenosine diphosphate

APP Amyloid precursor protein

bHLH Basic helix-loop-helix

ApoB-100 Apolipoprotein B-100

CVD Cardiovascular disease

DMAPP Dimethylallyl diphosphate

eNOS endothelial nitric oxide synthase

FPP Farnesyl pyrophosphate

FPPS Farnesyl pyrophosphate synthase

FTase Farnesyl transferase

FTIs Farnesyl transferase inhibitors

GPP Geranyl pyrophosphate

GGPP Geranylgeranyl pyrophosphate

GGTases Geranylgeranyl transferase

GGTIs Geranyl geranyltransferase inhibitors

HDL High-density lipoprotein

HIV-1 Human immunodeficiency virus 1

HMG-COA 3-Hydroxy-3-methylglutaryl-coenzyme

IPP Isopentenyl diphosphate

IPTG Isopropyl-beta-D-thiogalactopyranoside

KM Michaelis-Menten Constant

LBMD Low bone mineral density

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LDL Low-density lipoprotein

MDD Mevalonate 5-diphosphate decarboxylase

MVA Mevalonate, or mevalonic acid

MVAP Mevalonate 5-phosphate

MVAPP Mevalonate 5-diphosphate

MVK Mevalonate kinase

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazolium bromide

NADH (reduced) Nicotinamide adenine dinucleotide

N-BPs Nitrogen-containing bisphosphonates

NMR Nuclear magnetic resonance

PCR Polymerase chain reaction

PDB Protein data bank (http://www.rcsb.org/pdb/)

PKB Protein kinase B

PMK Phosphomevalonate kinase

PSQPP Presqualene diphosphate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

SMCs Smooth muscle cells

SREBPs Sterol regulatory element binding proteins

Stp Streptococcus pneumoniae

THF Tetrahydrofuran

THP Tetrahydropyranoxy group

TG Triglyceride

TLC Thin layer chromatography

VLDL Very low density lipoproteins

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LIST OF FIGURES, SCHEMES AND TABLES

Figure 1.1 The mevalonate pathway and the targets of inhibitors 2

Figure 1.2 Structures of end products of the mevalonate pathway 3

Figure 1.3 The mevalonate pathway: diseases and drugs 7

Figure 1.4 Detail Pathway of cholesterol biosynthesis 12

Figure 1.5 Cholesterol products 14

Figure 1.6 The structure and classification of the lipoproteins particles 16

Figure 1.7 Endogenously, exogenous and the transport pathways of cholesterol 17

Figure 1.8 Sterol regulatory element binding protein regulation of cholesterol metabolism 21

Figure 1.9 Diagram summarizing the membrane organization of lipid rafts and caveolae 24

Figure 1.10 Schematic of the life history of an atheroma 33

Figure 1.11 Stages in the development of atherosclerotic plaques 35

Figure1.12 Aggregation of the amyloid -peptide and subsequent plaque formation in Alzheimers disease 38

Figure 1.13 The relationship of Cav-1 and caveolae to tumour initiation and progression 40

Figure 1.14 Role of lipid rafts in fusion of human immunodeficiency virus 1to CD4+ T cells. 42

Figure 1.15 An overview of CAAX-protein processing 46

Figure 1.16 Distribution of drug targets 48

Figure 1.17 The reaction catalyzed by HMG-CoA reductase in mevalonate pathway 50

Figure 1.18 Statin inhibitors and the HMG-CoA reductase substrate 51

Figure 1.19 Ribbon diagram of the P. mevalonii HMG-CoA reductase 52

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Figure 1.20 Statin interactions mimic substrate binding in Pseudomonas mevalonii HMG-CoA reductase 53

Figure 1.21 Ribbon diagrams of human HMG-CoA reductase 55

Figure 1.22 Statins exploit the conformational flexibility of human HMG-CoA reductase 56

Figure 1.23 The pleiotropic effects of the statins 58

Figure 1.24 The reaction catalyzed by MVK 60

Figure 1.25 The inhibitors of MVK 61

Figure 1.26 The structures of Leishmania major, rat, and human MVK 62

Figure 1.27 Stereodiagram of the vicinity of the ligand binding site in rat MVK 64

Figure 1.28 A schematic of the proposed catalytic mechanism of mevalonate kinase 65

Figure 1.29 The reaction catalyzed by PMK 66

Figure 1.30 RIBBONS drawing of S. pneumoniae phosphomevalonate kinase 68

Figure 1.31 Stereo ribbon representation of human PMK 69

Figure 1.32 The inhibitors of PMK 70

Figure 1.33 The reaction catalyzed by MDD 71

Figure 1.34 Ribbon drawings of yeast MDD 72

Figure 1.35 The architecture of TbMDD and SaMDD 73

Figure 1.36 A mechanism for TbMDD and the chemical structures of compounds 74

Figure 1.37 Human MDD structure and models of substrate complexes 75

Figure 1.38 The inhibitors of MDD 77

Figure 1.42 The reaction catalyzed by FPPS in mevalonate pathway 80

Figure 1.40 The inhibitors of FPPS 81

Figure 1.41 X-ray crystallographic results for minodronate/FPPS 83

Figure 1.42 Structure of human farnesyl pyrophosphate synthase 85

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Figure 1.43 Illustration for the high in vivo efficacy of FPPS inhibition by N-BPs 86

Figure 1.44 Stereo illustration of the FPPS carbocation-binding site 87

Figure 1.45 The objective of our research 94

Figure 1.46 The planned synthetic multifunctional enzyme inhibitors 95

Figure 1.47 Study strategies and methods of the multifunctional enzyme inhibitors 96

Figure 2.1 SDS-PAGE analyses of rat and StpMVK, PMK, MDD, FPPS 115

Figure 2.2 A spectrophotometric assay method for monitoring the MVK, PMK, and MDD activity 119

Figure 2.3 Strategies for assaying FPPS activity through determination of PPi or Pi concentration 121

Figure 2.4 Absorbance spectra of the complexes 122

Figure 2.5 UV-Vis absorbance spectra and calibration curves 124

Figure 2.6 Absorbance spectra of Pi-molybdate complex formed in the presence of IPP 125

Figure 2.7 Absorbance spectra of Pi-molybdate complex formed in the presence of DMAPP 126

Figure 2.8 Colorimetric high throughput screening assay method for FPPS inhibitors 127

Figure 2.9 UV-Vis absorbance spectra of different concentration of inhibitors for FPPS 129

Figure 2.10 The color change of the assay system 129

Figure 2.11 MTT assay method for cell viability and proliferation 136

Figure 2.12 Competitive inhibition of compound 83 against Stp MVK 143

Figure 2.13 Compound 83 is uncompetitive with MVA against Stp MVK 144

Figure 2.14 Inhibition of Stp PMK by compound 55 145

Figure 2.15 Compound 55 is competitive with ATP against Stp PMK 146

Figure 2.16 Competitive inhibition of compound 55 against Stp PMK 147

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Figure 2.17 Inhibition of Stp MDD by compound 55 148

Figure 2.18 Competitive inhibition of compound 55 against Stp MDD 149

Figure 2.19 Competitive inhibition of compound 55 against Stp MDD 150

Figure 2.20 Protein structure of angiotensin converting StpMVK and PMK 151

Figure 2.21 The binding modes of the compounds to StpMVK and PMK predicted 153

Figure 2.22 Scatter plots of experimental IC50 versus predicted docking energies for StpMVK 156

Figure 2.23 Scatter Plots of experimental IC50 versus predicted docking energies for StpPMK 156

Figure 2.24 Docking of compounds 16 and 43 into the StpMVK 159

Figure 2.25 Docking of compounds 55 and 56 into the StpMVK 161

Figure 2.26 Docking of compounds 64 and 65 into the StpMVK 162

Figure 2.27 Docking of compounds 83 and 84 into the StpMVK 163

Figure 2.28 Docking of compounds 83 into the StpPMK with the best docked conformation 164

Figure 2.29 Various interactions of compound 83 with StpMVK 166

Figure 2.30 Various interactions of compound 84 with StpMVK 167

Figure 2.31 Various interactions of compound 83 with StpPMK 168

Figure 2.32 Various interactions of compound 84 with StpPMK 169

Figure 2.33 The alignment structures of the compounds with compound 83 as the template 171

Figure 2.34 The features of the compounds superimposed on the structure of an active compound 83 173

Figure 2.35 Compound 44 is competitive with ATP against rat MVK 178

Figure 2.36 Compound 44 is uncompetitive with MVA against rat MVK 179

Figure 2.37 Compound 84 is competitive with ATP against rat MVK. 180

Figure 2.38 Compound 84 is uncompetitive with MVA against rat MVK 181

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Figure 2.39 Compound 84 is competitive with ATP against rat PMK 182

Figure 2.40 Competitive inhibition of compound 84 against rat MDD 183

Figure 2.41 Compound 84 is competitive inhibition of rat MDD with ATP. 184

Figure 2.42 Competitive inhibition of compound 84 against rat MDD with MVAPP 185

Figure 2.43 Compound 55 is competitive inhibition of rat MDD with ATP. 186

Figure 2.44 Competitive inhibition of compound 55 against rat MDD 187

Figure 2.45 The binding modes of the compounds 83 and 84 to rat MVK, PMK, MDD, and FPPS 194

Figure 2.46 The binding modes of the compounds 64 and 65 to rat MVK, PMK, MDD, and FPPS 195

Figure 2.47 Various interactions of compound 83 with rat MVK 197

Figure 2.47 Various interactions of compound 84 with rat MVK 198

Figure 2.49 Various interactions of compound 83 with human PMK 199

Figure 2.50 Various interactions of compound 84 with human PMK 200

Figure 2.51 Various interactions of compound 83 with human MDD 201

Figure 2.52 Various interactions of compound 84 with human MDD 202

Figure 2.53 Various interactions of compound 83 with human FPPS 203

Figure 2.54 Various interactions of compound 84 with human FPPS 204

Figure 2.55 The binding modes of the bisphosphonate to rat MVK, PMK, MDD, and FPPS 207

Figure 2.56 Scatter plots of experimental IC50 versus MVD predicted binding energies of rat MVK 210

Figure 2.57 Scatter plots of experimental IC50 versus MVD predicted binding energies of rat PMK 210

Figure 2.58 Scatter plots of experimental IC50 versus MVD predicted binding energies of rat MDD 211

Figure 2.59 Scatter plots of experimental IC50 versus MVD predicted binding energies of rat FPPS 211

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Figure 2.60 Stereoview of aligned compounds with compound 83 as the template 213

Figure 2.61 The features of the compounds superimposed on the structure of an active compound 84. 215

Figure 2.62 Bisphosphonate analogues 83 and 84 inhibit the growth of Hela cancer cells 221

Figure 2.63 Bisphosphonate analogues inhibit the growth of Hela cancer cells 222

Figure 2.64 Effects of compound 84 on cell viability of Hela cancer cell line 223

Figure 2.65 Uptake of bisphosphonate analogue 83 by human Hela cells 225

Figure 2.66 Uptake of bisphosphonate analogue 84 by human Hela cells 226

Figure 2.67 Uptake of bisphosphonate analogue 84 by human Hela cells 227

Scheme 2.1 Structure of type I with P-C(R1)R2-P and type II with R1-P-C(H)R2-P 98

Scheme 2.2 Synthetic process and structures of the type I inhibitors 100

Scheme 2.3 Synthetic process and structures of the type II inhibitors 103

Scheme 2.4 Synthetic pathway for fluorinated phosphomevalonate analogues 104

Scheme 2.5 Synthetic pathway for blue fluorescent pyrophosphate analogue 106

Scheme 2.6 Synthetic pathway for blue fluorescent multifunctional enzyme inhibitors 108

Scheme 2.7 Synthesis of coumarin type red fluorescent bisphosphonate inhibitor 110

Scheme 2.8 Syntheses of substrates for MVK, PMK, MDD and FPPS 112

Scheme 2.9 Rat and S. pneumoniae gene expression and purification process of MVK, PMK, MDD and FPPS 114

Scheme 2.10 The binding orientation of small molecule drug to the protein targets 132

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Table 1.1 Cholesterol and sphingolipid binding proteins and their putative functions 25

Table 1.2 Diseases for which rafts and raft proteins are targets 28

Table 1.3 Genetic and environmental factors associated with atherosclerosis and coronary heart disease 30

Table 1.4 Enzyme inhibitors applied clinically in mevalonate pathway 49

Table 2.1 Kinetic date for inhibition of FPPS 130

Experimental results for bisphosphonate inhibition of Stp MVK, PMK, MDD, and FPPS 141 Table 2.2

Predicted binding energies and hydrogen bonds results for bisphosphonate against Stp MVK and PMK 154 Table 2.3

Similarity scores and the contributions of steric, H-bond donor and acceptor, negative charge, and ring of these compounds

172 Table 2.4

Experimental results for bisphosphonate inhibition of rat MVK, PMK, MDD, and FPPS 176 Table 2.5

Predicted binding energies results for bisphosphonate against rat MVK, PMK, MDD, and FPPS 208 Table 2.6

Similarity scores and the contributions of steric, H-bond donor and acceptor, negative charge, and ring of these compounds compared with compound 84

214 Table 2.7

The IC50 values for all bisphosphonates against human Hela cell line. 219 Table 2.8

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TABLE OF CONTENTS

Declaration i

Abstract ii

Thesis acceptance form vi

Acknowledgement vii

List of abbreviations ix

List of figures, schemes and tables xi

List of publications xviii

Table of contents xix

Chapter 1 Introduction 1

1.1 The mevalonate pathway 1

1.2 Therapeutic target in mevalonate pathway 5

1.3 Cholesterol, lipid rafts, and diseases 9

1.3.1 Sources of cellular cholesterol 9

1.3.1.1 The cholesterol biosynthetic pathway 10

1.3.1.2 Dietary cholesterol: The LDL receptor pathway 14

1.3.2 Regulation of cholesterol biosynthesis 18

1.3.3 Lipid rafts and caveolae 22

1.3.4 Cholesterol and lipid rafts related diseases 26

1.3.4.1 Atherosclerosis and cardiovascular disease 28

1.3.4.2 Alzheimer disease 37

1.3.4.3 Cancer 39

1.3.4.4 Viral infection 41

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1.4. Protein prenylation and cancer 45

1.5 Enzymes and inhibitors in mevalonate pathway. 48

1.5.1 Inhibition of HMG-CoA reductase: Statins 50

1.5.2 Mevalonate kinase and inhibitors 59

1.5.3 Phosphomevalonate kinase and inhibitors 66

1.5.4 Mevalonate pyrophosphate decarboxylase and inhibitors 71

1.5.5 Inhibition of farnesyl pyrophosphate synthase: Bisphosphonates 78

1.6 Summary and prospects of the mevalonate pathway 89

1.7 Objective of study 93

Chapter 2 Results and Discussion 97

2.1 Syntheses of multifunctional enzyme inhibitors 97

2.1.1 Syntheses and characterization of type I inhibitors 99

2.1.2 Syntheses and characterization of type II inhibitors 101

2.1.3 Syntheses of fluorinated phosphomevalonate analogues 104

2.1.4 Syntheses of fluorescent multifunctional enzyme inhibitors 105

2.1.4.1 Syntheses of blue fluorescent bisphosphonate inhibitors 105

2.1.4.2 Syntheses of red fluorescent bisphosphonate inhibitor 109

2.2 Syntheses of substrates for MVK, PMK, MDD, and FPPS 111

2.3 Expression and purification of MVK, PMK, MDD and FPPS 113

2.4 Assay methods of MVK, PMK, MDD, and FPPS 116

2.4.1 Assay method of ATP-dependent enzymes MVK, PMK, and MDD 118

2.4.2 Development of Colorimetric FPPS Assay Method 119

2.5 Computational molecular docking 132

2.6 Cell Viability Assay 135

2.7 Interaction study with multifunctional bisphosphonate inhibitors-Stp 137

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2.8 Interaction study with multifunctional bisphosphonate inhibitors-Rat 175

2.8.1 Bisphosphonates against rat MVK, PMK, MDD, and FPPS 175

2.8.2 3D-QSAR investigations 191

2.9 Study of human Hela cell with multifunctional inhibitors 217

2.10. Conclusions 229

Chapter 3 Materials and Methods 230

General Procedures. 230

3.1 Organic syntheses of multifunctional inhibitors 231

3.1.1 Syntheses of type I inhibitors with P-C(R1)R2-P. 231

3.1.2 Syntheses of type II inhibitors with P-C(R1)R2-P. 236

3.1.3 Syntheses of fluorinated phosphomevalonate analogues 246

3.1.4 Syntheses of blue fluorescent multifunctional enzyme inhibitors 249

3.1.5 Syntheses of red fluorescent multifunctional enzyme inhibitors 255

3.2 Substrates syntheses of MVK, PMK, MDD, and FPPS 258

3.3 Expression and purification of MVK, PMK, MDD and FPPS 263

3.3.1 rat and S. pneumonia mevalonate kinase 263

3.3.2 rat and S. pneumonia phosphomevalonate kinase 264

3.3.3 rat and S. pneumonia mevalonate 5-pyrophosphate decarboxylase 264

3.3.4 rat and S. pneumonia farnesyl pyrophosphate synthase 265

3.4. Enzymatic studies 265

3.4.1. Determination of IC50 of mevalonate kinase 265

3.4.2. Kinetic constant (Ki) of MVK with respect to ATP 265

3.4.3. Kinetic constant (Ki) of MVK with respect to MVA 266

3.4.4. Determination of IC50 of phosphomevalonate kinase 266

3.4.5. Kinetic constant (Ki) of PMK with respect to ATP 267

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3.4.6. Kinetic constant (Ki) of PMK with respect to MVAP 267

3.4.7. Determination of IC50 of MDD 268

3.4.8. Kinetic constant (Ki) of MDD with respect to ATP 268

3.4.9. Kinetic constant (Ki) of MDD with respect to MVAPP 269

3.4.10. Determination of IC50 of farnesyl pyrophosphate synthase 269

3.5 Molecular Docking 270

3.6 Hela Cell Culture 270

3.7 Cell Growth Inhibition Assays 270

References 272