Molecular Biology in Medicinal Chemistry

Lipases and Phospholipases in Drug Development......Page 4
Contents......Page 8
Preface......Page 16
List of Contributors......Page 18
1 Purification of Lipase......Page 22
1.2 Pre-purification Steps......Page 23
1.3 Chromatographic Steps......Page 24
1.4 Unique Purification Strategies......Page 28
1.5.1.1 Mobile Phase......Page 30
1.5.1.3 Boundary Conditions......Page 31
1.5.2 Solution......Page 32
1.5.3 Method of Moments......Page 34
1.5.4 Model Evaluation......Page 36
1.5.5.1 Effect of Feed Angle......Page 37
1.5.5.3 Effect of Rotation Rate......Page 38
1.6 Conclusions......Page 40
1.8 References......Page 41
2.1 Introduction......Page 44
2.2.2 Biochemical Characterization and Tissue Distribution......Page 48
2.2.4 Substrate Specificity......Page 50
2.2.5 Possible Functions......Page 51
2.3.1 Historical Aspects......Page 53
2.3.2 Characterization and Distribution......Page 54
2.3.4 Function......Page 55
2.4.1 Historical Aspects......Page 56
2.4.3 Substrate Specificity......Page 57
2.5.2 Characterization and Distribution......Page 58
2.6 References......Page 59
3.1 Introduction......Page 62
3.3 Lateral Microstructure of Lipid Bilayers and its Influence on sPLA(2)......Page 64
3.4.1 Liposomes Protected by Polymer Coating......Page 67
3.4.2 Biophysical Model Drug-delivery System to Study sPLA(2) Activity......Page 68
3.4.3 Effect of Lipid Composition on sPLA(2)-triggered Drug Release and Absorption......Page 69
3.4.4 Effect of Temperature on Liposomal Drug Release and Absorption by sPLA(2)......Page 70
3.4.5 Liposomal Drug Release as a Function of sPLA(2) Concentration......Page 71
3.6 Acknowledgments......Page 72
3.7 References......Page 73
4.1 Introduction......Page 76
4.2 Structure and Catalytic Mechanism of Mammalian Phospholipase D......Page 77
4.3 Cellular Locations of PLD1 and PLD2......Page 79
4.4 Post-translational Modification of PLD......Page 80
4.5.1 Role of PIP(2)......Page 81
4.5.2 Role of PKC......Page 82
4.6 Role of Rho Family GTPases......Page 85
4.7 Role of Arf Family GTPases......Page 86
4.10 Cellular Functions of PLD......Page 87
4.11 Role of PLD in Growth and Differentiation......Page 88
4.13 Role of PLD in Exocytosis and Endocytosis......Page 89
4.14 Role of PLD in Superoxide Formation......Page 90
4.15 Role in Actin Cytoskeleton Rearrangements......Page 91
4.17 Role of PA in Other Cellular Systems......Page 92
4.18 References......Page 93
5.1 Introduction and Scope......Page 100
5.2.1.1 Acid Sphingomyelinase (aSMase)......Page 101
5.2.1.3 Neutral, Mg(2+)-dependent Sphingomyelinases (nSMase)......Page 102
5.2.1.4 Mg(2+)-independent Neutral Sphingomyelinases......Page 105
5.2.2.2 Binding of Substrate......Page 106
5.2.2.3 Mechanism of Catalysis......Page 107
5.2.3 Sphingomyelinase Assay......Page 109
5.3 Sphingomyelinase–Membrane Interactions......Page 110
5.3.1 Lipid Effects on Sphingomyelinase Activity......Page 111
5.3.2.1 Effects on Membrane Lateral Organization......Page 112
5.3.2.2 Effects on Membrane Permeability......Page 114
5.3.2.3 Effects on Membrane Aggregation and Fusion......Page 115
5.5 References......Page 117
6.1 Introduction......Page 122
6.2 GPI Structure and Hydrolysis by Specific Phospholipases......Page 123
6.3 Diffusible Factors and the Regulation of GPI Levels......Page 125
6.4 IPG Structure and Biological Activities......Page 127
6.5 GPI/IPG Pathway and the Intracellular Signaling Circuit......Page 130
6.6 Acknowledgments......Page 133
6.7 References......Page 134
7.1.1 Lipases in Metabolism......Page 142
7.2 Lipases Show Unique Differences in Comparison to Other Drug Targets......Page 143
7.3 Lipase Assays......Page 144
7.4.1 Biological Role of HSL......Page 146
7.4.2 Characteristics of HSL......Page 147
7.4.3 Inhibitors of HSL......Page 149
7.6 References......Page 155
8.1 Introduction......Page 160
8.2 Structure of Endothelial Lipase......Page 161
8.3 Tissue Expression of Endothelial Lipase and Its Implications......Page 162
8.4 Enzymatic Activity and Effects on Cellular Lipid Metabolism of Endothelial Lipase......Page 163
8.5 Regulation of Endothelial Lipase Expression......Page 166
8.6 Physiology of Endothelial Lipase......Page 167
8.7 Variation in the Human Endothelial Lipase Gene......Page 170
8.9 References......Page 172
9.1 Introduction......Page 176
9.1.1 3-D Structure of Human Pancreatic Lipase......Page 177
9.1.2 3-D Structure of Human Gastric Lipase......Page 179
9.2 Methods for Lipase Inhibition......Page 180
9.2.2 Method B: Inhibition During Lipolysis......Page 183
9.2.3.2 Method D......Page 184
9.3 Inhibition of Lipases by E(600) and Various Phosphonates......Page 185
9.3.1 Inhibition of PPL, HGL and RGL by Radiolabeled E(600)......Page 186
9.3.3.1 Synthesis of New Chiral Organophosphorus Compounds Analogous to TAG......Page 188
9.3.3.2 The 2.46 Å Resolution Structure of the Pancreatic/Procolipase Complex Inhibited by a C(11) Alkylphosphonate......Page 191
9.3.3.3 Crystal Structure of the Open Form of DGL in Complex with a Phosphonate Inhibitor......Page 194
9.4.1 Introduction......Page 195
9.4.2.1 Inhibition of Gastric Lipases......Page 196
9.4.2.2 Inhibition of Pancreatic Lipases......Page 197
9.4.2.3 Kinetic Model Illustrating the Covalent Inhibition of HPL in the Aqueous Phase......Page 201
9.4.4 Inhibition of Digestive Lipases on Oil Emulsions “Poisoned” with Orlistat (Method C)......Page 202
9.4.5.1 Inhibition of Pancreatic Lipase on Emulsion “Pre-poisoned” with Orlistat......Page 205
9.4.5.3 Inhibition of Pancreatic Lipase on Oil Drop “Pre-poisoned” with Orlistat......Page 206
9.5 References......Page 208
10.1 Introduction......Page 216
10.2 Tissular and Cellular Origins of HGL and HPL......Page 217
10.3.1 Substrate Specificity......Page 220
10.3.2 Specific Activities of HGL and HPL......Page 221
10.3.4 Effects of Bile Salts on the Activity of HGL and HPL......Page 223
10.4 Gastrointestinal Lipolysis of Test Meals in Healthy Human Volunteers......Page 225
10.4.1 Test Meals......Page 226
10.4.4 Lipase Concentrations and Outputs......Page 228
10.4.5 Lipolysis Levels......Page 232
10.5 HGL and HPL Stability......Page 234
10.6 Potential Use of Gastric Lipase in the Treatment of Pancreatic Insufficiency......Page 236
10.7.1 The Lipase Inhibitor Orlistat......Page 237
10.7.2 Design of Clinical Studies for Quantification of Lipase and Lipolysis Inhibition......Page 238
10.7.3 HGL Inhibition by Orlistat......Page 239
10.7.4 HPL Inhibition by Orlistat......Page 240
10.7.5 Effects of Orlistat on Gastric Lipolysis......Page 241
10.7.8 Effects of Orlistat on Fat Excretion......Page 242
10.7.9 Weight Management by Orlistat in Obese Patients......Page 243
10.8 References......Page 245
11.1.1 Triacylglycerol and Energy Storage......Page 252
11.1.2 Lipolysis and Re-esterification......Page 255
11.1.3.1 Diabetes Mellitus and Metabolic Syndrome......Page 257
11.1.3.2.1 β-Cells......Page 259
11.1.3.2.2 Cardiac Myocytes......Page 260
11.1.3.2.3 Molecular Mechanisms......Page 261
11.1.3.3 Inborn Errors of TAG Storage and Metabolism......Page 262
11.2.1 TAG in Lipoproteins......Page 263
11.2.2 TAG in Adipose Cells......Page 264
11.2.2.1 Enzymes of TAG Synthesis......Page 265
11.2.2.2.1 Morphology and Lipid Composition......Page 267
11.2.2.2.2 Protein Composition......Page 269
11.2.2.2.3 Biogenesis......Page 273
11.3.1 cAMP......Page 280
11.3.2 Phosphorylation of HSL......Page 281
11.3.4 Intrinsic HSL Activity......Page 284
11.3.5.1 Mechanism......Page 285
11.3.5.2 Involvement of Perilipins......Page 287
11.3.5.3 Involvement of Lipotransin......Page 289
11.3.6.1 Feedback Inhibition......Page 291
11.3.6.2 Adipocyte Lipid-binding Protein......Page 293
11.3.7 Expression of HSL......Page 295
11.3.8.1 FA Transport......Page 296
11.3.8.2 Glycerol Transport......Page 297
11.3.8.3 Cholesterol Transport......Page 298
11.4.1 Muscle Contraction......Page 299
11.4.3.1.1 Molecular Mechanisms......Page 300
11.4.3.1.2 Desensitization......Page 302
11.4.3.2 Leptin......Page 303
11.4.3.5 TNF-α......Page 304
11.4.4 ASP......Page 306
11.4.5 Acipimox and Nicotinic Acid......Page 307
11.4.5.1 Mode of Action......Page 308
11.4.5.2 Molecular Mechanism......Page 309
11.4.5.3 Desensitization......Page 310
11.4.6 Glimepiride and Phosphoinositolglycans......Page 311
11.4.7 Differences in Regulation of TAG Storage and Mobilization between Visceral and Subcutaneous Adipocytes......Page 313
11.4.8.1 HSL......Page 315
11.4.8.2 ALBP......Page 317
11.4.8.3 Perilipin......Page 318
11.4.8.4 PKA......Page 320
11.4.8.5 ASP......Page 321
11.4.8.6 Caveolin......Page 322
11.5 Concluding Remarks......Page 323
11.6 References......Page 324
Subject Index......Page 354