Filling a real knowledge gap, this handbook and ready reference is both modern and forward-looking in its emphasis on the "bench to bedside" translational approach to drug development. Clearly structured into three major parts, the book stakes out the boundaries of peptide drug development in the preclinical as well as clinical stages. The first part provides a general background and focuses on the characteristic strengths and weaknesses of peptide drugs. The second section contains five cases studies of peptides from diverse therapeutic fields, and the lessons to be learned from them, while the final part looks at new targets and opportunities, discussing several drug targets and diseases for which peptide drugs are currently being developed.

Miguel Castanho is head of the Biochemistry department of the School of Medicine at the University of Lisbon (Portugal). He also leads the Physical Biochemistry research unit at the Institute of Molecular Medicine. His main field of research is the interaction of peptide drugs with lipids, in particular the role of lipid membranes in the mechanism of action of peptide drugs.

Nuno C. Santos is the leader of the Biomembranes Unit of the Institute of Molecular Medicine. His research is centered on the specific functions of biological membranes lipids and proteins, with a special focus on their relevance for pathologic processes ranging from viral or bacterial infection to cardiovascular diseases.

Together, Miguel Castanho and Nuno C. Santos have been involved in many collaborative drug discovery and development projects with industry partners in Europe, USA, Canada, and China.

Preface xiii

List of Contributors xv

Part I The Academia – Market Bouncing of Peptide Drugs – Challenges and Strategies in Translational Research with Peptide Drugs 1

1 Peptides as Leads for Drug Discovery 3
Paul J. Edwards, and Steven R. LaPlante

1.1 Introduction 3

1.2 Overview of Process for Transforming Peptides to Peptidomimetics 5

1.3 HCMV Protease 7

1.3.1 HCMV Protease: Identification and Characterization of Antiviral Inhibitors Targeting the Serine Protease Domain of the Human Cytomegalovirus (HCMV Protease) 7

1.3.2 Mapping Essential Elements of the Substrate Peptides and Determining Structures of Ligands Bound to HCMV 8

1.3.3 Improving Peptide Activity to Allow SAR Studies 10

1.3.4 Elucidation of the Binding Mode of the Optimized Peptidyl Segment 10

1.3.5 Ligand Adaptations upon Binding 12

1.3.6 Strategic Summary for HCMV Peptide Mimic Design Process 14

1.4 HCV Protease 15

1.4.1 HCV Protease as an Antiviral Target 15

1.4.2 NS3 Serine Protease Possesses a Chymotrypsin-Like Fold 16

1.4.3 Discovery of the Peptide DDIVPC as an Inhibitor of NS3 Protease 16

1.4.4 ‘‘Sensemaking’’ and Knowledge Building: Mapping of the Critical Binding Residues of the Peptide and Creation of an Inhibitor-Protease Model 18

1.4.5 Knowledge Building: Monitoring Ligand Flexibility in the Free-State and Changes Upon Binding – P3 Rigidification 18

1.4.6 N-Terminal Truncation and Improved P1, P2 and P5 Substituents 22

1.4.7 Macrocyclization: Linking the Flexible P1 Side-Chain to P3 25

1.4.8 HCV Protease Inhibitor BI00201335 29

1.5 Herpes Simplex Virus 32

1.5.1 Herpes Simplex Virus-Encoded Ribonucleotide Reductase Inhibitors 32

1.6 Renin 38

1.6.1 Aspartyl Protease Renin as a Target 38

1.7 HIV 45

1.7.1 HIV Protease Inhibitors 45

1.8 Conclusions 47

2 Marketing Antimicrobial Peptides: A Critical Academic Point of View 57
Eduard Bardajı´

2.1 Introduction 57

2.2 Basic Research: Antimicrobial Peptides 58

2.3 Patents 61

2.4 Potential Applications of AMPs 63

2.5 Technology Transfer: Valorization, Licensing, or Spin-Off Creation 64

2.6 Spin-Off Creation: An Academic Point of View 66

3 Oral Peptide Drug Delivery: Strategies to Overcome Challenges 71
Hamman, Josias H. and Steenekamp, Jan H.

3.1 Introduction 71

3.2 Challenges Associated with Oral Peptide Delivery 72

3.2.1 Transport Pathways Across the Intestinal Epithelium 72

3.2.2 Unfavorable Physicochemical Properties of Peptide Drugs 73

3.2.2.1 Molecular Size, Hydrophilicity, and Physical Stability 73

3.2.3 Physical Barriers of the Gastrointestinal Tract 73

3.2.3.1 Transcellular Pathway 73

3.2.3.2 Paracellular Pathway 75

3.2.4 Biochemical Barriers of the Gastrointestinal Tract 75

3.2.4.1 Luminal Enzymes 76

3.2.4.2 Brush Border Membrane Bound Enzymes and Intracellular Enzymes 76

3.2.5 Efflux Transport Systems 76

3.2.6 Gastrointestinal Transit Time and Site-Specific Absorption 77

3.3 Strategies to Overcome the Barriers of the Gastrointestinal Tract 77

3.3.1 Absorption Enhancing Agents 77

3.3.2 Chemical and Physical Modifications 78

3.3.3 Targeting Strategies 81

3.3.3.1 Targeting Specific Regions of the Gastrointestinal Tract 81

3.3.3.2 Targeting Receptors and Transporters 82

3.3.4 Formulation Strategies 83

3.3.4.1 Particulate Carrier Systems 83

3.3.4.2 Enzyme Inhibition 84

3.3.4.3 Mucoadhesive Systems 84

3.4 Conclusions 84

4 Rational Design of Amphipathic a-Helical and Cyclic b-Sheet Antimicrobial Peptides: Specificity and Therapeutic Potential 91
Wendy J. Hartsock and Robert S. Hodges

4.1 Introduction to Antimicrobial Peptides 91

4.2 Antimicrobial and Hemolytic Activities of Amphipathic a-Helical Antimicrobial Peptides: Mechanisms and Selectivity 92

4.3 Structure–Activity Relationship Studies of Amphipathic a-Helical and Cyclic b-Sheet Antimicrobial Peptides: Optimization of Pathogen Selectivity and Prevention of Host Toxicity 94

4.4 Commercialization of Antimicrobial Peptides 112

4.5 Therapeutic Potential 113

5 Conotoxin-Based Leads in Drug Design 119
Muharrem Akcan and David J. Craik

5.1 Introduction 119

5.1.1 Cone Snails 119

5.1.2 Conotoxin Discovery and Characterization (MS, cDNA, Peptide Sequencing) 120

5.1.3 Conotoxin Classification and Targets 121

5.1.4 Posttranslational Modifications (PTMs) 122

5.1.5 Prospects for Drug Discovery 124

5.2 Conotoxin Synthesis, Folding, and Structure 124

5.2.1 Synthesis 124

5.2.2 Folding 127

5.2.3 Structure by NMR and X-Ray 127

5.3 Conotoxins as Drug Leads 128

5.3.1 Overview of Conotoxins in Drug Design 128

5.3.2 o-Conotoxins (MVIIA, CVID) 129

5.3.3 a-Conotoxins (Vc1.1) 129

5.3.4 w-Conotoxins (MrIA) 130

5.3.5 Re-engineered Conotoxins in Drug Design 131

5.4 Conclusions 133

6 Plant Antimicrobial Peptides: From Basic Structures to Applied Research 139
Suzana M. Ribeiro, Simoni C. Dias, and Octavio L. Franco

6.1 Introduction 139

6.2 The Diversity of Plant Antimicrobial Peptides: Focusing on Tissue Localization and Plant Species Distribution 139

6.3 Possible Structural Folds Found in Plant AMPs to Date 140

6.4 New Biotechnological Products Produced from Plant Peptides 144

Part II Peptide Drugs’ Translational Tales – Peptide Drugs Before, Through and After Industry Pipelines 157

7 Omiganan Pentahydrochloride: A Novel, Broad-Spectrum Antimicrobial Peptide for Topical Use 159
Evelina Rubinchik and Dominique Dugourd

7.1 Omiganan: A Novel Anti-Infective Agent for Topical Indications 159

7.2 Structure and Mechanism of Action 160

7.3 Spectrum of Activity 163

7.4 Preclinical Efficacy Studies 163

7.5 Preclinical Toxicology Studies 164

7.6 Clinical Studies 165

7.7 Conclusions 167

8 Turning Endogenous Peptides into New Analgesics: The Example of Kyotorphin Derivatives 171
Marta M.B. Ribeiro, Isa D. Serrano, and So´nia Sa´ Santos

8.1 Introduction 171

8.2 Peptides as Future Drug Candidates 171

8.3 Central Nervous System Analgesic Peptides 172

8.4 Endogenous Opioid System 173

8.5 Strategies to Deliver Analgesic Peptides to the Brain 174

8.6 Development of New Opioid-Derived Peptides 175

8.7 Kyotorphin – the Potential of an Endogenous Dipeptide 177

8.8 New KTP Derivatives 178

8.9 Assessing BBB Permeability with Peptide – Membrane Partition Studies 179

8.10 Kyotorphins: Partition to the Membrane and Enhanced Analgesic Activity 179

8.11 Academia and Pharmaceutical Industry: Friends or Foes? 183

9 The Development of Romiplostim – a Therapeutic Peptibody Used to Stimulate Platelet Production 189
Graham Molineux and Ping Wei

9.1 Introduction 189

9.2 Thrombopoietin and c-Mpl 189

9.3 Discovery and Optimization of Romiplostim 192

9.4 Pharmacodynamics (PD) and Pharmacokinetics (PK) of Romiplostim 194

9.5 A Brief ITP Primer 199

9.5.1 Diagnosis and Treatment 199

9.5.2 Thrombopoietin and ITP 200

9.6 Romiplostim Clinical Data 201

9.7 Safety and Other Insights Gained from Romiplostim Design and Development 203

10 HIV vs. HIV: Turning HIV-Derived Peptides into Drugs 209
Henri G. Franquelim, Pedro M. Matos, and A. Salome´ Veiga

10.1 Introduction 209

10.2 HIV-1 Envelope Protein 209

10.3 HIV Entry and Its Inhibition 210

10.4 HIV-1 Fusion Inhibitors: from Bench to Clinical Administration 211

10.5 New Strategies for Creating New HIV Fusion Inhibitor Peptides 215

10.5.1 Increasing Helicity and Binding to gp41 216

10.5.2 Isomeric Peptides and Resistance to Proteolysis 219

10.5.3 Bacterially Expressed Peptides 220

10.5.4 Modification of Peptides by Derivatization with Lipids or Proteins 220

10.6 Drug-Resistance and Combination Therapy 222

10.7 Concluding Remarks 223

11 Sifuvirtide, A Novel HIV-1 Fusion Inhibitor 231
Xiaobin Zhang, Hao Wu, and Fengshan Wang

11.1 Ideal Drug Target HIV-1 gp41 231

11.2 Structure-Based Drug Design of Sifuvirtide 232

11.3 High Potency of Sifuvirtide 234

11.4 Limited Drug Resistance 235

11.5 Enhancement of the Efficiency of Sifuvirtide by Biomembrane Selectivity 236

11.6 Pharmacokinetics of Sifuvirtide with Long Half-Life 237

11.7 Stratification of Monotherapy 238

11.8 20 mg Sifuvirtide Once Daily vs. 100 mg T20 Twice Daily 239

11.9 Conclusions and Discussion 240

Part III Whither Peptide Drugs? Peptides Shaping the Future of Drug Development 245

12 Endogenous Peptides and Their Receptors as Drug Discovery Targets for the Treatment of Metabolic Disease 247
Mary Ann Pelleymounter, Yuren Wang, and Ning Lee

12.1 Centrally Secreted Neuropeptide Systems 248

12.1.1 Corticotropin Releasing Factor (CRF) Peptides 248

12.1.2 Melanin Concentrating Hormone (MCH) 249

12.1.3 Melanocortins 250

12.1.4 Neuropeptide Y (NPY) 252

12.1.5 Neuromedin U (NMU) and Neuromedin S (NMS) 254

12.1.6 Opioids 255

12.1.7 QRFP 256

12.2 Peripherally Secreted Neuropeptides 256

12.2.1 Amylin 256

12.2.2 Bombesin-Like Peptides (Bombesin and Gastrin-Releasing Peptide) 257

12.2.3 Cholecystokinin (CCK) 258

12.2.4 Ghrelin 259

12.2.5 Glucagon-like Peptide-1 260

12.2.6 Leptin 261

12.2.7 Oxyntomodulin (OXM) 262

12.2.8 PYY3-36 and PP 262

12.3 Summary 263

13 Translation of Motilin and Ghrelin Receptor Agonists into Drugs for Gastrointestinal Disorders 269
Gareth J. Sanger, John Broad, and David H. Alpers

13.1 Introduction 269

13.1.1 Similarities and Differences Between Motilin and Ghrelin 269

13.1.2 Clinical Potential of Motilin and Ghrelin Receptor Agonists 270

13.2 Motilin and Ghrelin Receptor Agonists Under Development 271

13.3 Translational Value of Preclinical Assays 275

13.3.1 Motilin 271

13.3.1.1 Assays Relevant to the Therapeutic Mechanism of Action 271

13.3.1.2 Assays Relevant to Possible Non-GI Activity 275

13.3.2 Ghrelin 276

13.3.2.1 Assays Relevant to the Therapeutic Mechanism of Action 276

13.3.2.2 Assays Relevant to Non-GI Activity 276

13.4 Clinical Translation: Selecting the ‘‘Right’’ Patient Population 277

13.4.1 Critically Ill Patients with Delayed Gastric Emptying 279

13.4.2 Patients with Gastroparesis 279

13.4.2.1 Diabetic Gastroparesis 281

13.4.2.2 Parkinson’s Disease 281

13.4.2.3 Cyclic Nausea and Vomiting 282

13.4.2.4 Migraine 282

13.4.2.5 Functional Dyspepsia (FD) 282

13.4.2.6 Gastroesophageal Reflux Disease (GERD) 283

13.4.2.7 Anorexia and Decreased Appetite (Ghrelin Agonists Only) 284

13.5 Clinical Development of Motilin and Ghrelin Receptor Agonists 284

13.6 Conclusions 285

14 Of Mice and Men: Translational Research on Amylin Agonism 295
Jonathan D. Roth, Christine M. Mack, James L. Trevaskis, and David G. Parkes

14.1 Overview of Amylin Physiology 295

14.2 Pramlintide: An Amylin Agonist 296

14.3 Amylin Agonism: Translational Research in Insulin-Dependent Diabetes 297

14.3.1 Post-Prandial Hyperglucagonemia and Diabetes 297

14.3.2 Amylin Agonism and Glucagon: Preclinical and Clinical Studies 297

14.3.3 Gastric Emptying and Diabetes 298

14.3.4 Amylin Agonism and Gastric Emptying: Preclinical and Clinical Studies 298

14.4 Amylin Agonism: Translational Research in Obesity 299

14.4.1 Food Intake and Body Weight: Role of Endogenous Amylin 299

14.4.2 Food Intake and Body Weight: Pre-clinical Studies 300

14.4.3 Food Intake and Body Weight: Clinical Studies 302

14.4.4 Combination Studies 304

14.4.5 Amylin Agonism and Small Molecule Agents 304

14.4.6 Combined Amylin and Leptin Agonism 305

14.4.7 Future Areas for Amylin Agonism-Based Translational Research 307

15 Peptides and Polypeptides as Immunomodulators and Their Consequential Therapeutic Effect in Multiple Sclerosis and Other Autoimmune Diseases 313
Ruth Arnon, Michael Sela, and Rina Aharoni

15.1 Introduction 313

15.2 Peptides as Antigens and Vaccines 314

15.3 Peptides as Immunomodulators 315

15.4 Development of Copolymer 1 – a Polypeptide Immunomodulator Drug for the Treatment of Multiple Sclerosis 316

15.4.1 Clinical Studies with Cop 1 in MS Patients 317

15.4.2 Immunological Mechanisms Involved in the Mitigation of Disease by Cop 1 318

15.4.3 Immunomodulation by Cop 1 in the CNS 320

15.4.4 Neuroprotection and Augmentation of Neurotropic Factors in the Brain 321

15.4.5 Myelin Repair and Neurogenesis 323

15.4.6 The Effect of Cop 1 on Another Autoimmune Disease – Inflammatory Bowel Disease 326

15.5 Additional Immunomodulatory Peptides as Drug Candidates 327

15.5.1 Peptide Therapy for Type 1 Diabetes 327

15.5.2 Myasthenia Gravis (MG) 328

15.5.3 A Novel Tolerogenic Peptide for the Specific Treatment of Systemic Lupus Erythematosus 328

15.6 Summary and Concluding Remarks 329

16 Development of Antibody Fragments for Therapeutic Applications 337
Sofia Coˆrte-Real, Frederico Aires da Silva, and Joa˜o Gonc- alves

16.1 Antibodies 337

16.1.1 Antibody Structure 338

16.1.2 Antibody Fragments 341

16.1.3 Single-Domain Antibodies 343

16.1.4 Engineering Multivalent, Bispecific, and Bifunctional Fragments 345

16.1.5 Intracellular Antibodies (Intrabodies) 347

16.1.5.1 Immunogenicity of Engineered Antibodies 348

16.1.5.2 Engineering New Protein Scaffolds 349

16.2 Conclusions 350

Index 357