Aspartic Acid Proteases as Therapeutic Targets E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Serie: Methods and Principles in Medicinal Chemistry
• Sprache: Englisch

Seitenzahl: 1179

Veröffentlichungsjahr: 2011

Beschreibung

In this ground-breaking practical reference, the family of aspartic acid proteases is described from a drug developer's perspective. The first part provides a general introduction to the family of aspartic acid proteases, their physiological functions, molecular structure and inhibition. Parts two to five present various case studies of successful protease inhibitor drug design and development, as well as current and potential uses of such inhibitors in pharmaceutical medicine, covering the major therapeutic targets HIV-1 protease, renin, beta-secretase, gamma-secretase,plasmepsins and fungal proteases.
A ready reference aimed primarily at professionals in the pharmaceutical industry, as well as for anyone studying proteases and their function.

Leseprobe

Contents

List of Contributors

Preface

A Personal Foreword

Part One Overview of Aspartic Acid Proteases

Chapter 1: Introduction to the Aspartic Proteinase Family

1.1 Introduction

1.2 Sequence Alignment and Family Tree

1.3 Three-Dimensional Structure

1.4 Conferences and Progress

1.5 Exploration of the Active Sites of Aspartic Proteinase and Relation to Drug Discovery

References

Chapter 2: Aspartic Proteases: Structure, Function, and Inhibition

2.1 Introduction

2.2 Structures of Aspartic Proteases

2.3 Catalytic Mechanism and Substrate Specificity

2.4 Zymogen Activation

2.5 Inhibition and the Development of Inhibitor Drugs

2.6 Perspectives

2.7 Information Resource on Aspartic Proteases

References

Chapter 3: Human Aspartic Proteinases

3.1 Introduction

3.2 Human Aspartic Proteinases

3.3 New Human Aspartic Proteinases

3.4 Concluding Remarks

References

Chapter 4: Structure-Based Drug Design Strategies for Inhibition of Aspartic Proteinases

4.1 Introduction

4.2 Associated Disease States

4.3 Development of Aspartic Proteinase Inhibitors

4.4 General Strategies for Design of Renin Inhibitors

4.5 Structural Studies of Renin Complexed with Inhibitors

4.6 Structure-Based Drug Design for the HIV Proteinase

4.7 Structure-Based Drug Design for Other Aspartic Proteinases

4.8 Possible New Leads from Macromolecular Inhibitor Complexes and Zymogen Structures

4.9 Conclusions

References

Part Two HIV-1 Protease as Target for the Treatment of HIV/AIDS

Chapter 5: HIV-1 Protease: Role in Viral Replication, Protein–Ligand X-Ray Crystal Structures and Inhibitor Design

5.1 Introduction

5.2 HIV-1 and the AIDS Pandemic

5.3 PR Interactions with Peptidic Inhibitors and Design of First Antiviral Inhibitors

5.4 PR–Inhibitor Interactions Revealed in Atomic Resolution Structures

5.5 The Challenge of Drug Resistance in HIV

5.6 Future Perspectives

References

Chapter 6: First-Generation HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS

6.1 Introduction

6.2 Structure of HIV Protease

6.3 Design of Inhibitors

6.4 Viral Resistance to First-Generation Protease Inhibitors

6.5 Perspectives

References

Chapter 7: Second-Generation Approved HIV Protease Inhibitors for the Treatment of HIV/AIDS

7.1 Introduction

7.2 Second-Generation Protease Inhibitors

7.3 Conclusion

References

Chapter 8: Darunavir, a New PI with Dual Mechanism: From a Novel Drug Design Concept to New Hope Against Drug-Resistant HIV

8.1 Introduction

8.2 Molecular Insights from Protein–Ligand X-Ray Crystal Structures of Early PIs and Design of Novel PIs Inspired by Nature

8.3 Design of Novel Protease Inhibitors Containing Cyclic and Polycyclic Ether Templates

8.4 Development of PIs Based Upon Our “Backbone Binding Concept” to Combat Drug-Resistant HIV

8.5 Antiviral Activities, Resistance Profiles of TMC 126 (20), and Relevance to “Backbone Binding Concept”

8.6 Selection of TMC114 and Its Subsequent Development to Darunavir

8.7 High-Resolution X-Ray Structure of Darunavir-Bound HIV-1 Protease

8.8 A Unique Dual Mode of Action: Darunavir also Inhibits Dimerization of HIV-1 Protease

8.9 Optically Active Synthesis of Bis-THF Ligand for Initial Clinical Studies and Beyond

8.10 Pharmacological Profile of Darunavir: ADME Properties

8.11 Therapeutic Evaluation of DRV

8.12 Conclusion

References

Chapter 9: Development of HIV-1 Protease Inhibitors, Antiretroviral Resistance, and Current Challenges of HIV/AIDS Management

9.1 Introduction

9.2 Targeting the Viral Protease

9.3 The Role of PIs and the Challenges Faced by HAART

9.4 “Boosting”: A Critical Modification to the Clinical Efficacy of PIs

9.5 HIV-1 Variants Resistant to Conventional PIs

9.6 New Generation PIs with Activity against Drug-Resistant HIV-1

9.7 A Novel Modality of HIV-1 Protease Inhibition: Dimerization Inhibition

9.8 HIV-1 Resistance to Darunavir

9.9 When to Start HAART with Protease Inhibitors

9.10 Conclusions

References

Part Three Renin as Target for the Treatment of Hypertension

Chapter 10: Discovery and Development of Aliskiren, the First-in-Class Direct Renin Inhibitor for the Treatment of Hypertension

10.1 Introduction

10.2 The History of Renin and Aliskiren

10.3 The Renin–Angiotensin–Aldosterone System

10.4 History of the Renin Inhibitor Project in Ciba–Geigy/Novartis

10.5 New Concept Toward (P3–P1) Topological Peptidomimetic Inhibitors

10.6 Early Preclinical Leads

10.7 Scalable Synthesis Development of Aliskiren

10.8 Properties of Aliskiren

10.9 Clinical Efficacy of Aliskiren

10.10 Conclusions and Outlook

References

Chapter 11: Evolution of Diverse Classes of Renin Inhibitors Through the Years

11.1 Introduction

11.2 Mechanism and Structural Biology of Renin

11.3 Screening and Animal Models

11.4 Peptidomimetic Renin Inhibitors

11.5 Nonpeptidomimetic Renin Inhibitors

11.6 Conclusions

References

Part Four γ-Secretase as Target for the Treatment of Alzheimer’s Disease

Chapter 12: γ-Secretase: An Unusual Enzyme with Many Possible Disease Targets, Including Alzheimer’s Disease

12.1 Introduction

12.2 Presenilin: From a Zymogen to the Subunit of a Complex Enzyme

12.3 γ-Secretase: A Promiscuous Enzyme That Mediates Regulated Intramembrane Proteolysis

12.4 Structure of γ-Secretase

12.5 Mechanism of γ-Secretase

12.6 Pharmacology of γ-Secretase

12.7 Concluding Remarks

References

Chapter 13: γ-Secretase Inhibition: An Overview of Development of Inhibitors for the Treatment of Alzheimer’s Disease

13.1 Introduction

13.2 APP Genetics

13.3 Amyloid Cascade Hypothesis

13.4 γ-Secretase Inhibitors: Compounds and Clinical Results

13.7 Conclusion

References

Part Five β-Secretase as Target for the Treatment of Alzheimer’s Disease

Chapter 14: BACE: A (Almost) Perfect Target for Staving Off Alzheimer’s Disease

14.1 Introduction: APP Cloning, α-Secretase Pathway, and β-Secretase Hypothesis

14.2 Cell Biology Evidence for β-Secretase

14.3 BACE-Deficient Mice: Effect on Aβ Production

14.4 Other BACE Substrates

14.5 BACE Structure and BACE Inhibitors

14.6 Conclusions

References

Chapter 15: The Discovery of β-Secretase and Development Toward a Clinical Inhibitor for AD: An Exciting Academic Collaboration

15.1 Introduction

15.2 Discovery of Memapsin 2 and Its Identification as β-Secretase (by Jordan Tang)

15.3 Design of the First Substrate-Based Inhibitors: A Remarkable Scientific Adventure Between Two Laboratories (by Jordan Tang and Arun Ghosh)

15.4 Crystal Structure of Memapsin 2 and Binding of Inhibitors (by Lin Hong)

15.5 Evolution of Memapsin 2 Inhibitors (by Arun Ghosh)

15.6 Perspective

References

Chapter 16: Peptidomimetic BACE1 Inhibitors for Treatment of Alzheimer’s Disease: Design and Evolution

16.1 Introduction

16.2 Substrate-Based (Peptidic) Inhibitors

16.3 Peptidomimetic Statine and Homostatine Inhibitors

16.4 Hydroxyethylamine-Based Inhibitors

16.5 Other Peptidomimetics

16.6 Conclusions

References

Chapter 17: Nonpeptide BACE1 Inhibitors: Design and Synthesis

17.1 Introduction

17.2 Preliminary Peptidomimetic BACE1 Inhibitors

17.3 Acyl Guanidine-Based Inhibitors

17.4 Aminoimidazolone-Based Inhibitors

17.5 2-Aminopyridine and Pyrimidine-Based Inhibitors

17.6 Aminoimidazopyrimidine-Based Inhibitors

17.7 2-Aminoquinazoline-Based Inhibitors

17.8 Piperidine and Piperazine-Based Inhibitors

17.9 Other Miscellaneous Scaffolds

17.10 Conclusions

References

Part Six Plasmepsins and Other Aspartic Proteases as Drug Targets

Chapter 18: The Plasmepsin Family as Antimalarial Drug Targets

18.1 Introduction

18.2 Plasmepsins In Vivo

18.3 Plasmepsins In Vitro

18.4 Plasmepsin Family Structures

18.5 Plasmepsin Inhibitors

18.6 Conclusions

References

Chapter 19: Plasmepsins Inhibitors as Potential Drugs Against Malaria: Starving the Parasite

19.1 Introduction

19.2 The Plasmepsin Family of Enzymes

19.3 Inhibitors of the Plasmepsin Family of Enzymes

19.4 Transition-State Isostere-Based Inhibitors

19.5 Nonpeptidic Inhibitors

19.6 “Double-Drugs”

19.7 Conclusions

References

Chapter 20: Fungal Aspartic Proteases as Possible Therapeutic Targets

20.1 Introduction

20.2 Biochemical Properties of Fungal Aspartic Proteases (Table 20.1)

20.3 Phylogeny of Fungal Aspartic Proteases

20.4 Production of Fungal Aspartic Proteases

20.5 Biological Functions of Aspartic Proteases in Fungi

20.6 Secreted Aspartic Proteases in Virulence of C. albicans

20.7 Functions of Aspartic Protease in C. albicans Virulence Processes

20.8 Secreted Aspartic Proteases in the Virulence of Candida Species Other Than C. albicans

20.9 Secreted Aspartic Proteases During Infection by Opportunistic Filamentous Fungi

20.10 Fungal Aspartic Proteases as Possible Drug Targets

20.11 Conclusions

References

Index

Methods and Principles in Medicinal Chemistry

Edited by R. Mannhold, H. Kubinyi, G. FolkersEditorial BoardH. Timmerman, H. van de Waterbeemd, T. Wieland

Previous Volumes of this Series:

Ecker, Gerhard F./Chiba, Peter (Eds.)Transporters as Drug CarriersStructure, Function, Substrates2009ISBN: 978-3-527-31661-8Vol. 44

Faller, Bernhard/Urban, Laszlo (Eds.)Hit and Lead ProfilingIdentification and Optimization of Drug-like Molecules2009ISBN: 978-3-527-32331-9Vol. 43

Sippl, Wolfgang/Jung, Manfred (Eds.)Epigenetic Targets in Drug Discovery2009ISBN: 978-3-527-32355-5Vol. 42

Todeschini, Roberto/Consonni, VivianaMolecular Descriptors for ChemoinformaticsVolume I: Alphabetical Listing/Volume II: Appendices, References2009ISBN: 978-3-527-31852-0Vol. 41

van de Waterbeemd, Han/Testa, Bernard (Eds.)Drug BioavailabilityEstimation of Solubility, Permeability, Absorption and Bioavailability Second, Completely Revised Edition2008ISBN: 978-3-527-32051-6Vol. 40

Ottow, Eckhard/Weinmann, Hilmar (Eds.)Nuclear Receptors as Drug Targets2008ISBN: 978-3-527-31872-8Vol. 39

Vaz, Roy J./Klabunde, Thomas (Eds.)AntitargetsPrediction and Prevention of Drug Side Effects2008ISBN: 978-3-527-31821-6Vol. 38

Mannhold, Raimund (Ed.)Molecular Drug PropertiesMeasurement and Prediction2007ISBN: 978-3-527-31755-4Vol. 37

Wanner, Klaus/Höfner, Georg (Eds.)Mass Spectrometry in Medicinal ChemistryApplications in Drug Discovery2007ISBN: 978-3-527-31456-0Vol. 36

Hüser, Jörg (Ed.)High-Throughput Screening in Drug Discovery2006ISBN: 978-3-527-31283-2Vol. 35

Series Editors

Prof. Dr. Raimund MannholdMolecular Drug Research GroupHeinrich-Heine-UniversitätUniversitätsstrasse 140225 Dü[email protected]

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Prof. Dr. Gerd FolkersCollegium HelveticumSTW/ETH Zurich8092 [email protected]

Volume Editors

Prof. Dr. Arun K. GhoshPurdue UniversityDept. of Chemistry560 Oval DriveWest LafayetteIN 47907-2084USA

Cover Description:X-ray structure of darunavir-bound HIV-1 protease (Tie, Y.; et al. 2004). Darunavir carbons are shown in green. Hydrogen bonds are shown as dotted lines.

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Preface

The digestive enzymes pepsin and chymosin (formerly called rennin), both being known since long, are members of the aspartic protease family. While these enzymes cleave many proteins and peptides in a relatively nonspecific manner, the substrate-specific aspartyic protease renin converts angiotensinogen to angiotensin I, the precursor of the hypertensive peptide angiotensin II. For this purpose, research on renin inhibitors as potential antihypertensive drugs started in the early 1970s, based on Pauling’s concept of transition-state mimics as effective enzyme inhibitors. Umezawa’s discovery of pepstatin, a picomolar to nanomolar inhibitor of several aspartic proteases, confirmed the importance of a replacement of the scissile amide bond by such a transition state-imitating group. Some early peptide-like renin inhibitors showed nanomolar activities but lacked sufficient bioavailability, a problem that continued for the next decades. In the meantime, the search for aspartic proteases inhibitors was significantly stimulated by the discovery that HIV protease also belongs to the aspartic protease family, like pepsin and renin. Research on this new target was supported by the concept of chemogenomics, that is, by transferring successful design strategies to the new target. Based on the accumulated experience from the search for renin inhibitors, saquinavir, indinavir, and ritonavir resulted as first drugs in the mid-1990s. These peptide-like compounds were followed by some nonpeptidic analogues, all of them bearing a transition state partial structure. Finally, after thousands of man years in research in different companies, the first bioavailable renin inhibitor, aliskiren, was approved and marketed in 2007. Much effort and hope go now into the search for β- and γ-secretase inhibitors as drugs to prevent the development and progression of Alzheimer’s disease, as well as plasmepsin inhibitors to treat malaria. This book by Arun K. Ghosh treats all these topics in much detail, starting from the function and physiological role of the corresponding protease, discussing its structural biology, and finally the medicinal chemistry of ligand and drug design.

For several years, it has been our goal to include monographs on therapeutically relevant targets and their modulators in our book series “Methods and Principles in Medicinal Chemistry.” After volumes on G protein-coupled receptors (volume 24), voltage-gated ion channels (volume 29), ligand design for GPCRs (volume 30), antitargets (volume 38), nuclear receptors (volume 39), epigenetic targets (volume 42), and drug transporters (volume 44), this monograph is the first one in an important family of therapeutically relevant enzymes, the aspartic proteases.

We would like to express our appreciation for Arun Ghosh for his enthusiasm and perseverance in bringing this project to fruition. We wish to thank Joseph Vacca for his initial efforts toward this goal. A team of leading scientists discusses the abovementioned aspartic proteases, as well as some others, and their inhibitors. We are very grateful to all these authors for their excellent contributions, as well as to Frank Weinreich and Nicola Oberbeckmann-Winter for their ongoing engagement in our series “Methods and Principles in Medicinal Chemistry,” in which this book will surely be as well accepted as all previous volumes.

April 2010

Raimund Mannhold, DüsseldorfHugo Kubinyi, Weisenheim am SandGerd Folkers, Zürich

List of Contributors

Daniel BurActelion Pharmaceuticals Ltd.CH-4123 AllschwilSwitzerland

Matthew G. BursavichMedical ChemistryMyriad Pharmaceuticals305 Chipeta WaySalt Lake City, UT 84108USA

Bruno D. ChapsalPurdue UniversityDepartment of Chemistry560 Oval DriveWest Lafayette, IN 47907-2084USA

Derek C. ColeMedical Chemistry DepartmentTakeda San Diego10410 Science Center DriveSan Diego, CA 92121-1119USA

Jon B. CooperUCL Department of MedicineCentre for Amyloidosis and AcutePhase ProteinsLaboratory for Protein CrystallographyRoyal Free CampusRowland Hill StreetLondon NW3 2PFUK

Jared N. CummingSchering-Plough Research Institute2015 Galloping Hill RoadKenilworth, NJ 07033USA

Ben M. DunnUniversity of Florida College of MedicineDepartment of Biochemistry &Molecular BiologyP.O. Box 100245Gainesville, FL 32610-0245USA

Ernesto FreireJohns Hopkins UniversityDepartment of Biology3400 North Charles StreetBaltimore, MD 21218USA

Sandra GemmaUniversità degli Studi di SienaDipartimento Farmaco ChimicoTecnologicovia Aldo Moro, No. 253100 SienaItaly

Arun K. GhoshPurdue UniversityDepartments of Chemistry andMedicinal Chemistry560 Oval DriveWest Lafayette, IN 47907-2084USA

Christopher L. HamblettMerck Research Laboratories33 Avenue Louis PasteurBoston, MA 02115USA

and

Cowen and Company, LCCEquity Research2 International Place, 27th FloorBoston, MA 02100USA

Richard Heidebrecht, Jr.The Broad Institute7 Cambridge CenterCambridge, MA 02142USA

Lin HongCoMentis, Inc.865 Research ParkwayOklahoma City, OK 73104USA

Ulrich IserlohSchering-Plough Research Institute2015 Galloping Hill RoadKenilworth, NJ 07033USA

Olivier JoussonUniversity of TrentoCentre for Integrative Biology38100 TrentoItaly

John KayCardiff UniversitySchool of BiosciencesMuseum AvenueCardiff CF10 3US, WalesUK

Yoshiaki KisoKyoto Pharmaceutical UniversityDepartment of Medicinal ChemistryCenter for Frontier Research inMedicinal ScienceYamashina-kuKyoto 607-8412Japan

Urban LendahlMedical Nobel InstituteKarolinska InstituteDepartment of Cell and MolecularBiologySE 171 77 StockholmSweden

Johan LundkvistCNS/PAIN, Astrazeneca R&DNeuroscience DepartmentSE 151 85 SödertäljeSweden

Jürgen MaibaumNovartis Pharma AGInstitutes for BioMedical ResearchNovartis CampusCH-4056 BaselSwitzerland

Hiroaki MitsuyaNational Cancer InstituteCenter for Cancer ResearchHIV & AIDS Malignancy BranchThe Experimental Retrovirology SectionBuilding 10, Room 5A119000 Rockville PikeBethesda, MD 20892USA

and

Kumamoto University School of MedicineDepartment of Hematology & InfectiousDiseasesKumamoto 860-8556Japan

Michel MonodCentre Hospitalier UniversitaireVaudoisLaboratoire de Mycologie, BT422Service de Dermatologie1011 LausanneSwitzerland

Benito MunozMerck Research Laboratories33 Avenue Louis PasteurBoston, MA 02115USA

and

The Broad Institute7 Cambridge CenterCambridge, MA 02142USA

Utz ReichardUniversity Hospital of GoettingenDepartment of Medical Microbiologyand National Reference Center forSystemic Mycoses37075 GoettingenGermany

Adam J. RubenSanaria Inc.Vaccine Stabilization & Logistics9800 Medical Center DriveSuite 209ARockville, MD 20850USA

Sanjiv ShahMerck Research Laboratories33 Avenue Louis PasteurBoston, MA 02115USA

Suresh B. SinghVitae Pharmaceuticals502 West Office Center DriveFort Washington, PA 19034USA

Sukanto SinhaActiveSite Pharmaceuticals, Inc.1456 Fourth Street, Unit CBerkeley, CA 94710USA

Peter StaibLeibniz Institute for Natural ProductResearch and Infection Biology – HansKnoell InstituteJunior Research Group “FundamentalMolecular Biology of Pathogenic Fungi”D-07745 JenaGermany

Jordan TangOklahoma Medical ResearchFoundation and University of OklahomaHealth Science Center825 NE 13th StreetOklahoma City, OK 73104USA

Colin M. TiceVitae Pharmaceuticals502 West Office Center DriveFort Washington, PA 19034USA

Scott C. VirgilDirectorCalifornia Institute of TechnologyDepartment of ChemistryCaltech Center for Catalysis and Chemical Synthesis164-30, 1200 East California Blvd.Pasadena, CA 91125USA

Yuan-Fang WangGeorgia State UniversityDepartment of BiologyMolecular Basis of Disease ProgramP.O. Box 4010Atlanta, GA 30302-4010USA

Irene T. WeberGeorgia State UniversityDepartment of BiologyMolecular Basis of Disease ProgramP.O. Box 4010Atlanta, GA 30302-4010USA

Jeanette M. WoodS_BIO Pte Ltd1 Science Park Rd, #05-09The Capricorn, SingaporeScience Park IISingapore 117528Singapore

A Personal Foreword

The human genome features a family of 15 active aspartic acid proteases that play a central role in the pathogenesis of many human diseases. The therapeutic inhibition of these aspartic acid proteases could conceivably provide a basis for novel treatment of human diseases. The disease targets include renin for hypertension, pepsin for gastric ulcers, HIV protease for AIDS, β- and γ-secretases for Alzheimer’s disease, plasmepsins for malaria, cathepsin D and E for neoplastic diseases, and candida proteases for fungal infections. The catalytic aspartic acids in the active site motif are responsible for polarizing a bound water molecule that affects the proteolysis of the scissile bond of the substrate through a tetrahedral transition-state intermediate. Subsequently, mechanism-based design of nonhydrolysable dipeptide isosteres, mimicking the transition-state cleavage the scissile bond led to the evolution of potent substrate-derived inhibitors. Early studies on X-ray structures of fungal aspartic proteases bound to inhibitors, especially pepstatin-related structures, provided important molecular insights into the ligand-binding site interactions. This, in turn, provided important impetus for medicinal chemistry efforts toward the design and synthesis of a variety of transition-state isostere-derived peptidomimetic aspartyl protease inhibitors. Early efforts in the development of aspartic protease inhibitor drugs focused on the design of renin inhibitors for the treatment of hypertension. However, first-generation inhibitors were peptide-like, and due to their unfavorable pharmacological properties, early renin inhibitors never made it to clinical development. With the advent of AIDS and the discovery of human immunodeficiency virus (HIV) as the etiological agent, HIV-protease was recognized to play a critical role in the HIV life cycle. This led to massive research efforts in both academic and industrial laboratories in the quest for orally bioavailable protease inhibitor (PI) drugs as effective chemotherapeutics for AIDS. By the mid-1990s, the first aspartic acid protease inhibitor drug, saquinavir, received regulatory approval for the treatment of HIV/AIDS in combination with reverse transcriptase inhibitors. Soon after, a number of other first-generation HIV protease inhibitors were approved for use in highly active antiretroviral therapy (HAART). This was indeed a turning point in the management of HIV/AIDS. HAART treatment regimens significantly reduced mortality and improved the quality of life for HIV/AIDS patients. However, the emergence of multidrug-resistant HIV strains required the search for a better long-term treatment with novel drugs. New-generation PIs have been developed to address the issue of drug resistance.

The clinical success in the realm of HIV protease inhibitor drugs stimulated an intense effort to find new inhibitor drugs for other aspartic protease targets. The search for a renin inhibitor drug was reinvigorated. The design, development, and approval of the first orally bioavailable renin inhibitor, aliskiren, marked the beginning of a new treatment for hypertension. This development has intensified further design and development of novel classes of renin inhibitors. The design of γ-secretase and β-secretase inhibitors for the treatment of Alzheimer’s disease (AD) has also been a very active area of research in academic and pharmaceutical laboratories and several inhibitors are now in clinical development. Furthermore, drug design efforts against plasmepsins for malaria treatment are being actively made in many laboratories.

The X-ray crystal structures of numerous human aspartic acid proteases have now been determined at high resolution. In addition, detailed kinetic and subsite specificity studies provided in-depth knowledge of their catalytic mechanisms. These studies immensely contributed to the structure-based design and synthesis of a broad range of aspartic acid protease inhibitors for the treatment of many human diseases. Today, aspartic acid protease enzymes, as well as medicinal chemistry in the quest of novel inhibitor drugs, have become one of the most prolific areas of research.

This book is a comprehensive collection aimed at providing a thorough overview of the field of medically important aspartic acid proteases. It covers broad research areas with particular emphasis on the chemical and biological relevance of the targets, design of molecular probes, and problems and resolutions related to selectivity, toxicity, and oral bioavailability. Furthermore, it encompasses structure–activity studies, critical molecular insights from protein-ligand X-ray structures, and the development of various design tools. The first four chapters provide a general overview of aspartic acid proteases and their relevance to various disease targets. Chapters 5–9 describe the design, synthesis, X-ray structures, and clinical development of the first- and second-generation HIV-1 protease inhibitors for the treatment of HIV/AIDS. Chapter 9 provides a clinical perspective on protease inhibitors and their use in the treatment of HIV infection and AIDS. Chapters 10 and 11 cover the design and development of aliskiren as a first-in-class treatment of hypertension and current drug design efforts in renin inhibitors. The chemistry and biology of γ-secretase and inhibitors of γ-secretase for the treatment of AD are described in Chapters 12 and 13. The next four chapters address the emergence of β-secretase as a possible target for Alzheimer’s disease intervention, drug design efforts, development of tools for selectivity, issues of blood–brain barrier penetration, and the design of extensive structural classes of inhibitors. The plasmepsins as antimalarial targets and related inhibitor design efforts are described in Chapters 18 and 19. The last chapter provides an overview of fungal aspartic acid proteases and their relevance as targets for antifungal agents.

This book is written by the foremost experts in the field of aspartic acid proteases. Each chapter is well illustrated and provides an up-to-date account of the subject materials. This book will be an excellent resource to medicinal chemists, biochemists, pharmacologists, and to those working in related fields. I am very grateful to all authors for their insightful contribution to this work. I hope that their efforts and research expertise will pave the way for further advancement in the aspartic acid protease research field. I very much enjoyed working with Dr. Frank Weinreich, Dr. Nicola Oberbeckmann-Winter, and the Wiley-VCH editorial team. I personally want to express my sincere appreciation for their help, support, toward the completion of this work. I would like to thank Dr. Joseph Vacca for his initial efforts and help on this book. I am grateful to Dr. Jordan Tang, my friend and collaborator, for help and suggestions. I would also like to thank my research colleagues, Dr. Bruno Chapsal, Dr. Sean Fyvie, Mr. Zach Dawson, and Mr. David Anderson for their help with proofreading of chapters and Ms. Heather Miller for her help with manuscript preparation and organization. Finally, I wish to thank my family, my wife Jody, and my three children for their love, support, and inspiration. They are my spirit.

January 2010

Arun K. Ghosh, Purdue University

Part One

Overview of Aspartic Acid Proteases

1

Introduction to the Aspartic Proteinase Family

Ben M. Dunn

Abbreviations

BACE

β-amyloid cleaving enzyme

C-terminal

carboxyl terminal

D-G-I-L-G-L

amino acid sequence using single-letter code

HIV

human immunodeficiency virus

Nle

norleucine

Nph

para-nitrophenylalanine

N-terminal

amino terminal

PDB

Protein Data Bank

PfPM2

Plasmodium falciparum plasmepsin 2

Symbols

kcat/Km

characterizes the kinetics of cleavage of an enzyme–substrate pair

Kcat

turnover number of an enzyme

Km

Michaelis–Menten constant

1.1 Introduction

All cells, tissues, and organisms require proteolysis for the control of metabolism and growth. Even a virus, the smallest nucleic acid-based self-replicating organism, typically requires either host cell proteolysis or enzymes coded by its own genetic material to provide processing of initial viral gene products. Proteolysis is required to activate prohormones and other precursor molecules, to invade into cells and tissues, to release membrane-bound molecules, to provide a cascade effect in a variety of rapid response systems, to regulate cell growth, tissue homeostasis, remodeling, and renewal, and to stimulate cell division, to name but a few vital functions. This applies to pathogenic organisms as well as to normal cells and tissues.

Thus, it is not surprising that proteolytic enzymes from pathogens are targets for drug discovery or that creating molecules that will selectively block the activity of an enzyme from a pathogen, while not harming normal cellular function, is an ongoing endeavor in many pharmaceutical companies and academic labs around the world.

Classically, proteolytic enzymes have been divided into four groups based on their catalytic apparatus: aspartic, cysteine, metallo-, and serine proteases. However, recently, three new systems have been defined: the threonine-based proteosome system [1], the glutamate–glutamine system of eqolisin [2], and the serine–glutamate–aspartate system of sedolisin [3]. It remains to be seen if the proteases mentioned here represent the total spectrum of proteolytic mechanisms available in biology.

This book focuses on the aspartic proteinase family of proteolytic enzymes and specifically on several enzymes that are currently being pursued as drug targets for therapeutic intervention in humans. This chapter will describe several general features of these enzymes to provide a context in which to compare the specific examples found in the following chapters.

The aspartic proteinase family has a long and complicated history. In fact, one of the first processing involving enzyme action was discovered by accident. Legend has it that around 7000 BC, an Arabic traveler placed milk in a pouch made from the stomach of an animal, possibly a sheep or a cow, and set off on a journey across a desert. Different versions of the legend suggest that the traveler was either male or female. Upon reaching the destination, the traveler opened the pouch to find that the milk had coagulated and separated into curd and whey. Sampling the curd, the traveler decided that the process had been productive and it became possible to capture the essential nutrients of milk in a more concentrated and thus easier to carry form. Many centuries later, it was discovered that the enzyme renin, which is contained in the cells lining the stomach cavity of many ruminant animals, was responsible for the cleavage of kappa-casein proteins to cause precipitation that leads to the curd.