Apicomplexan Parasites -  - ebook

Apicomplexan Parasites ebook

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Opis

This handbook is the first dealing with the discovery of drugs directed against Apicomplexan Parasites. Amongst others, this group of endoparasites includes the causative agents of Malaria, Toxoplasmosis, and Babesiosis, the latter occurring mainly in animals. Written by renowned scientific experts from academia and industry, the book focuses on currentdrug development approaches for all apicomplexan diseases making it appealing to a large audience, ranging from research labs in academia to the human and veterinarian pharmaceutical industry. This work is the second volume of the new book series 'Drug Discovery in Infectious Diseases', edited by Prof. Dr Paul M. Selzer.

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Contents

Cover

Titles of the Series “Drug Discovery in Infectious Diseases”

Title Page

Copyright

Foreword

Preface

List of Contributors

Part One: Screening, Bioinformatics, Chemoinformatics, and Drug Design

Chapter 1: Drug Discovery Approaches Toward Anti-Parasitic Agents

Abstract

Drug Discovery Initiatives to Accelerate the Development of Novel Anti-Parasitic Drugs for Humans and Animals

Innovation from Anti-Parasitic Drug Discovery Approaches

The Process of Target-Based Drug Discovery

Examples of Successful Target-Based, Anti-Parasitic Drug Discovery Programs

Conclusion

References

Chapter 2: New Bioinformatic Strategies Against Apicomplexan Parasites

Abstract

Introduction

Databases and Methods

Conclusion

References

Chapter 3: Sorting Potential Therapeutic Targets in Apicomplexa

Abstract

Introduction

Protein Sorting in Apicomplexa: Ontogenesis and Phylogenesis

Conclusion

References

Chapter 4: Alternatives to Drug Development in the Apicomplexa

Abstract

Introduction

Infection and Immunity

Vaccination

Available Vaccines

Conclusions

Acknowledgments

References

Part Two: Metabolic Pathways and Processes Addressed by Current Drug-Discovery Approaches

Chapter 5: Energy Metabolism as an Antimalarial Drug Target

Abstract

Abbreviations

Introduction

Conclusion

Acknowledgments

References

Chapter 6: Polyamines in Apicomplexan Parasites

Abstract

Introduction

Conclusion

References

Chapter 7: The Reducing Milieu of Parasitized Cells as a Target of Antimalarial Agents: Methylene Blue as an Ethical Drug

Abstract

Introduction

Different Goals Require Different Antimalarial Agents

Pro-Oxidant Mechanisms for Preventing and Curing Malaria as an Acute Disease

Pharmaceutical Agents as Inhibitors and/or Substrates of Disulfide Reductases

Discussion

Conclusion

Acknowledgments

References

Chapter 8: Lipids as Drug Targets for Malaria Therapy

Abstract

Introduction

Malarial Lipids: Why are Glycerophospholipids Not Dispensable?

Glycerolipid Acquisition

How Crucial are these Metabolic Pathways?

Lipid-Based Antimalarial-Chemotherapy: Pharmacological Validation

Pharmacological Activity Performances of Choline Analogs

Mechanism by Which Choline Analogs Exert Their Antimalarial Activity

Current Status of Development

Conclusion

Acknowledgments

References

Chapter 9: Targeting Apicoplast Pathways in Plasmodium

Abstract

Introduction

Housekeeping Functions

Metabolic Pathways

Conclusion

References

Chapter 10: Lipoic Acid Acquisition and Glutathione Biosynthesis in Apicomplexan Parasites

Abstract

Introduction

The Biological Role of LA

Conclusion

References

Chapter 11: Antimalarial Drugs and Molecules Inhibiting Hemozoin Formation

Abstract

Introduction

Drugs/Molecules Inhibiting Hemozoin Formation

Conclusion

References

Chapter 12: Exploiting the Vitamin Metabolism of Apicomplexa as Drug Targets

Abstract

Introduction

Vitamin B1 Metabolism: A Novel Drug Target?

Vitamin B6 Metabolism in Apicomplexa

Conclusion

Acknowledgments

References

Chapter 13: Vitamin Biosynthetic Pathways, the PLP Synthase Complex, and the Potential for Targeting Protein–Protein Interaction

Abstract

Introduction

Protein–Protein Interaction

Isothermal Titration Calorimetry

PLP Synthase and Characterization of its Protein Interfaces by ITC

Conclusion: Chances for Drug Design

Acknowledgments

References

Chapter 14: Targeting Prokaryotic Enzymes in the Eukaryotic Pathogen Cryptosporidium

Abstract

Introduction

The Nucleotide Biosynthetic Pathways of Cryptosporidium

CpIMPDH is a Target for Anticryptosporidial Drugs

Targeting the Pyrimidine Nucleotide Pathways

Conclusion

References

Part Three: Drug Targets in Apicomplexan Parasites

Chapter 15: Novel Apicomplexan Phosphatases and Immunophilins as Domain-Specific Drug Targets

Abstract

Introduction

“Classical” Protein Phosphatases and their Inhibitors

Unique, Authenticated Phosphatases: PP3, PP5, PP7, PPα, and PP2C

Classical Immunophilins: CyP and FKBP

A Potentially Novel Structural Center in PfFKBP35

Novel Dual-Family Immunophilin, TgFCBP57

Conclusion

References

Chapter 16: Dehydrogenases and Enzymes of the Mitochondrial Electron Transport Chain as Anti-Apicomplexan Drug Targets

Abstract

Introduction

Enzymes of the Electron Transport Chain

Dehydrogenases of the TCA Cycle as Drug Targets

Conclusion

References

Chapter 17: Calcium-Dependent Protein Kinases as Drug Targets in Apicomplexan Parasites

Abstract

Introduction

Biological Functions of the Individual CDPKs in the Plasmodial Life Cycle

Regulatory Controls of CDPKs

Protein Kinase Inhibitors Against CDPKs

Conclusion

References

Chapter 18: Protein Acylation: New Potential Targets for Intervention Against the Apicomplexa

Abstract

Introduction

Protein Acylation: N-Myristoylation and S-Palmitoylation

Potential of N-Myristoylation and S-Palmitoylation as Targets for Intervention

Conclusion

Acknowledgments

References

Chapter 19: Drugs and Drug Targets in Neospora caninum and Related Apicomplexans

Abstract

Introduction

Mode of Action and Drug Targets

Conclusion

References

Part Four: Compounds

Chapter 20: Subversive Substrates of Glutathione Reductases from Plasmodium falciparum-Infected Red Blood Cells as Antimalarial Agents

Abstract

Introduction

Antimalarial Drugs

The Dual Prodrug Approach

Conclusion

Acknowledgments

References

Chapter 21: Ferroquine: A Concealed Weapon

Abstract

Introduction

From A Ferrocene Strategy to Ferroquine

Antimalarial Activity of Ferroquine

Ferroquine as a New Antimalarial

Conclusion

Acknowledgments

References

Chapter 22: Current Aspects of Endoperoxides in Antiparasitic Chemotherapy

Abstract

Introduction

First-Generation Endoperoxides

Second-Generation Endoperoxides

Mechanism of Endoperoxide Action

Endoperoxide-Based Combinations

Endoperoxide Resistance

Conclusion

References

Chapter 23: Plasmodium Hsp90 as an Antimalarial Target

Abstract

Introduction

Biochemistry and Structure of Hsp90

Conclusion

References

Chapter 24: Drug Discovery Against Babesia and Toxoplasma

Abstract

Introduction

Current Status of Chemotherapy

Chemotherapeutic Targets for Babesia and Toxoplasma

Conclusion

References

Chapter 25: Search for Drugs and Drug Targets against Babesia bovis, Babesia bigemina, Babesia caballi, and Babesia (Theileria) equi

Abstract

Introduction

The First Approaches Start at the Bench

Compounds Successfully Tested In Vitro

Recently Discovered or Proposed Drug Targets

Conclusion

References

Chapter 26: Orlistat: A Repositioning Opportunity as a Growth Inhibitor of Apicomplexan Parasites?

Abstract

Repositioning of Actives: New, But Not Really New

Chemistry and Synthesis of Orlistat

Orlistat's Modes of Action

Orlistat's Effect on Plasmodium falciparum and Toxoplasma gondii

Bioinformatic Analysis for Potential Orlistat Targets in Apicomplexan Parasites

Conclusion

Acknowledgment

References

Chapter 27: Recent Drug Discovery Against Cryptosporidium

Abstract

Introduction

The Discovery of the First Treatments

Conclusion

References

Index

Titles of the Series “Drug Discovery in Infectious Diseases”

Selzer, P. M. (ed.)

Antiparasitic and Antibacterial Drug Discovery

From Molecular Targets to Drug Candidates

2009

ISBN: 978-3-527-32327-2

Forthcoming Topics of the Series

HelminthsKinases in ParasitesKinetoplastida

Related Titles

Sansonetti, P. (ed.)

Bacterial Virulence

Basic Principles, Models and Global Approaches

2010

ISBN: 978-3-527-32326-5

Kaufmann, S. H. E., Walker, B. D. (eds.)

AIDS and Tuberculosis

A Deadly Liaison

2010

ISBN: 978-3-527-32270-1

Krämer, R., Jung K. (eds.)

Bacterial Signaling

2010

978-3-527-32365-4

Schaible, U. E., Haas, A. (eds.)

Intracellular Niches of Microbes

A Pathogens Guide Through the Host Cell

2009

ISBN: 978-3-527-32207-7

The Editors

Volume Editor:

Prof. Dr. Katja Becker

University of Giessen

Nutritional Biochemistry

Heinrich-Buff-Ring 26-32

35392 Gießen

Germany

[email protected]

Series Editor:

Prof. Dr. Paul M. Selzer

Intervet Innovation GmbH

BioChemInformatics

Zur Propstei

55270 Schwabenheim

Germany

[email protected]

Cover

Microscopic image of the malaria parasite Plasmodium falciparum: with kind permission of the Institut Pasteur, Paris, France.

Ribbon representation of the plasmodial flavoenzyme glutathione reductase - a drug target of Plasmodium falciparum - with the bound cofactor flavin adenine dinucleotide in ball and stick representation (PDB-Code 1ONF): picture courtesy of Dr. Richard J. Marhöfer and Prof. Dr. Paul M. Selzer, Intervet Innovation GmbH, Schwabenheim, Germany.

The parasite and the protein structure are connected by protein crystals of P. falciparum glutathione reductase: courtesy of Prof. Dr. Katja Becker, Justus Liebig University, Giessen, Germany.

Limit of Liability/Disclaimer of Warranty: While the publisher and authors have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.

© 2011 WILEY-VCH Verlag & Co. KGaA,

Boschstr. 12, 69469 Weinheim, Germany

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley's global Scientific, Technical, and Medical business with Blackwell Publishing.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978-3-527-32731-7

Epub ISBN: 978-3-527-63390-6

Foreword

The Apicomplexa are obligate intracellular eukaryotic parasites that infect metazoans, and bear a huge impact on both animal and human health worldwide. Since both the parasites and the hosts in which they cause disease belong to the eukaryotic kingdom – and despite the fact that astounding chemotherapeutic success has been achieved, notably with chloroquine prior to the emergence and spread of resistance in malarial parasites which rendered the drug useless in most parts of the world – it has been argued that selective intervention based on small molecules would be more difficult to achieve than in the case of bacterial pathogens. Phylogenetic research conducted during recent decades, however, has revealed that the Apicomplexa diverged very early from the main branch of Eukaryotes, and arose from a complex evolutionary path that involved secondary endosymbiosis. It is now well established that the enormous phylogenetic distance between these parasites and their metazoan hosts is reflected by apicomplexan-specific features in terms of organelle complement, biochemical pathways, and the properties of specific enzymes (even in cases where orthologous molecules are present in the vertebrate host).

The widely encompassing collection of chapters that constitute the present volume provides a comprehensive snapshot of ongoing drug discovery activities aimed at exploiting such divergences. The topics covered range from bioinformatics and chemogenomics approaches for target selection, to the characterization of compounds with established parasiticidal activity. A large part of the book is composed of chapters in which apicomplexan features not found in vertebrates are discussed from the perspective of their potential as targets for chemotherapy. These include the apicoplast, the remnant of a photosynthetic organelle retaining pathways that are absent from mammalian cells, and the calcium-dependent protein kinases, which possess a domain organization found only in plants and Alveolates, the phylum that includes the Apicomplexa and Ciliates – are discussed from the perspective of their potential as targets for chemotherapy. Other chapters are devoted to the exploitation of more subtle differences between orthologous enzymatic systems between host and parasite, for example heat shock proteins or the machineries pertaining to redox metabolism or cell cycle control.

Undoubtedly, much remains to be uncovered. It is natural that drug-discovery efforts are largely focused on those enzymes that can be recognized as potential targets. This, however, leaves behind the large fraction of the parasite proteomes (approximately 60% in the case of Plasmodium, though a similar situation applies to other Apicomplexa) currently annotated as “hypothetical proteins,” and which may hide prime targets, despite the fact that chemogenomics approaches fill this gap to some extent.

Nevertheless, the contents of this book provides glimmers of hope. Some compounds – for example, ferroquine and bi-cationic inhibitors of phospholipid metabolism – have entered clinical trials against malaria, although whether or not novel drugs based on these compounds will reach the market in the not-too-distant future remains to be seen. It is to be hoped that other projects discussed in this book, many of which are in the exploratory/discovery phase, will be pursued to development. The relatively recent implementation of specific institutions to promote the translation of early drug discovery projects into drug development for previously neglected diseases, such as the Medicines for Malaria Venture (MMV) and the Drugs for Neglected Diseases Initiative (DNDi) Public-Private Partnerships, is playing an important part in this endeavor.

Whilst the apicomplexan parasites remain formidable foes, the variety of approaches and targets addressed in this book demonstrate that the “anti-apicomplexan drug discovery pipeline” is well primed, largely as a result of efforts devoted to parasite genomics during the previous decades, and testifies to the vitality of ongoing research in the “arms race” against these pathogens. May this book stimulate interest and dedication of the research community, and thus contribute to successes in this race.

Lausanne

September 2010

Christian Doerig

Preface

Ever since the discovery of apicomplexan parasites by Anthony von Leeuwenhoek, who first detected the oocysts of Eimeria in a rabbit in 1674, the term Apicomplexa has comprised a growing number of protists causing severe and often neglected diseases in humans and animals worldwide. The Apicomplexa possess a unique plastid-like organelle, the apicoplast, and an apical complex structure that is involved in host cell invasion. Many of these parasites undergo complex developmental cycles, and exist in both intracellular and extracellular forms in various hosts. Diseases caused by apicomplexan parasites include, among others, malaria, toxoplasmosis, cryptosporidiosis, coccidiosis, babesiosis, and theileriosis. Worldwide, Plasmodium accounts for up to 500 million clinical cases of malaria each year, with 1–2 million deaths, mainly in young children, while Toxoplasma gondii infects approximately one-third of the human population. Although most Toxoplasma infections are benign, severe opportunistic diseases affect immunodeficient or immunosuppressed individuals or children infected in utero. Likewise, Cryptosporidium causes infections that are especially harmful for immunocompromised individuals. Coccidiosis, a disease mainly of chicken and other poultry, caused by Eimeria spp., is a major problem for the global food industry, not least in developed countries. In this case, the annual global loss has been estimated to be in the range of US$0.3–3 billion, and improved strategies for the effective control of eimerian parasites are required. Other parasites in this family include Babesia and Theileria, which cause a hemolytic disease resembling malaria in cattle and sheep, and a severe and often fatal lymphoproliferative disease in cattle, respectively.

For many parasitologists it is difficult to believe that it has not yet been possible to combat apicomplexan parasites – in spite of intense drug discovery and vaccine development efforts, of numerous available target molecules, novel highly active drug candidates, and approved drugs for which antiparasitic action could be an additional indication. Whilst it has always been difficult for scientists to influence political and industrial priorities and decision making, it is nonetheless possible for the scientific community to make continuous joint efforts and suggestions, with the hope of improving the situation for patients suffering from infections. Over the past years, these efforts have been increasingly supported by initiatives not only to commonly share genomic, transcriptomic, proteomic, and structural data, but also to share and exchange materials and methods as well as chemical compound libraries, data acquired from inhibitor screens, and the results of chemical genetics studies.

This book represents the second volume of a series dealing with drug discovery and development approaches against infectious diseases. Whereas, the first volume “Antiparasitic and Antibacterial Drug Discovery: From Molecular Targets to Drug Candidates” dealt with general drug discovery approaches towards diseases caused by protozoans, multicellular parasites, and bacteria, the present volume is solely dedicated to the Apicomplexa. This decision was made not only on the basis of the worldwide importance of the respective organisms and the diseases they cause, but also because the apicoplast harbors unique enzymes and metabolic pathways that represent highly attractive drug targets. Both aspects strongly suggest the coordination of drug discovery and development strategies against different Apicomplexa.

We are grateful to all authors who contributed articles to this book, for their excellent work and their constructive cooperation. We furthermore are indebted to the Justus Liebig University Giessen, Germany and the Intervet Innovation GmbH, Schwabenheim, Germany for their support and inspiration over the past months. In particular, we wish to thank Timothy Bostick for his excellent editorial assistance.

Giessen and Schwabenheim

September 2010

Katja Becker and

Paul M. Selzer

List of Contributors

Heike Adler

University of Heidelberg

Biochemistry Center

Im Neuenheimer Feld 328

69120 Heidelberg

Germany

Uday Bandyopadhyay*

Indian Institute of Chemical Biology

Division of Infectious Diseases and

Immunology

4, Raja S. C. Mullick Road, Jadavpur

Kolkata 700032

India

E-mail: [email protected]

Sailen Barik*

University of South Alabama

College of Medicine

Department of Biochemistry and

Molecular Biology, MSB 2370

307 University Blvd

Mobile, AL 36688-0002

USA

Present address:

Cleveland State University

Center for Gene Regulation in Health

and Disease

Room SR351, 2121 Euclid Avenue

Cleveland, OH 44115

USA

E-mail: [email protected]

Stefan Baumeister

University of Marburg

FB Biology/Parasitology

35043 Marburg

Germany

Katja Becker*

Justus-Liebig-University

Interdisciplinary Research Center

Chair of Nutritional Biochemistry

Heinrich-Buff-Ring 26-32

35392 Giessen

Germany

E-mail: [email protected]

Christophe Biot*

Université de Lille 1

Unité de Catalyse et Chimie du Solide

UMR CNRS 8181, ENSCL

Bâtiment C7, B.P. 90108

59652 Villeneuve d.Ascq Cedex

France

E-mail: [email protected]

and

Université de Lille1

Unité de Glycobiologie Structurale et

Fonctionnelle

CNRS UMR 8576, IFR 147

59650 Villeneuve d.Ascq Cedex

France

Subir Biswas

Central Drug Research Institute

Division of Molecular and Structural

Biology M.G. Road

226001 Lucknow

India

Sabine Bork-Mimm*

Bavarian Health and Food Safety

Authority (Bayerisches Landesamt für

Gesundheit und

Lebensmittelsicherheit, LGL)

Department Oberschleissheim

(Dienststelle Oberschleissheim)

Veterinaerstrasse 2

85764 Oberschleissheim

Germany

E-mail: [email protected]

Conor R. Caffrey

University of California San Francisco

Department of Pathology and the

Sandler Center for Drug Discovery

Byers Hall, CA 94158

USA

Sergio Caldarelli

Université Montpellier 2

Institut des Biomolécules Max

Mousseron

UMR 5247 CNRS-UM1&2

CC1705, Place E. Bataillon

34095 Montpellier Cedex 5

France

Nicholas D.P. Cosford

Sanford Burnham Medical Research

Institute

10901 North Torrey Pines Road

La Jolla, CA 92037

USA

Thomas Dandekar*

University of Würzburg

Department of Bioinformatics

Biocenter

Am Hubland

97074 Würzburg

Germany

E-mail: [email protected]

Elisabeth Davioud-Charvet*

Biochemistry Center of the University of Heidelberg

Im Neuenheimer Feld 328

69120 Heidelberg

Germany

and

University of Strasbourg

European School of Chemistry,

Polymers and Materials (ECPM)

UMR7509 CNRS

25 rue Becquerel

67087 Strasbourg Cedex 2

France

E-mail: [email protected]

Bianca Derrer

University Hospital Heidelberg

Department of Infectious Diseases,

Parasitology

Im Neuenheimer Feld 326

69120 Heidelberg

Germany

Sumanta Dey

Indian Institute of Chemical Biology

Division of Infectious Diseases and

Immunology

4, Raja S. C. Mullick Road, Jadavpur

Kolkata 700032

India

Daniel Dive

Université Lille Nord de France

Institut Pasteur de Lille

UMR CNRS 8024, CIIL, Inserm U 1019

1 rue du Pr. Calmette

59019 Lille Cedex

France

Laurent Fraisse

Sanofi-Aventis Recherche et

Developpement

Unité thérapeutique des maladies infectieuse

195 route d.Espagne

31036 Toulouse

France

Karin Fritz-Wolf

Max Planck Institute for Medical

Research

Department of Biophysics

Jahnstraße 29

69120 Heidelberg

Germany

Gilles Gargala*

University of Rouen

Faculty of Medicine & Pharmacy

Department of Parasitology

Rouen

France

E-mail: [email protected]

Suresh Kumar Gorla

Brandeis University

Department of Biology

Waltham, MA 02454

USA

Robin Das Gupta

University of Bern

Institute of Cell Biology

Baltzerstrasse 4

3012 Bern

Switzerland

Saman Habib*

Central Drug Research Institute

Division of Molecular and Structural

Biology M.G. Road

226001 Lucknow

India

E-mail: [email protected]

Anja R. Heckeroth

Intervet Innovation GmbH

Profiling Antiparasitic

Zur Propstei

55270 Schwabenheim

Germany

Christian Hedberg

Department of Chemical Biology

Max Planck Institute of Molecular

Physiology

Otto-Hahn-Strasse 11

44227 Dortmund

Germany

Lizbeth Hedstrom*

Brandeis University

Departments of Biology and Chemistry

Waltham, MA 02454

USA

E-mail: [email protected]

Andrew Hemphill*

University of Berne

Institute of Parasitology

Länggass-Strasse 122

3012 Berne

Switzerland

E-mail: [email protected]

Jan A. Hiss*

Eidgenössische Technische Hochschule

(ETH)

Department of Chemistry and Applied

Biosciences

Zürich

Switzerland

E-mail: [email protected]

Ikuo Igarashi*

Obihiro University of Agriculture and

Veterinary Medicine

National Research Center for Protozoan

Diseases

Obihiro, Hokkaido 080-8555

Japan

E-mail: [email protected]

Corey Johnson

Brandeis University

Graduate Program in Chemistry

Waltham, MA 02454

USA

Esther Jortzik

Justus-Liebig-University

Interdisciplinary Research Center

Heinrich-Buff-Ring 26-32

35392 Giessen

Germany

Barbara Kappes*

University Hospital Heidelberg

Department of Infectious Diseases,

Parasitology

Im Neuenheimer Feld 326

69120 Heidelberg

Germany

E-mail: [email protected]

Denis Matovu Kasozi

University of Gießen (Justus-Liebig-University)

Interdisciplinary Research Center

Chair of Nutritional Biochemistry

Heinrich-Buff-Ring 26-32

35392 Giessen

Germany

Jihan Khan

Brandeis University

Graduate Program in Chemistry

Waltham, MA 02454

USA

Dominik Kugelstadt

University Hospital Heidelberg

Department of Infectious Diseases

Parasitology

Im Neuenheimer Feld 326

69120 Heidelberg

Germany

and

Genzyme Virotech GmbH

Löwenplatz 5

65428 Rüsselsheim

Germany

Don Antoine Lanfranchi

University of Strasbourg

European School of Chemistry,

Polymers and Materials (ECPM)

UMR7509 CNRS

25 rue Becquerel

67087 Strasbourg Cedex 2

France

Kai Lüersen

Westfalian Wilhelms University

Institute for Zoophysiology

Hindenburgplatz 55

48143 Münster

Germany

Richard J. Marhöfer

Intervet Innovation GmbH

BioChemInformatics

Zur Propstei

55270 Schwabenheim

Germany

Peter Meissner

University of Ulm

Department of Pediatrics

89075 Ulm

Germany

Thorsten Meyer

Intervet Innovation GmbH

Lead Optimization

Zur Propstei

55270 Schwabenheim

Germany

Christian Miculka

Intervet Innovation GmbH

Zur Propstei

55270 Schwabenheim

Germany

Present address:

Merial Limited

3239 Satellite Boulevard

Duluth, GA 30096

USA

Snober S. Mir

Central Drug Research Institute

Division of Molecular and Structural

Biology M.G. Road

226001 Lucknow

India

Ingrid B. Müller*

Bernhard-Nocht-Institute for Tropical

Medicine

Biochemical Parasitology

Bernhard-Nocht-Str. 74

20359 Hamburg

Germany

E-mail: [email protected]

Joachim Müller

University of Berne

Institute of Parasitology

Länggass-Strasse 122

3012 Berne

Switzerland

Norbert Müller

University of Berne

Institute of Parasitology

Länggass-Strasse 122

3012 Berne

Switzerland

Sylke Müller*

University of Glasgow

Institute of Infection, Immunity and

Inflammation

College of Medical, Veterinary and Life

Sciences

120 University Place

Glasgow G12 8TA

UK

E-mail: [email protected]

Eva-Maria Patzewitz

University of Glasgow

Institute of Infection, Immunity and

Inflammation

College of Medical, Veterinary and Life

Sciences

120 University Place

Glasgow G12 8TA

UK

Diana Penarete

Université Montpellier 2

Dynamique des Interactions

Membranaires Normales et

Pathologiques

UMR 5235 CNRS-UM2

CC107, Place E. Bataillon

34095 Montpellier Cedex 5

France

Suzanne Peyrottes

Université Montpellier 2

Institut des Biomolécules Max

Mousseron

UMR 5247 CNRS-UM1&2

CC1705, Place E. Bataillon

34095 Montpellier Cedex 5

France

Bruno Pradines

Institut de Recherche Biomédicale des

Armées, Antenne de Marseille

Unité de Recherche en Biologie et

Epidémiologie Parasitaires

URMITE-UMR 6236

Allée duMédecin Colonel Jamot, Parc le

Pharo, BP 60109

13262 Marseille Cedex

France

G. Sridhar Prasad

CalAsia Pharmaceuticals, Inc.

6330 Nancy Ridge Drive, Suite 102

San Diego, CA 92121

USA

Stefan Rahlfs

Justus-Liebig-University

Interdisciplinary Research Center

Chair of Nutritional Biochemistry

Heinrich-Buff-Ring 26-32

35392 Giessen

Germany

Andreas Rohwer

Intervet Innovation GmbH

BioChemInformatics

Zur Propstei

55270 Schwabenheim

Germany

Jean-François Rossignol

Stanford University School of Medicine

Division of Gastroenterology & Hepatology

Department of Medicine

Stanford, CA

USA

and

University of Oxford

Exeter College

Glycobiology Institute

Department of Biochemistry

Oxford

UK

Joana M. Santos

University of Geneva

CMU

Department of Microbiology and

Molecular Medicine

1 Rue Michel-Servet

1211 Geneva 4

Switzerland

Theo P.M. Schetters*

Microbiology R&D Department

Intervet/Schering-Plough Animal

Health

Wim de Korverstraat 35

5831 Boxmeer

The Netherlands

E-mail: [email protected]

and

University Montpellier I

LBCM, UFR Pharmacy

Av. Charles Flahault 15

34093 Montpellier Cedex 5

France

R. Heiner Schirmer*

University of Heidelberg

Biochemistry Center

Im Neuenheimer Feld 328

69120 Heidelberg

Germany

E-mail: [email protected]

Gisbert Schneider

Eidgenössische Technische Hochschule

(ETH)

Department of Chemistry and Applied

Biosciences

Zürich

Switzerland

Frank Seeber

Robert-Koch-Institut

Nordufer 20

13353 Berlin

Germany

Paul M. Selzer*

Intervet Innovation GmbH

BioChemInformatics

Zur Propstei

55270 Schwabenheim

Germany

E-mail: [email protected]

J. Edward Semple

University of Oxford

Exeter College

Glycobiology Institute

Department of Biochemistry

Oxford

UK

Lisa Sharling

University of Georgia

Center for Tropical and Emerging

Global Diseases

500 D.W. Brooks Drive

Athens, GA 30602

USA

Irmgard Sinning

Biochemiezentrum der Universität

Heidelberg BZH

Im Neuenheimer Feld 328

69120 Heidelberg

Germany

Dominique Soldati-Favre*

University of Geneva

CMU

Department of Microbiology and

Molecular Medicine

1 Rue Michel-Servet

1211 Geneva 4

Switzerland

E-mail: [email protected]

Andrew V. Stachulski

University of Oxford

Exeter College

Glycobiology Institute

Department of Biochemistry

Oxford

UK

Janet Storm

University of Glasgow

Institute of Infection, Immunity and

Inflammation

College of Medical, Veterinary and Life

Sciences

120 University Place

Glasgow G12 8TA

UK

Boris Striepen

University of Georgia

Center for Tropical and Emerging

Global Diseases and the Department of

Cellular Biology

500 D.W. Brooks Drive

Athens, GA 30602

USA

Xin Sun

Brandeis University

Graduate Program in Biochemistry

Waltham, MA 02454

USA

Mohamad Alaa Terkawi

Obihiro University of Agriculture and

Veterinary Medicine

National Research Center for Protozoan

Diseases

Obihiro, Hokkaido 080-8555

Japan

Ivo Tews*

University of Southampton

School of Biological Sciences

Institute for Life Sciences (IfLS)

B85, Highfield Campus

Southampton SO17 1BJ

UK

E-mail: [email protected]

Hon Q. Tran

Intervet Innovation GmbH

BioChemInformatics

Zur Propstei

55270 Schwabenheim

Germany

Henri J. Vial*

Université Montpellier 2

Dynamique des Interactions

Membranaires Normales et

Pathologiques

UMR 5235 CNRS-UM2

CC107, Place E. Bataillon

34095 Montpellier Cedex 5

France

E-mail: [email protected]

Rolf D. Walter

Bernhard-Nocht-Institute for Tropical

Medicine

Biochemical Parasitology

Bernhard-Nocht-Str. 74

20359 Hamburg

Germany

Sharon Wein

Université Montpellier 2

Dynamique des Interactions

Membranaires Normales et

Pathologiques

UMR 5235 CNRS-UM2

CC107, Place E. Bataillon

34095 Montpellier Cedex 5

France

Carsten Wrenger*

Bernhard-Nocht-Institute for Tropical

Medicine

Biochemical Parasitology

Bernhard-Nocht-Str. 74

20359 Hamburg

Germany

E-mail: [email protected]

Ke Xiao

University of Würzburg

Department of Bioinformatics,

Biocenter

Am Hubland

97074 Würzburg

Germany

Kathleen Zocher

Justus-Liebig-University

Interdisciplinary Research Center

Chair of Nutritional Biochemistry

Heinrich-Buff-Ring 26-32

35392 Giessen

Germany

Part One

Screening, Bioinformatics, Chemoinformatics, and Drug Design

Chapter 1

Drug Discovery Approaches Toward Anti-Parasitic Agents

Andreas Rohwer, Richard J. Marhöfer, Conor R. Caffrey, and Paul M. Selzer1

1Corresponding author

Abstract

Parasitic diseases afflict hundreds of millions of people worldwide, and are a major issue in animal health. Because most drugs available today are old and have many limitations, novel drugs for the treatment of human and animal parasitic diseases are urgently needed. Modern research disciplines such as genomics, proteomics, metabolomics, chemogenomics, and other “-omics” technologies improve the quality of the drug discovery process and influence the design of novel anti-parasitic agents. These include the application of high-throughput technologies such as DNA/RNA sequencing, microarrays, mass spectrometry, high-throughput screening, and bio/chemoinformatics. Here, an overview is provided of the drug discovery workflow, and the steps employed to generate novel drug candidates with anti-parasitic activity are briefly described.

Drug Discovery Initiatives to Accelerate the Development of Novel Anti-Parasitic Drugs for Humans and Animals

Infectious diseases, including those caused or transmitted by parasites, are responsible for substantial morbidity and mortality worldwide and affect several billion people globally, particularly in developing countries [1]. Until recently, infectious diseases were viewed as a problem of the past; however, the emergence of drug-resistant organisms makes the need for new drugs or vaccines more important than ever before. Moreover, as these diseases predominantly afflict inhabitants of poor countries, drug discovery efforts are minimal due to the lack of returns on investment. Accordingly, diseases such as malaria, leishmaniasis, Chagas disease, elephantiasis, or schistosomiasis are often called “neglected diseases” [2].

More recently, the growing realization of the humanitarian and economic consequences of neglected diseases in poor countries has spurred the establishment of new organizations specifically focused on novel anti-parasitic drug development [3–5]. Collaborations between the pharmaceutical industry, specialized academic drug discovery centers, and public–private partnerships (PPPs) have been initiated to support anti-parasitic drug discovery and development programs (Figure 1.1).

Figure 1.1 Research and development activities to fight neglected diseases. Anti-parasitic research and development programs were initiated by academic R&D centers, public–private partnerships, and the pharmaceutical industry. Intensive collaboration is key to optimizing R&D output.

Public–private partnerships focus to combine the skills and resources of academia, the pharmaceutical industry, and contract research teams, with the goal of generating independent research and development (R&D) consortia. Well-known initiatives are the World Health Organization's Special Program for Research and Training in Tropical Diseases (WHO/TDR; http://www.who.int/tdr), the Drugs for Neglected Diseases initiative (DNDi; http://www.dndi.org), the Institute for One World Health (iOWH; http://www.oneworldhealth.org), and the Bill & Melinda Gates Foundation (B&MGF; http://www.gatesfoundation.org). For instance, the DNDi has built a virtual, not-for profit R&D organization for developing new drugs against kinetoplastid diseases, which include human African trypanosomiasis, visceral leishmaniasis, and Chagas disease. The partners of DNDi are Doctors Without Borders, the Oswaldo Cruz Foundation of Brazil, the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health in Malaysia, the Pasteur Institute in France, and the WHO/TDR. DNDi has already registered two products; in 2007 and 2008, respectively, the antimalarial drugs fixed-dose artesunate-amodiaquine (AS/AQ) and fixed-dose artesunate-mefloquine (AS/MQ) were launched [2].

Development projects organized by PPPs are often supported by the pharmaceutical industry itself. One particular project in the public eye is the Accelerating Access Initiative (AAI; http://www.ifpma.org/health/hiv/health_aai_hiv.aspx), a global initiative to broaden access to and ensure affordable and safe use of drugs for HIV/AIDS-related illnesses. Related programs, such as the Global Alliance to Eliminate Lymphatic Filariasis (GAELF; http://www.filariasis.org) and the Medicines for Malaria Venture (MMV; http://www.mmv.org), exist for many parasitic diseases. In addition, the pharmaceutical industry also invests directly in anti-parasitic research activities, with many companies having established state-of-the-art research facilities that concentrate exclusively on the development of drugs and vaccines for neglected diseases. Prominent research centers include the Novartis Institute for Tropical Diseases (NITD; http://www.novartis.com/research/nitd), the GlaxoSmithKline Drug Discovery Center for Diseases of the Developing World (DDW; http://www.gsk.com), or the MSD Wellcome Trust Hilleman Laboratories (http://www.hillemanlaboratories.in).

Needless to say, another major source of novel drugs in anti-parasitics stems from the extensive research activities in academic facilities. A relatively recent trend has been the foundation of academic drug discovery centers that focus exclusively on R&D in the field of neglected diseases. These aim to translate basic biomedical research into candidate medicines for neglected diseases. Examples are the Drug Discovery Unit of the University of Dundee (DDU; http://www.drugdiscovery.dundee.ac.uk), the Sandler Center for Drug Discovery (formerly Sandler Center for Basic Research in Parasitic Diseases) at the University of California San Francisco (http://www.sandler.ucsf.edu), and the Seattle Biomedical Research Institute (SBRI; http://www.sbri.org).

It is worth noting that anti-parasitic drug R&D programs are not confined to human medicine. Indeed, the animal health industry also performs intensive research on novel anti-parasitics [6–8]. This is important, as the situation of people in developing countries suffering from neglected diseases is aggravated by drastic economic losses in agriculture due to parasitic infections in farm animals. In this context, some animal health companies support developing countries in the framework of corporate social responsibility activities. For example, Intervet/Schering-Plough Animal Health (ISPAH; http://www.intervet.com) has an ongoing cooperation with the Indian non-governmental organization Bharatiya Agro Industries Foundation (BAIF; http://www.baif.org.in), whereby poor farmers in India have access to ISPAH's range of livestock products, including vaccines and anti-parasitic agents. It is expected that more than two million rural families could benefit from this project.

Innovation from Anti-Parasitic Drug Discovery Approaches

A parasite is defined as an animal that lives completely at the expense of plants, other animals, or humans [9]. In general, parasites are much smaller than their hosts, show a high degree of specialization for their mode of life, and reproduce more quickly and in greater numbers than their hosts. Parasites belong to a wide range of biologically diverse organisms and, based on their interactions with their hosts, are often classified into three categories: parasitic protozoa; endo-parasites; and ecto-parasites [7, 9].

1. Parasitic protozoa are unicellular microorganisms that infect humans or animals and either live extra- or intracellularly. Representatives include Plasmodium falciparum, Trypanosoma cruzi, and Leishmania donovani; causing malaria, Chagas disease, and leishmaniasis, respectively [9].

2. Endo-parasites are mainly multicellular helminthes that have adapted to live in the host's gastrointestinal tract, or systemically. Well-known endo-parasites are the nematode Brugia malayi, the causative agent of lymphatic filariasis, and the blood-fluke Schistosoma mansoni, which causes schistosomiasis [9, 10].

3. Ecto-parasites are parasitic organisms that live on the surface of their hosts. In most cases, ecto-parasites do not cause fatal maladies by themselves but affect the health of their hosts by transmitting pathogenic viruses, bacteria, or protozoa [9, 11]. Taxonomically, the majority of ecto-parasites belong to the phylum Arthropoda, and include important organisms such as fleas, flies, and ticks.

Historically, all marketed anti-parasitic products have been discovered by screening synthetic and natural compounds against intact parasites, either in culture or in animal models [12]. Such physiology-based assays, bioassays, or phenotypic assays involve parasites cultured in vitro, and exist for many different protozoa, endo-parasites, and ecto-parasites [13–16]. The main benefit of testing candidate compounds directly on whole organisms is that compounds with anti-parasitic activity are immediately apparent, suggesting that they possess the physico-chemical properties that allow them to penetrate the membrane barriers of the parasites in order to reach their molecular targets [17]. Since the simultaneous optimization of lead compounds for optimal anti-parasitic activity and bioavailability is one of the major hurdles in the lead optimization process, the advantage of bioassays should not be underestimated. Bioassays continue to play an important role in today's drug discovery process, particularly during the identification and optimization of novel anti-parasitic compounds [7].

On the other hand, the use of bioassays as the sole screening platform has a disadvantage. Those potential drugs with a high activity against attractive anti-parasitic target molecules, but no activity in bioassays (e.g., due to disadvantageous physico-chemical properties), are discarded. For this reason, alternative target- or mechanism-based drug screening strategies have been developed [8].

The Process of Target-Based Drug Discovery

In contrast to physiology-based drug discovery screens, the target-based approach starts with the identification of a protein that is used to search for new active compounds in in vitro screens [18, 19]. The goal of the target-based approach, as with every drug discovery workflow, is to provide drug candidates for the downstream development process, finally ending with a newly registered drug (Figure 1.2). In principle, the target-based approach consists of four major steps: (i) target identification; (ii) target validation; (iii) lead discovery; and (iv) lead optimization, including the in vitro profiling of the optimized lead structures (Figure 1.2). Target-based drug discovery is a highly technology-driven process, which particularly benefits from advances in modern “omics” research areas. Omics is a neologism which refers to a broad area of study in biology of fields ending in the suffix “-omics,” such as genomics, transcriptomics, proteomics, or metabolomics (http://omics.org). Omics sciences apply large-scale experiments in order to analyze complete biological entities such as genomes, proteomes, metabolomes, and so on. They are enabled by major advances in modern high-throughput technologies such as DNA/RNA sequencing, microarrays, mass spectrometry, high-throughput screening, or combinatorial and medicinal chemistry – technologies that are increasingly common and affordable [20]. These technologies have already started to improve the quality and quantity of the drug discovery process [21].

Figure 1.2 Drug discovery and development workflow. The workflow is organized as a stage-gate model; that is, a product development process is divided into stages separated by gates. At each gate, the continuation of the development process is decided by the organization. The drug discovery stage typically consists of target identification and validation, lead discovery and optimization, and profiling.

Target Identification

Target identification starts with the discovery of a relevant drug target believed to be essential for the survival of a parasitic organism [22]. In order to avoid or minimize potential toxicity effects prior to the development of a new anti-parasitic drug, an optimal drug target would be absent from the host [23]. However, experience has shown that many of the existing anti-parasitic drugs act on target molecules which also exist in the host organisms [10, 24, 25].

Common methods for the selection of potential drug targets are classical biochemistry and molecular biology techniques. For example, the reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to verify the expression of a potential target protein in the critical life stages of a parasite [19, 26]. Alternatively, information that can be “mined” in genome and drug target databases such as EuPathDB (http://w1.eupathdb.org/eupathdb) [27], GeneDB (http://www.genedb.org) [28], and the TDR target database (http://tdrtargets.org) [29], enables the identification of new drug targets. Such databases contain a wealth of data relating to parasite genes, proteins, homologs, transcript expression, single nucleotide polymorphisms (SNPs), cellular localization, and putative functions. It is expected that the data content in these databases will continue to increase due to advances in high-throughput technologies, and their ever-greater data output. For example, the area of functional genomics already enables the determination of complete genomic protein functions by utilizing high-throughput experiments such as microarrays, serial analysis of gene expression (SAGE), ChIP-on-chip experiments, or proteomics [30]. Moreover, the emergence of competitive second- or next-generation DNA-sequencing techniques [31] and further advances in single-molecule DNA-sequencing technologies [32] will lead to the sequencing of additional genomes, including those of parasites [33]. Currently, over 1000 bacterial and 120 eukaryotic genomes have been reported as completely sequenced, and many more are ongoing (http://www.genomesonline.org) [34]. In examining the phylum Apicomplexa, approximately 50 genome sequencing projects have now been initiated, of which nine have already been published (Table 1.1). The availability of genome datasets for parasites, their vectors, and hosts provides the basis for another highly effective target identification method, the bioinformatic comparison of genomes [10, 35–38]. Such comparative genomics strategies aim to compare simultaneously two or more genomes in order to identify similarities and differences, and hence identify potential drug targets [18, 19].

Table 1.1 Published genomes of apicomplexan organisms.

ParasiteTaxonomyLinkBabesia bovis T2BoAconoidasidaGenbank accession AAXT00000000Theileria annulata str. AnkaraAconoidasidahttp://www.sanger.ac.uk/Projects/Theileria parva str. MugugaAconoidasidaGenbank accession AAGK00000000Cryptosporidium hominis TU502Coccidiahttp://cryptodb.org/cryptodb/Cryptosporidium parvum IowaCoccidiahttp://cryptodb.org/cryptodb/Plasmodium yoelii str. 17XNLAconoidasidahttp://plasmodb.org/plasmo/Plasmodium falciparum 3D7Aconoidasidahttp://plasmodb.org/plasmo/Toxoplasma gondii ME49Coccidiahttp://toxodb.org/toxo/Toxoplasma gondii TgCkUg2Coccidiahttp://toxodb.org/toxo/

Target Validation

When a particular protein has been identified as a potential drug target, the validation of its function is mandatory (Box 1.1) [39]. This involves the demonstration that affecting the target will be sufficient for obtaining a significant anti-parasitic effect, and this can be accomplished by genetic studies that include the generation of loss-of-function (Knock-out) and gain-of-function (Knock-in) mutants in animal models [40]. Further common target validation methods are RNA interference, antisense RNA, and antibody-mediated inhibition experiments. Alternatively, the validation of a drug target is performed using chemical compounds [26, 41]. In such cases, experimental compounds with well-understood modes of action are tested directly on parasites and screened for anti-parasitic phenotypes. With positive results, it is inferred that the phenotypic effect is due to the interaction of the chemical compound with its known target. Chemical validation is a reliable form of target validation, although it cannot be excluded that the phenotypes resulting from the chemical validation experiment are in fact due to an interaction of the compounds with secondary, unknown, or multiple targets. One benefit in employing chemical validation is that, simultaneously, the druggability of a molecular target –that is, the ability of a target to interact with a small compound that modulates its function – is analyzed [42]. Experience has shown that this is a key prerequisite to successful drug discovery [42]. Since both genetic and chemical validation approaches have their benefits and drawbacks, a drug target is best validated using a combination of the two.

Box 1.1 Features of Optimal Anti-Parasitic Targets

A validated target:

has a clear biological functionhas an essential role for the growth or survival of the parasiteis expressed during the relevant life stagesis druggablecan be screened in a biochemical or cellular assay.

A potential drug target should fulfill additional criteria, including the ease of recombinant expression and purification, and “assayability” in automated biochemical or cellular assays (including their miniaturization) (Box 1.1) [26]. Drug target validation is a complex process that often produces ambiguous results. Accordingly, target validation is a risk-adjusted decision on the overall value of the target protein [43]. However, since the downstream steps in target-based drug discovery include very expensive and time-consuming processes, it is important that potential target molecules are validated in as large a quantity as possible.

Lead Discovery

The next step after the validation of a drug target is to identify compounds that interfere with its function [44]. A general lead discovery workflow is shown in Figure 1.3; this consists of a series of steps of hit identification, hit exploration, hit-to-lead, and lead selection [45]. The compounds should be amenable to chemical optimization, finally leading to a drug candidate. Such compounds are called “leads,” and the process is called “lead discovery” [17].

Figure 1.3 A typical lead discovery workflow.

Hit Identification

In order to identify small chemical compounds that interact with the target molecule, a screening campaign (often high-throughput screening, HTS) is performed [46]. Before an HTS can be started, biochemical or cellular assays must first be developed and miniaturized for optimal robotics and throughput [18]. The HTS assays are usually validated for their suitability and robustness by screening a small subset of compounds before the actual high-throughput screen is started. Depending on the type of assay, the HTS can typically involve the examination of more than one million compounds within a few weeks [47, 48]. The primary screen during the hit identification process is carried out on the initial validated target molecule, while secondary screens may be performed on further targets. For example, orthologous targets from additional parasites may be screened if the goal of the drug discovery project is ultimately to produce a compound that exhibits broad anti-parasitic activity, as is often the case in the animal health industry. Another possibility is to consider orthologs from host organisms, and to include only those compounds that show a high activity on the target molecules of the parasite. Thus, potentially toxic compounds may be filtered out in a very early stage [7]. A complementary approach is to involve chemoinformatics to identify hit compounds (Figure 1.3) [49]. Such in silico approaches – which are also known as “virtual screening” – deal with the automatic evaluation of large virtual compound libraries in order to prioritize compound subsets [50]. Compared to HTS, virtual screening has two major advantages: (i) the speed and throughput of in silico screens are much greater than in experimental set-ups; and (ii) more importantly, virtual screening is not limited to existing in-house compound collections, and provides a fast and cheap way to explore unknown parts of the chemical space [49]. Accordingly, virtual screening can generate target-focused and activity-enriched datasets, which can eventually be tested in experimental HTS assays [49, 51].

Hit Exploration

Hit exploration can be divided into “hit verification” and “hit confirmation,” and mainly involves filtering processes to separate appropriate from inappropriate molecules (Figure 1.3) [17]. Hit verification concentrates on the experimental validation of the effectiveness of a compound by measuring the half-maximal inhibitory concentration (IC50) in the case of antagonists, and the half-maximal effective concentration (EC50) for agonists [46]. During hit confirmation, the stability of compounds is proofed. For example, compound solutions are freshly prepared from solid stocks and measured again in order to exclude artifacts from compound degradation in liquid stocks. Hit confirmation also includes the verification of the compound structures using techniques such as mass spectrometry (MS) and nucleic magnetic resonance (NMR) [19, 46]. The result of the hit exploration process is termed a “confirmed hit” (Figure 1.3).

Hit-To-Lead

The next phase in a lead discovery project is the “hit-to-lead” (H2L) process [52]. This is characterized by less filtering and a broader knowledge of the hits for a subsequent prioritization [17]. During the H2L process, data regarding toxicity, bioactivity, and intellectual property are assembled (Figure 1.3). One of the first actions in the H2L process is usually to purchase or synthesize structurally related compounds which are then tested together with the confirmed hits for their target activity and, by screening in bioassays against intact parasites in culture, for their anti-parasitic activity. In this way, data related to the on-target activity and initial structure–activity relationships (SARs) of the compound classes are generated. The experiments also provide the first hints regarding the bioactivity of the compound classes. If the compounds are inactive in the bioassays, then data arising from solubility, lipophilicity, or permeability experiments may explain the lack of bioactivity. Other typical H2L activities are the evaluation of toxicity data from cytotoxicity and genotoxicity tests [53], and an understanding of the patent literature for the corresponding compound classes (Figure 1.3) [54]. The H2L process concludes with a list of lead candidates that fulfill clearly defined criteria (Box 1.2), and from which a lead is selected for chemical optimization.

Box 1.2 Definition of a Lead

A lead:

possesses specific activity in functional target assaysexhibits a particular SARshows no indication for genotoxicityhas adjustable physico-chemical propertiesalready features some bioactivity in parasitic bioassays, or at least offers favorable physico-chemical properties needed for bioactivity.

Lead Optimization and Profiling

Leads display certain effects and properties of active drugs, but not with all of the necessary attributes. The missing properties are subsequently introduced in the lead optimization phase, a process that aims to transform an active lead compound into a drug candidate [55]. Lead optimization is a multi-parametric process aimed at simultaneously optimizing several features, such as on-target activity, bioactivity, and stability (Figure 1.4) [7, 56]. Therefore, lead optimization is highly complex, and generally takes the most time in drug discovery projects, largely because the necessary medicinal chemistry is a major bottleneck in the process [57].

Figure 1.4 Multiparametric lead optimization. Starting with an in vitro lead, a chemical compound is optimized until it meets specific criteria defined for drug candidates. The process is multiparametric, and several sometimes conflicting requirements (e.g., high on-target activity, anti-parasite efficacy, low toxicity) must be accomplished simultaneously during the chemical optimization.

The lead optimization workflow can be divided into different phases connected with decision points, any of which might bring an end to the project (Figure 1.4). During the first phase, mainly in vitro target assays are used to control the optimization progress, and the emphasis is on improving on-target activity; this is fundamental for achieving biological activity [7]. During this phase, several thousand derivatives might be synthesized, a situation made possible by the major advances in medicinal and combinatorial chemistry that have helped to increase both diversity and yields [58]. With such large-scale synthesis, a clear SAR for a compound class can be determined. In SAR studies, the lead compounds are typically divided into specific regions, after which each in turn is chemically modified. Figure 1.5 shows the results of a SAR experiment with anthelmintic thienopyrimidine analogs, and demonstrates how different substitutions can affect nematocidal activity [59]. It is important to note that the lead optimization steps can be efficiently supported by chemoinformatic tools [60]; this is especially true if the protein structure of a target complex is available. Then, modern structure-based drug design methods can be applied to support rational lead optimization [61, 62]. Finally, it is essential to check the toxicity potential of interesting compounds in this first phase of lead optimization, usually by performing in vitro toxicity tests [63].

Figure 1.5 Structure–activity relationship (SAR) of thienopyrimidine analogs. The lead structure was divided into three regions, with all regions being chemically modified sequentially, varying only one motif at a time: first the aromatic head (region 1), followed by the central diamine linker (region 2), and finally the thienopyrimidine core (region 3). The anthelmintic potencies were determined in bioassays. This figure summarizes the nematocidal SARs of compound analogs. For example in region 1, substitution of the para position on the aromatic motif led to analogs with superior activity. Of these substituents, methyl, trifluoromethyl, chloride, and phenyl were observed to be the best. Reproduced with permission from Ref. [59]; © 2009, Wiley-VCH, Weinheim.

Yet, because on-target activity alone is not sufficient to achieve anti-parasitic activity, it is also essential to consider the biological activity and to ensure that the physico-chemical properties are within the desired range. This is achieved during the second phase of lead optimization, which focuses on monitoring biological activity using in vitro parasite models [13–15]. Critical to the biological activity, and thus to the success of any potential drugs, are the ADME parameters (this is an acronym for absorption, distribution, metabolism, and excretion) [64]. ADME deals with the disposition of a drug within organisms, with each of the four criteria having an important influence on the efficiency and pharmacological activity of a compound as a drug [65].

The third phase of lead optimization goes a step further, and includes animal models to assist in making the transition from in vitro assays to in vivo conditions [7]. For this, the compounds are profiled in model organisms such as mice or rats [66]. In any animal model it is necessary to generate pharmacokinetic, pharmacodynamic, and toxicity profiles. Pharmacokinetics describes the time course of the drug in the body (namely, its ADME behavior [67]) while, in contrast, pharmacodynamics specifies the effect versus concentration relationship. In simple words, pharmacodynamics explores what a drug does to the body over time, whereas pharmacokinetics is the study of what the body does to the drug [68]. Often, the intricacies and expense of animal models limits the numbers of compounds that can be tested annually [7]. Following the successful completion of a lead optimization project, a compound is then considered to be a suitable drug candidate and transferred into a drug development program. In human health, a drug candidate has already been profiled in model organisms, whereas animal health research can go a step further and prepare a clinical profile in target animals.

Examples of Successful Target-Based, Anti-Parasitic Drug Discovery Programs

Proteases are validated as targets for therapy of a number of parasitic diseases, including malaria, leishmaniasis, African trypanosomiasis, and schistosomiasis [5, 25, 69, 70]. Several chemical structures have already been identified as protease inhibitor leads [1]; for example, promising inhibitor leads targeting the falcipain protease for the treatment of P. falciparum infection have been discovered by a collaboration between the pharmaceutical company GlaxoSmithKline plc and the University of California San Francisco, supported by the Medicines for Malaria Venture. Although these compounds are far along in the drug development process, their structures remain proprietary [69]. A related example of a target-based drug discovery workflow is the identification of the cysteine protease inhibitor, N-methyl-piperazine-phenylalanyl-homophenylalanyl-vinylsulfone-phenyl (K11777 or K777) as a small-molecule therapy of Chagas disease by targeting the parasite's cathepsin L-like cysteine protease, cruzain [24, 71]. The vinyl sulfone class of molecules was originally identified in the mid-1990s in a curtailed industrial drug discovery program (at Khepri Pharmaceuticals) to target bone loss, but the parent molecule of K777 (K11002 or K02) was subsequently transferred (including the intellectual property rights) to an anti-parasitic drug discovery program conducted at the University of California, San Francisco. Following the modification of K02 to improve its bioavailability, K777 was put through a standard development workflow which included on-target mechanism of action studies incorporating crystallography, both in vitro and in vivo anti-parasitic activity profiling (the latter in mice and dogs), and a suite of ADME and (acute and chronic) toxicity (studies in rodents and dogs). As of early 2010, a dossier is being prepared for filing K777 as an Investigational New Drug (IND) at the US Food and Drug Administration in advance of clinical trials in humans. A structure-guided medicinal chemistry program is also ongoing to identify “back-up” compounds (Figure 1.6) [72].

Figure 1.6 Ribbon (a) and surface (b) representations of the cysteine protease cruzain from T. cruzei, complexed with the inhibitor K11777 [72].

Another recent success story in the development of novel anti-parasitic drugs is the identification of a new class of anthelmintics, the amino-acetonitrile derivatives (AADs) [73]. In veterinary medicine, there is an urgent need for novel drugs against parasitic worms, as some nematodes have developed drug resistance against all available anthelmintic drugs; even worse, some multidrug-resistant worms have appeared [74, 75]. Thus, the development of the AADs, which were discovered in a physiological-based screen, proved to be most welcome [76]. Consequently, an extensive lead optimization program is ongoing which, to date, has resulted in over 600 compounds with different anthelmintic activities, both in vitro and in vivo, in different hosts [77]. Moreover, the compounds are effective against a wide range of livestock helminths, including several drug-resistant parasites [78]. This indicates a new mode of action for the AADs and, indeed, genetic experiments have shown that they act on unique, nematode-specific subunits of the acetylcholine receptors [79]. If the excellent pharmacokinetic properties and tolerability of the AADs in ruminants can be extended to humans, the class may offer an alternative anthelmintic for human medical practice [73].

Conclusion

The discovery of novel drugs for parasitic diseases is a high-risk, expensive, and lengthy process [22]. The past few years have seen increased financial and infrastructural support for drug discovery and development for parasitic diseases by academic institutes, PPPs, and the pharmaceutical industry [3, 5]. Already this has led to imaginative, comprehensive and dynamic drug discovery and development pipelines (even in the face of sometimes modest financial backing) when compared to those diseases directly impacting developed countries [1]. A closer look, however, at the portfolios of some of the PPPs reveals a plethora of early discovery projects for parasitic diseases that have yet to translate into late development leads. The enormous expense and considerable expertise required to develop late leads and drug candidates (involving medicinal and combinatorial chemistry, and in-life animal studies such as A DME and toxicology) remain major bottlenecks. This is especially true for academic institutions which, with their relatively finite resources, concentrate either on the biology or chemistry of the drug discovery workflow. Here, a closer collaboration between biology and chemistry groups may lead to an increased efficiency, and indeed a number of specialized academic centers have already arisen specifically focused on R&D for neglected diseases. Most importantly, the pharmaceutical industry, through a variety of internal and external R&D programs, has re-entered the business of drug development for infectious diseases. This is vital, given its decades-long know-how on furthering compounds through to the market. In summary, the continued collaboration between academic groups, PPPs, and the pharmaceutical industry is the key to optimizing R&D output and, eventually, the registration of novel and badly needed anti-parasitic drugs.

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