AntiparasiticandAntibacterialDrugDiscovery · 7 Lessons Learned from Target-Based Lead Discovery 99 Michael Gassel, Jörg Cramer, Christopher Kern, Sandra Noack, and Wolfgang Streber

Antiparasitic and Antibacterial Drug Discovery

From Molecular Targets to Drug Candidates

Edited byPaul M. Selzer

57268File Attachmentcover.jpg

Antiparasitic and Antibacterial

Drug Discovery

Edited by

Paul M. Selzer

Book SeriesDrug Discovery in Infectious DiseasesForthcoming Topics:

� Viruses� Helminths� Apicomplexa

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Antiparasitic and Antibacterial Drug Discovery

From Molecular Targets to Drug Candidates

Edited byPaul M. Selzer

The Editor

Prof. Dr. Paul M. SelzerBioChemInformaticsIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

[email protected]

CoverLight microscopic image of the helminth Schistosomamansoni–with a male hosting a female in the canalisgynaecophorus: courtesy of Dr. Conor R. Caffrey,University of California San Francisco, USA.Scanning electron microscopic image of the gram-negative bacteria Mannheimia heamolytica: courtesyof Prof. Dr. Lothar H.Wieler, Freie Universität Berlin,Dr. Heike Kaspar, and Dr. Christoph Schaudinn,Robert Koch Institut Berlin, Germany. The chemicalstructure is taken from chapter 19 authored byThorsten Meyer et al., figure 19.9.

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Foreword

It is ironic that three decades ago infectious diseases were viewed as a problem of thepast. Malaria and tuberculosis were going to be eradicated, effective vaccines wereavailable for major childhood infections, and an armamentarium of antibioticswas available for common community and hospital-acquired infections. Youngphysicians were advised not to enter infectious disease specialties because theywere becoming irrelevant. The AIDS epidemic was the first wakeup call thatinfectious diseases would again become a major global health problem. Drug-resistant malaria and tuberculosis are now almost ubiquitous and new and emer-ging infectious diseases are almost a weekly staple of the popular press. Indeed theneed for new drugs for infectious diseases has never been greater. Global industryand global travel means that formerly exotic diseases can rapidly establish them-selves at any port of entry. Effective vaccines against the most prevalent infectiousdiseases like AIDS and malaria have proven difficult to develop. Multidrug-resistantorganisms are an issue in any clinical setting. This publication provides a window onnew approaches to drug discovery and development targeting infectious diseases.Fortunately, technology and training in new methodologies of drug discovery haveexpanded rapidly in the past 10 years. The challenge is how to effectively apply thistechnology to the thorny problems of global infections and to maintain a drugdevelopment pipeline for infectious diseases in light of the immense cost nowassociated with bringing new drugs to market.

San Francisco, USA James H. McKerrowNovember 2008

V

Contents

Foreword VPreface XIList of Contributors XIII

Part One Drug Discovery Approaches 1

1 Target Identification and Mechanism-Based Screening for Anthelmintics:Application of Veterinary Antiparasitic Research Programs to Search forNew Antiparasitic Drugs for Human Indications 3Timothy G. Geary�, Debra J. Woods, Tracey Williams, and Solomon Nwaka

2 Anthelmintic Resistance as a Guide to the Discovery of New Drugs? 17Georg v. Samson-Himmelstjerna�, Roger K. Prichard, andAdrian J. Wolstenholme

3 Drug Discovery for Neglected Diseases: View of A Public–PrivatePartnership 33Rob Don� and Eric Chatelain

4 Bioinformatics and Chemoinformatics: Key Technologies in theDrug Discovery Process 45Andreas Krasky, Andreas Rohwer, Richard J. Marhöfer, and Paul M. Selzer�

5 Target Identification and Validation in Antiparasitic Drug Discovery 59Christian Wolf and Nikolas Gunkel�

6 Selective Drug Targets in Parasites 75Peter Köhler� and Richard J. Marhöfer

VII

7 Lessons Learned from Target-Based Lead Discovery 99Michael Gassel, Jörg Cramer, Christopher Kern, Sandra Noack,and Wolfgang Streber�

8 Approaches Towards Antiparasitic Drug Candidates forVeterinary Use 117Christophe Chassaing� and Harald Sekljic

9 Learning to Relate Structural Space to Property Space 135Michael Berger, Jörg Cramer, Michael Hinz, Christina Mertens,Christian Miculka�, Trevor Newton, Jörg Schröder, and Harald Sekljic

10 Recruiting the Host Defense Mechanisms: Roles for Vaccinesand Chemotherapeutics 159Theo P.M. Schetters�

Part Two Protozoan Parasites 175

11 Proteases of Parasitic Protozoa – Current Status and Validation 177Mohammed Sajid�, Michael J. Blackman, Patricia Doyle, Chen He,Kirkwood M. Land, Cheryl Lobo, Zachary Mackey, Momar Ndao,Sharon L. Reed, Brian Shiels, Ryan Swenerton, and William Weir

12 In Search of Trypanocidal Drugs 211Leopold Flohé�

13 Trypanosomatid Protein Kinases As Potential Drug Targets 227Martin Wiese, Andrew Morris, and Karen M. Grant�

14 Targeting the Malaria Kinome: Discovering Kinase Inhibitorsas Novel Antimalarial Agents 249Dayadevi Jirage, Susan M. Keenan, and Norman C. Waters�

15 Malaria and Antimalarials – a Focused View 277Frank Seeber�

Part Three Multicellular Parasites 299

16 Chemotherapeutic Development Strategies for Schistosomiasis 301Conor R. Caffrey�, David L. Williams, Matthew H. Todd, David L. Nelson,Jennifer Keiser, and Jürg Utzinger

VIII Contents

17 Searching New Antiparasitics in Virtual Space 323Frank Oellien, Kristin Engels, Jörg Cramer, Richard J. Marhöfer,Christopher Kern, and Paul M. Selzer�

18 Cyclooctadepsipeptides – an Anthelmintically Active Classof Compounds Exhibiting a Novel Mode of Action 339Achim Harder�, Kathryn Bull, Marcus Guest, Lindy Holden-Dye,and Robert Walker

19 Chemical Optimization of Anthelmintic Compounds – A Case Study 357Thorsten Meyer�, Jörg Schröder, Manfred Uphoff, Sandra Noack,Anja R. Heckeroth, Michael Gassel, Petra Rohrwild, and Thomas Ilg

Part Four Bacteria 373

20 Pathogenomics: Identification of Novel Drug Targets and VaccineCandidates in Bacteria 375Knut Ohlsen�, Martin Eckart, Ulrich Dobrindt, Heike Bruhn,and Jörg Hacker

21 Tuberculosis Drug Discovery: Issues, Gaps and the Way Forward 415Véronique Dartois�, Franz Joel Leong, and Thomas Dick

22 Decreasing the Number of Gaps in the Draft Assembly of theMannheimiahaemolytica M7/2 Genome Sequence 441Hon Q. Tran, Mathias Beig, Volker Spehr, Andreas Rohwer,Gottfried Unden, and Paul M. Selzer�

23 Total Synthesis and Configurational Assignment of Pasteurestin A and B,a Natural Product with Antimicrobial Activity on Pasteurellaceae 453Marion Kögl, Lothar Brecker, Ralf Warrass, and Johann Mulzer�

Index 473

Contents IX

Preface

In the age of antibiotics, vaccines, and drugs, we might be lulled into a sense ofcomplacency regarding infectious diseases and that there is ‘‘a cure for everything’’.This sense of security is maintained at our peril, however. One has only to considerthe growing devastation caused by such big-name diseases as influenza, HIV-AIDS,tuberculosis, and malaria to see that the struggle to treat and control infectiousdiseases is truly titanic and indeed becoming more perilous with the ever-evolvingdevelopment and spread of drug resistance compounded by the greater freedom andspeed of movement of goods, animals, and people. Aside from the recently perceivedsecurity threat to the health and business structures of the developed world caused bythese and a plethora of other infectious disease, billions living in developing countriesmust endure the daily struggle of diseases. In contrast to most human health-relatedpharmaceutical companies, academic institutions, veterinary science, and animalhealth companies remain very much focused on infectious diseases, including thosecaused by bacteria and parasites. As illustrated in Figure 1, the animal health sectorremains profitable, and thankfully so, as history has shown that therapies produced inthis sector often prove invaluable for treatment of similar infectious diseases ofhumans – the application of anthelmintics being a case in point.The improvedunderstandingof the resilience ofdisease-causing agents to therapies,

their expanding diseasemenace in the era of ‘‘globalization,’’ and the balance providedby the opportunities for cross-sector exchange of ideas and applications spurred thepreparation of this book. Also, thebook serves to highlight the importance and visibilityof drug discovery efforts for infectious diseases of both animals and humans.Though it is not possible to address every aspect, disease, or approach within a

single volume, this book sets forth a series of case studies and review articles thatfocus on bacterial and parasitic diseases in order to showcase how scientists in thedifferent disciplines strive to move drug discovery forward. The contributing authorsare experts drawn from drug discovery units of the pharmaceutical industry, acade-mia, and nonprofit organizations in an effort to offer a global and balanced insightinto the issues and problems at stake and their possible solutions.Writing this has been a rewarding task for everybody involved. My heartfelt thanks

go to the contributing authors for their excellent work performed within a short time-frame. In addition, I am grateful to Intervet/Schering-Plough Animal Health andits Drug Discovery Unit for their unreserved support, inspiration, and motivation

XI

during the preparation of this book. I also thank the members of Intervets Bio-ChemInformatics Unit for their excellent technical backing and team spirit. Finally, Iam very grateful to Ms SimoneMaus-Gilbert for her outstanding editorial assistance.

Schwabenheim, Germany Paul M. SelzerNovember 2008

Figure 1 The world animal health market basedon data from 2006. The first row depicts theproportion of antiparasitics and antiinfectives inthe whole animal health market. Rows two andthree represent the antiparasitics andantiinfectives market, respectively. From left toright the individual proportions are broken downaccording to regional sales (ROW = rest of

world), sales per animal species, and sales perchemical class. The area of the individual piecharts is not size-adjusted. Original data werederived by Wood Mackenzie and kindly providedby Linda Franken-Horspool, InternationalMarketing, Intervet/Schering-Plough AnimalHealth.

XII Preface

List of Contributors

XIII

Mathias BeigIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Michael BergerIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Michael J. BlackmanDivision of ParasitologyNational Institute for Medical ResearchThe RidgewayMill HillLondonNW7 1AAUK

Lothar BreckerInstitut für Organische Chemie derUniversität WienWähringer Strasse 381090 WienAustria

Heike BruhnUniversity of WürzburgInstitute forMolecular Infection BiologyRöntgenring 1197070 WürzburgGermany

Kathryn BullSchool of Biological SciencesUniversity of SouthamptonBassett Crescent EastSouthamptonSO16 7PXUK

Conor R. Caffrey�

Department of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesByers HallUniversity of California San FranciscoCA [email protected]

Christophe Chassaing�

Intervet Innovation GmbHZur Propstei55270 [email protected]

� Corresponding author

Eric ChatelainDrugs for Neglected Diseases Initiative(DNDi)15 Chemin Louis-Dunant1202 GenevaSwitzerland

Jörg CramerIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Véronique Dartois�

Novartis Institute for Tropical Diseases10 Biopolis Road05-01 ChromosSingapore [email protected]

Thomas DickNovartis Institute for Tropical Diseases10 Biopolis Road05-01 ChromosSingapore 138670Singapore

Ulrich DobrindtUniversity of WürzburgInstitute forMolecular Infection BiologyRöntgenring 1197070 WürzburgGermany

Rob Don�

Drugs for Neglected Diseases Initiative(DNDi)15 Chemin Louis-Dunant1202 [email protected]

Patricia DoyleDepartment of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesUniversity of California San FranciscoCA 94158USA

Martin EckartUniversity of WürzburgInstitute for Molecular InfectionBiologyRöntgenring 1197070 WürzburgGermany

Kristin EngelsIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Leopold Flohé�

Molisa GmbHBrenneckestraße 2039118 [email protected]

Michael GasselIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Timothy G. Geary�

Institute of ParasitologyMcGill University21111 Lakeshore RoadSte-Anne-de-BellevueQC Canada H9X [email protected]

XIV List of Contributors

Karen M. Grant�

School of Health & MedicineDivision of MedicineFaraday Building, Lancaster UniversityLancasterLA1 [email protected]

Marcus GuestSchool of Biological SciencesUniversity of SouthamptonBassett Crescent EastSouthamptonSO16 7PXUK

Nikolas Gunkel�


Jörg HackerRobert-Koch-InstituteNordufer 2013353 BerlinGermany

Achim Harder�

Bayer HealthCare AGAnimal Health GmbHCRD-ParasiticidesAlfred-Nobel-Strasse 5040789 [email protected]

Chen HeUCSD Medical Center200 West Arbor DriveSan DiegoCA 92103-8416USA

Anja R. HeckerothIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Michael HinzIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Lindy Holden-DyeSchool of Biological SciencesUniversity of SouthamptonBassett Crescent EastSouthamptonSO16 7PXUK

Thomas IlgIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Dayadevi JirageDivision of Experimental TherapeuticsWalter Reed Army Institute of Research503 Robert Grant AvenueSilver SpringMD 20910USA

Susan M. KeenanSchool of Biological SciencesUniversity of Northern Colorado501 20th StreetGreeleyCO 80501USA

List of Contributors XV

Jennifer KeiserDepartment of Medical Parasitology andInfection BiologySwiss Tropical InstituteP.O. Box4002 BaselSwitzerland

Christopher KernIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Marion Kögl (passed away)Institut für Organische Chemie derUniversität WienWähringer Strasse 381090 WienAustria

Peter Köhler�

University of ZürichInstitute of [email protected]

and

Niederdorf 8338132 HintereggSwitzerland

Andreas KraskyIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Franz Joel LeongNovartis Institute for Tropical Diseases10 Biopolis Road05-01 ChromosSingapore 138670Singapore

Kirkwood M. LandDepartment of Biological SciencesUniversity of the PacificStocktonCA 95211USA

Cheryl LoboLab of Blood-Borne ParasitesThe Lindsley Kimball Research InstituteNew York Blood Center310 East 67th StreetNew YorkNY 10021USA

Zachary MackeyDepartment of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesByers HallUniversity of California San FranciscoCA 94158USA

Richard J. MarhöferIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

James H. McKerrow�

Department of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesByers HallUniversity of California San FranciscoCA [email protected]

XVI List of Contributors

Christina MertensIntervet International BVWim de Körverstraat 355830 AA BoxmeerThe Netherlands

Thorsten Meyer�


Christian Miculka�

Intervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Present address:Merial Inc.3239 Satellite Blvd.Duluth, GA [email protected]

Andrew MorrisInstitute for Science and Technology inMedicineHuxley BuildingKeele UniversityKeele, Newcastle-under-LymeST5 5BGUK

Johann Mulzer�

Institut für Organische Chemie derUniversität WienWähringer Strasse 381090 [email protected]

Momar NdaoNational Reference Centre forParasitologyDepartment of MedicineDivision of Infectious DiseasesResearch Institute of the McGillUniversity Health CentreMontreal General, Hospital/ResearchInstitute R3-131625 Pine Ave WestMontreal H3G 1A4Quebec Canada

David L. NelsonDepartamento de AlimentosFaculdade de FarmáciaUniversidade Federal de Minas Gerais31270-901 Belo HorizonteMinas GeraisBrazil

Trevor NewtonBASF SEGVA/HC – B00967056 LudwigshafenGermany

Sandra NoackIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Solomon NwakaSpecial Programme for Research andTraining in Tropical Diseases (TDR)World Health Organization20 Avenue Appia1211 Geneva 27Switzerland

List of Contributors XVII

Frank OellienIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Knut Ohlsen�

University of WürzburgInstitute forMolecular Infection BiologyRöntgenring 1197070 Wü[email protected]

Roger K. PrichardInstitute of ParasitologyMcGill University21111 Lakeshore RoadSainte Anne-de-BellevueH9X 3V9QuebecCanada

Sharon L. ReedUCSD Medical Center200 West Arbor DriveSan DiegoCA 92103-8416USA

Petra RohrwildIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Andreas RohwerIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Georg von Samson-Himmelstjerna�

Institute for ParasitologyUniversity of Veterinary MedicineBuenteweg 1730559 [email protected]

Mohammed Sajid�

Department of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesUniversity of California San FranciscoCA [email protected]

and

Leiden University Medical CentreLeiden Malaria Research Group, afd.ParasitologieAlbinusdreef 2Kamer P4-352333 ZA [email protected]

Jörg SchröderIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Theo P.M. Schetters�

Intervet/Schering-PloughAnimalHealthKörverstraat 355830 AA BoxmeerThe [email protected]

XVIII List of Contributors

Frank Seeber�

Molecular ParasitologyInstitute for BiologyHumboldt UniversityPhilippstraße 1310115 [email protected]

Harald SekljicIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Paul M. Selzer�


Brian ShielsInstitute of Comparative MedicineVet SchoolUniversity of GlasgowBearsden RoadGlasgowG61 1QHUK

Volker SpehrIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Wolfgang Streber�


Ryan SwenertonDepartment of Pathology andthe Sandler Center for Basic Researchin Parasitic DiseasesUniversity of California San FranciscoCA 94158USA

Matthew H. ToddSchool of ChemistryUniversity of SydneyNSW 2006Australia

Hon Q. TranIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Robert WalkerSchool of Biological SciencesUniversity of SouthamptonBassett Crescent EastSouthamptonSO16 7PXUK

Ralf WarrassIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Norman C. WatersDivision of Experimental TherapeuticsWalter Reed Army Institute of Research503 Robert Grant AvenueSilver SpringMD [email protected]

and

List of Contributors XIX

Australian Army Malaria InstituteWRAIR laboratoryGallipoli BarracksWeary Dunlop DriveEnoggeraQLD 4051Australia

William WeirInstitute of Comparative MedicineVet SchoolUniversity of GlasgowBearsden RoadGlasgowG61 1QHUK

Martin WieseStrathclyde Institute of Pharmacy andBiomedical Sciences (SIPBS)John Arbuthnott Building27 Taylor StreetGlasgowG4 0NRUK

David L. WilliamsDepartment of Biological SciencesIllinois State UniversityNormalIL 61790USA

Tracey WilliamsPfizer Animal Health7000 Portage RoadKalamazooMI 49001USA

Christian WolfIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Adrian J. WolstenholmeDepartment of Biology andBiochemistryUniversity of BathClaverton DownBathBA2 7AYUK

and

Department of Infectious DiseasesUniversity of GeorgiaCollege of Veterinary MedicineAthensGA 30602USA

Debra J. WoodsPfizer Animal Health7000 Portage RoadKalamazooMI 49001USA

Manfred UphoffIntervet Innovation GmbHZur Propstei55270 SchwabenheimGermany

Gottfried UndenInstitut für Mikrobiologie undWeinforschungUniversity of MainzBecherweg 1555128 MainzGermany

Jürg UtzingerDepartment of Public Health andEpidemiologySwiss Tropical InstituteP.O. Box 4002 BaselSwitzerland

XX List of Contributors

Part OneDrug Discovery Approaches

1Target Identification and Mechanism-Based Screeningfor Anthelmintics: Application of Veterinary AntiparasiticResearch Programs to Search for New Antiparasitic Drugsfor Human IndicationsTimothy G. Geary�, Debra J. Woods, Tracey Williams, and Solomon Nwaka

Abstract

Anthelmintic discovery in the veterinary pharmaceutical industry has succeededonly through screening synthetic compounds and fermentation products againstwhole parasites in culture or in host animals. Following trends in the parent, andmuch larger, human pharmaceutical industry, many programs have been develo-ped in the past 20 years to exploit mechanism-based screening strategies for theidentification of new leads in this therapeutic area. This strategy relies on therobust identification of parasite proteins as targets for chemotherapeutic inter-vention and their subsequent validation. Expanding access to sequenced genomesof parasitic nematodes will facilitate identification of genes that encode putativedrug targets. Of particular relevance will be those that are shared amongnematodes of veterinary and human importance. These targets offer the bestchance for finding new molecules with potential utility in both arenas andprovide an opportunity for collaboration and synergy between the two sectors.Validation of these gene products as drug targets will require advances infunctional genomics methods for parasites. Expanded capacities for parasite-basedphysiological and biochemical experiments are also likely to be needed. Whilemechanism-based approaches remain an attractive alternative to organism-basedstrategies for broad-spectrum anthelmintic discovery, proof-of-concept for theplatform is still needed.

Introduction

Screening for antiparasitic drugs as a scientific exercise can be traced to the earlywork of Ehrlich, who screened a collection of synthetic dyes for trypanocidalactivity in mice with the aim of allowing the importation of European horses and

j3

cattle into the African colonies of Germany prior to 1900 (see Refs. [1, 2]). This wasperhaps the first example of a screen of a collection of chemicals for anytherapeutic indication; Ehrlichs efforts at drug discovery seem to have begunwith a veterinary parasite as target, but led to the introduction of the first anti-infective drugs for use in humans. Thus, the process by which drugs introducedinto veterinary practice for parasite control were adopted for use in humans has along history.The motivation to discover modern antiparasitic drugs for the animal health

industry can be traced to the introduction of sulfaquinoxaline for the prevention ofmortality and morbidity due to poultry coccidiosis in the late 1940s, phenothiazine(1930s) and piperazine (1950s) as veterinary anthelmintics, and the chlorinatedhydrocarbons and organophosphates as ectoparasiticides in the 1940s and 1950s.Diethylcarbamazine was discovered as an agent for use in human filariasis withinthe same time-frame, being developed for veterinary practice for heartwormprevention some time later. It is important to note that all these drugs were firstused in humans – not necessarily for parasites – prior to being adopted forveterinary use. Their utility for controlling parasites in animals paved the way forthe institution of systematic screening of chemical collections for new syntheticantiparasitic drugs for veterinary application. Their use in clinical settings provedthat chemotherapeutic control of parasites which plagued livestock and poultrywas economically rewarding for the manufacturer, the veterinarian, and thefarmer.The general flow of antiparasitic drugs from human to veterinary application

(Table 1.1) reversed over time. For anthelmintics, the reversal began with thediscovery of thiabendazole for veterinary medicine in the 1960s (Table 1.2), whichwas later introduced for treatment of various gastrointestinal (GI) nematodes inhumans. This pattern was repeated for the nicotinic cholinergic agonists (pyrantel,levamisole), the second generation benzimidazoles, particularly albendazole andmebendazole, and ivermectin (and, potentially, related macrocyclic lactones). Incontrast, antiprotozoal drugs have notmoved as easily between the sectors (Table 1.2)or continue to flow in the opposite direction, a situation that primarily reflects the

Table 1.1 Antiparasitic drugs introduced to veterinary practice from human medical interest.

Drug Human use Veterinary use

Diethylcarbamazine Filariasis Heartworm prophylaxisArsenicals Trypanosomiasis, onchocerciasis Heartworm therapyPiperazine Gout (discontinued) GI nematodesPhenothiazine Malaria/mosquito control GI nematodesSulfa antibiotics Bacteria Poultry coccidiosisMetronidazole Anaerobic microbes GiardiasisBuparvoquone Malaria TheileriosisHalofuginone Malaria Poultry coccidiosis

4j 1 Target Identification and Mechanism-Based Screening for Anthelmintics

differences in the major species of protozoal pathogens of animals compared tohumans (see below).

Potential for Veterinary ! Human Transfer of new AntiparasiticDrugs

Like most of the pharmaceutical industry, animal health companies underwent aconsiderable reduction in abundance over the past 20 years from mergers andacquisitions [3, 4]. This led to a net reduction in investment in antiparasitic drugdiscovery, with a consequent focus of efforts on the most profitable sectors of theanimal health market [5]. As a result, priorities for veterinary parasite control nowdiverge more extensively from those of human medicine. A summary of currentemphasis on typesofparasites targeted fordrugdiscovery for humanversus veterinaryapplications is shown in Box 1.1. It is worth noting in this context that there may be a

Table 1.2 Antiparasitic drugs introduced into human use from veterinary interest.

Drug Veterinary indication Human indication

Benzimidazoles Anthelmintic/antiprotozoal Nematodes, protozoaPyrantel/levamisole GI Nematodes, Lungworms NematodesPraziquantel Cestodes SchistosomiasisIvermectin Heartworm prophylaxis FilariasisNitazoxanide Sarcocystis in horses Protozoa, nematodesMoxidectin Nematodes, ectoparasites Onchocerciasis (in development)Emodepside Nematodes Onchocerciasis (investigation)

Box 1.1: Areas of synergism/overlap based on current trends in discovery investment

Apicomplexan protozoa: human ", veterinary #Kinetoplastids: human ", veterinary #Giardia/ameba/Cryptosporidium: human #, veterinary #Trematodes: human $, veterinary $Filarial nematodes: human ", veterinary $GI nematodes: human #, veterinary "

This box illustrates the potential forflowof compounds in eachdirection as discovery effortscontinue.

": relatively high interest and activity in discovering new drugs.$: modest interest/activity.#: minimal or declining interest/activity.

Potential for Veterinary ! Human Transfer of new Antiparasitic Drugs j5

resumption of drug transfer for parasites from the human to the veterinary side in thefuture. This situation may benefit both areas, as described below.

Protozoan Parasites

A renaissance has occurred in the attention of public and private funders to thediscovery of new drugs for protozoal parasites that infect humans. The primarytargets for chemotherapy include the Apicomplexanmalaria parasites (Plasmodiumspp.), kinetoplastids such as Leishmania spp., Trypanosoma brucei and T. cruzi,Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis,and Cryptosporidium parvum. Based on prevalence and pathogenicity, these drugdiscovery efforts are considerably weighted to malaria and the kinetoplastids [6–12]. In contrast, the primary protozoal target for veterinary medicine is a distinctgroup of Apicomplexans, the Eimeria spp. of poultry, with additional interest inphylogenetically related parasites (Neospora caninum, Sarcocystis neurona) and inGiardia spp. and Cryptosporidium spp. [3]. However, dedicated antiprotozoaldiscovery programs are no longer common in the animal health pharmaceuticalindustry (vaccine discovery is more prevalent at this time), and so future drugs forthese infections will likely flow from human to veterinary use. Current work in thisarea on the human side is heavily focused on mechanism-based, as opposed towhole-organism, high-throughout screening. The extent of target overlap is likely tobe reasonably good across the human/veterinary species divide, though targetchoice in the human-focused projects does not routinely include an assessment ofrelevance for parasite species of strictly veterinary importance. Inclusion of thisfactor as a criterion for prioritization could provide a for-profit component thatwould appeal to potential animal health partners, with benefits similar to thoseanticipated in the anthelmintic arena (see below).

Ectoparasites

Indications for the use of ectoparasiticides in humanmedicine are far fewer than forveterinary clientele, which in turn is a much smaller market than agriculturalapplications. The flow of these compounds has typically been from agriculture toanimal health to human applications, with the exception of DDT, which was firstdeveloped for use in humans. The use of ivermectin for the treatment of head lice andscabies is an example of an ectoparasiticide developed for animals being adopted forhumans. However, the current economic driving force for this arena is so small thatdiscovery programs in animal health sectors typically do not include a componentthat addresses possible human uses. From the human medical perspective, thetemporally limited (as opposed to chronic) use of these products and the relatively lownumber of infestations in the West make the cost–benefit analysis in terms ofregistration unrewarding. This situationmay change if head lice and scabies developmore extreme resistance to available ectoparasiticides, including ivermectin.


Trematodes

These considerations suggest that the primary influence of animal health drugdiscovery research on human medicine will continue to be in the anthelminticarena. More specifically, this will be largely restricted to drugs that primarily affectnematodes. The only flatworm of economic significance in veterinary medicine isthe liver fluke, Fasciola hepatica. This parasite is important in some areas, but is notenough of a production problem in livestock to warrant dedicated screening in mostanimal health companies, even though resistance is emerging to the best availabledrug, triclabendazole (which is not even registered in the USA). AlthoughF. hepatica is a significant human pathogen in some regions, it has not provento be sufficiently prevalent to elicit a dedicated discovery effort for it. Instead, workon flatworms in the human sector focuses on Schistosoma spp., currently controlledby a single drug (praziquantel). In the absence of rigorously documented cases ofpraziquantel resistant schistosomes, investment in new antischistosomal drugdiscovery has been somewhat limited compared to the efforts mounted againstprotozoa. This situation may be changing in light of the Helminth Drug Initiativerecently developed by WHO/TDR [13], which aims to reinforce and advancescreening for new antischistosomal drugs. As for protozoan parasites, this effortmay discover compounds that can be adopted for use against liver flukes inveterinary medicine.

Nematodes

Further analysis of the impact of veterinary antiparasitic drug discovery programswill be restricted to nematocides.Historically, the discovery of nematocides for use inanimals or humans was based on low-throughput systems that employed infectedanimals as the primary screen. These assays were labor-intensive, slow to attain thefinal read-out and used relatively large amounts of experimental compounds. Evenso, it remains true that at least the prototype of every available anthelmintic class,including emodepside, the paraherquamides, and the newest class, the AADs, wasdiscovered by screening in infected animals or worms in culture. Nonetheless, therehas been a marked shift of strategy in the animal health industry to emphasizediscovery programs based on targets, or high-throughput, mechanism-basedscreening [14].The initial change from screening in infected animals to tests run on organisms

in culture was motivated primarily by the need for animal health operations to fitinto the evolving discovery paradigms adopted by their parent companies. Thismeant a marked reduction in the amount of chemicals used in a screen (to adapt tonew parameters for compound synthesis inmedicinal chemistry programs) as wellas a reduction in animal use and in labor costs associated with screening. Inaddition, there were concerns that in vivo screens might fail to detect trulyinteresting actives that are false negatives due to insufficient potency or pharma-ceutical inadequacy.

Potential for Veterinary ! Human Transfer of new Antiparasitic Drugs j7

Unfortunately, screening against parasites or the free-living speciesCaenorhabditiselegans in culture, while vastly increasing throughput and radically diminishing theamount of compound needed for primary screening, was not very successful inrevealing new anthelmintic templates. Indeed, these procedures led to a very highrate of false positives, as many compounds were noted to kill nematodes in culture,but very few (almost none) were subsequently found to be active in infected animalmodels. As resources were typically insufficient to permit experiments designed todetermine why in vitro actives routinely failed in vivo, improvements in the screeningstream designed to reduce the incidence of false positives were not possible. A newapproach was clearly needed, and it was incompatible with standard industrypractices to return to the era of screening in infected animals. In keeping with drugdiscovery for human medicine, mechanism-based approaches came into vogue [14].The drive to move from organism to target-based screens was based on several

factors. One factor was the motivation to capitalize on biology-based intellectualproperty (IP); screens using infected animals or organisms in culture barred fewcompetitors from an area and can only exploit chemistry-based IP.Mechanism-basedscreens can restrict the discovery activities of competitors by taking advantage ofinvestments made in understanding the physiology and molecular pathology ofdiseases and infectious pathogens that dominate Western human medicine. Inaddition, advances in chemical technology, such as combinatorial chemistry, made itpossible to synthesize thousands ofmolecules at a time in small amounts; organism-based screens were ill-equipped to test either the number or the small amount ofavailable compounds. Advances in computational chemistry and structure-baseddrug design meant that the traditional whole-organism blind-screen approachesbecame seen as intellectually unchallenging and out of step with the times. Finally,whole-organism approaches are labor-intensive compared to mechanism-basedstrategies; the incorporation of mechanism-based screening allowed a relativelysmall team of screeners to evaluate hundreds of thousands of compounds againstmultiple targets in amatter ofweeks. The combination of vastly increased throughputwith lowered labor costs made this an irresistible strategy, validated by expertscientific opinion. To date, however, it is undeniably true that the adoption of thisoverall strategy has not led to an increase in the number of new chemical entitiesregistered for use in humans. For our topic, it is crucial to stress again that at least theprototype of all commercially available anthelmintics was discovered in whole-organism assays, despite at least two decades using the more modern approachesto discovery. What does this bode for the switch to more modern screeningplatforms? We can use the neuropeptide area as an example (see page 12).

Discovery Synergies

Discovery programs in animal health companies can contribute to the discovery anddevelopment of drugs for use in humans in several ways. The most obvious is the


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AntiparasiticandAntibacterialDrugDiscovery · 7 Lessons Learned from Target-Based Lead Discovery 99 Michael Gassel, Jörg Cramer, Christopher Kern, Sandra Noack, and Wolfgang Streber