Mechanistic Studies of Anti-Leishmanial Arylimidamides

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MECHANISTIC STUDIES OF ANTI-LEISHMANIAL ARYLIMIDAMIDES

DISSERTATION

Presented in Partial Fulfillments of the Requirements

for the Degree

Doctor of Philosophy

from the Graduate School of The Ohio State University

Trupti Pandharkar, M.S.

******

Graduate Program in Medicinal and Pharmaceutical Chemistry

The Ohio State University

2012

Dissertation Committee:

Karl A. Werbovetz, Ph.D., Advisor

Esperanza Carcache de Blanco, Ph.D.

Mark E. Drew, Ph.D.

Juan D. Alfonzo, PhD.

Trupti Pandharkar

2012

ABSTRACT

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus

Leishmania . With the estimated global incidences of 0.7 to 1.2 million for CL cases and

0.2 to 0.4 million VL cases cases per year, leishmaniasis is estimated to cause the ninth

largest burden of disease. In the absence of effective treatment, visceral leishmaniasis is

most often fatal and cutaneous and other forms of this disease often result in severe

disfigurement and can be debilitating. Given the issues with existing treatment options,

like toxicity, prohibitive cost and loss of effectiveness due to emergence of drug resistant

strains, the need for the new drugs is urgent.

With the mission to develop cheaper, safer and more efficacious drugs, the Consortium

for Parasitic Drug Development (CPDD) has synthesized and evaluated series of

diamidine analogs for their anti-parasitic activity against several pathogens including

Leishmania. Medicinal chemistry efforts to improve the efficacy of diamidines resulted in

discovery of new class of molecules called ‘arylimidamides (AIAs)’ with extraordinary

activity, especially against intracellular pathogens like T. cruzi and Leishmania . The anti- leishmanial efficacy of an AIA, DB766 (2,5-bis[2-(2-propoxy)-4-(2-

ii pyridylimino)aminophenyl]furan hydrochloride) that displayed outstanding activity against the intracellular form of Leishmania donovani [IC 50 = 0.036 µM] and oral efficacy in murine and hamster models of visceral leishmaniasis [71% and 89% reduction in liver parasitemia at 100 mg/kg/day × 5, respectively] was recently reported. Despite intensive lead optimization efforts that permitted exhaustive analysis of the AIA structure activity relationship, attempts to improve the efficacy of AIAs have met with limited success. The lack of knowledge about the parasite drug target and the host toxicity mechanism has precluded further pre-clinical development of AIAs. In the present study, we employed three different approaches, 1) a 2 dimensional difference in gel electrophoresis-mass spectrometry (2D-DiGE-MS) assisted comparative proteomics analysis to study changes in the Leishmania proteome post-treatment with the lead AIA-

DB766, 2) transmission electron microscopy to study the ultrastructural alterations caused by DB766 treatment in Leishmania and 3) generation and characterization of a

Leishmania cell line that is over 10-fold resistant to DB766 through stepwise increases in the concentration of the compound to identify the target and understand the anti- leishmanial mechanism of action of AIAs to facilitate future anti-leishmanial drug discovery efforts.

The DiGE approach used in this study led to the identification of 19 proteins that were differentially modulated in DB766 treated Leishmania. Most of the downregulated proteins like RNA helicase, mitochondrial tryparedoxin peroxidase (mTXNPx), HSP60,

ATP synthase and ATPase were nuclear encoded mitochondrial proteins and four

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upregulated proteins corresponding to HSP60 were mitochondrial precursors, leading to

the evaluation and confirmation of mitochondrial involvement in response to DB766

treatment in Leishmania . However, in the absence of the specific target information and considering the fact that mitochondrial involvement could be a secondary event downstream of a primary target, more detailed investigation employing other approaches was undertaken.

Comparison of the ultrastructural effects of AIAs with the diamidine DB1111 in

Leishmania donovani axenic amastigotes indicated that, unlike DB1111, DB766 treatment did not result in any change in the mitochondrial morphology of these parasites.

However, dramatic ultrastructural alterations were noted in other organelles. The similarities in the ultrastructural profile induced by DB766 to those produced by sterol biosynthesis inhibitors and protease inhibitors in Leishmania led to evaluation of

hypotheses that AIAs act via protease inhibition and/or disturbances in sterol metabolism

in Leishmania . In fluoregenic serine protease substrate based assays, DB766 failed to

show marked inhibition of oligopeptidase B like enzyme activity in Leishmania lysates,

leading to cessation of its pursuit as a target of the AIAs. However, GC-MS analysis of

Leishmania sterols indicated important changes in the sterol profile of DB766 treated

Leishmania, consistent with the ‘disturbances in sterol metabolism’ hypothesis .

In an attempt to identify mutations that cause resistance and in the hope of obtaining

additional mechanistic information, a L. donovani axenic amastigotes cell line over 10-

iv fold resistant to DB766 was developed and characterized. In vitro susceptibility assays revealed that these DB766 resistant parasites (766R) are hypersensitive to miltefosine

(over 2-fold more sensitive) and anti-fungal azoles (over 1000-fold more sensitive).

Systematic studies to test the hypothesis that resistance to DB766 is associated with perturbations in Leishmania sterol metabolism and enzymes in the sterol biosynthetic pathway were undertaken. Western blot analysis of 766R parasites indicated dramatically reduced expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania , in the DB766 resistant parasites. GC-MS analysis of sterols extracted from DB766 sensitive and resistant parasites indicated that the reduced expression of CYP5122A1 was associated with changes in the levels of sterol intermediates without affecting the ergosterol content. Susceptibility assays demonstrated that CYP5122A1 single knockout (HKO) Leishmania donovani promastigotes are

significantly less susceptible to DB766 and more susceptible to ketoconazole than their

wild type counterparts, consistent with the observations in DB766 resistant parasites.

Our studies demonstrate that 1) DB766 disrupts sterol metabolism in Leishmania and synergizes the anti-leishmanial activity of posaconazole 2) CYP5122A1 plays an important role in governing susceptibility and resistance to DB766 in this parasite, and 3)

CYP5122A1 also modulates the susceptibility of Leishmania to antifungal azoles. These results support our hypothesis that DB766 and other AIAs disrupt sterol metabolism by targeting CYP5122A1, in Leishmania.

DEDICATED TO

MY DAUGHTER AMBAR, HUSBAND ROHIT TIWARI

AND MY PARENTS, DILIP AND SANDHYA PANDHARKAR

ACKNOWLEDGMENTS

The pursuit of my PhD has been a long journey. It wouldn’t have been possible for me to complete my studies without invaluable help and support from numerous people during this journey.

Beginning with my advisor, I would like to give my special thanks to Karl Werbovetz for providing me with intellectual insights, guidance and encouragement throughout my graduate career. If not for his patience with me, faith in me, flexibility, genuine care and concern, I would not have been able to attend to demands of life outside the scientific career. He has never judged me and has always gently encouraged me, knowing that I need to juggle priorities.

I am also grateful to Richard Burchmore, University of Glasgow, UK, for his help and discussion with the initial proteomics experiment. I also sincerely appreciate Frederick Buckner and members of his group at University of Washington, Seattle, for their help with the GC-MS analysis of Leishmania sterol samples and for providing me anti-CYP51 antibody for Western blot analysis. I thank Chandrima Shaha and members of her group at National Institute of Immunology, India for their help with drug susceptibility studies with genetically modified Leishmania cell line and also for providing me with anti- CYP5122A1 antibody for Western blot analysis and CYP5122A1 expression plasmid for future studies. I am also grateful to Mark Drew and members of his group for helping me with flow cytometry, Robert Curley for helping with GC instrumentation, Werner Tjarks and members of his group for helping me with HPLC instrumentation and Thomas Schmittgen and members of his group for helping me with Real Time PCR assays.

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I am incredibly grateful for the financial support from the Bill and Melinda Gates Foundation. I am also grateful for scientific feedbacks and invaluable insights from members of my academic committee: Pui-Kai (Tom) Li, Esperanza Carcache de Blanco, Mark Drew and Juan Alfonzo.

I also extend my heartfelt gratitude to my fellow graduate students and post docs in both Werbovetz and Li labs, especially Xiaohua, Carolyn, Sihui, Molla, Julian, Shanshan, Jason, Bulbul, Deepak, Nick, Som, Jonathan and Justin. Their presence helped to lighten my stress and keep the lab environment fun and sane. I have a learned a lot from all of them during the humorous marathon discussions that we had on topics like science, politics, culture, spirituality and many more. I also give my special thanks to Swati Dhar and Dakshayini Rao for playing the part of a friend and confidante and providing support and care.

In addition, I give my sincere thanks to people for their assistance with some of the experiments described in this dissertation: Kathy Wolken and Richard Montione at Campus Microscopy Instrumentation Facility for processing Leishmania samples for transmission electron microscopy and Jocelyn Hach and Kari-Green Church for processing samples for proteomic studies at OSU Shared Proteomic Resources.

My acknowledgement would not be complete without also giving special thanks to my 4 ½ year old daughter Ambar, my husband, Rohit Tiwari and both mine and Rohit’s parents for supporting, having faith in and enduring with me. This enabled me to pursue my scientific career and allowed me to have an impetus to finish my PhD. I will be grateful to them forever!

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VITA

August 18, 1979 ...... Born – Nasik, India

2001...... B. Pharmacy, University of Pune, India

2001-2003 ...... M.Pharm., UICT, Mumbai, India

2003-2005………...... Research assistant, ACTREC, Mumbai, India

2007-2012……………………………….Graduate Teaching and Research Associate,

The Ohio State University

PUBLICATIONS

1. Endeshaw, Molla; Zhu, Xiaohua; He, Shanshan; Pandharkar, Trupti ; Cason, Emily; Mahasenan, Kiran V.; Agarwal, Hitesh; Li, Chenglong; Munde, Manoj; Wilson, David W.; Bahar, Mark; Doskotch, Raymond W.; Kinghorn, Douglas A.; Kaiser, Marcel; Brun, Reto; Drew, Mark E.; Werbovetz, Karl A. J Nat Prod . 2012 (Published online )

2. Reid, Carolyn S.; Farahat, Abdelbasset A.; Zhu, Xiaohua; Pandharkar, Trupti ; Boykin, David W.; Werbovetz, Karl A.; Bioorg Med Chem Lett . 2012 , 22(22), 6806-6810.

3. Zhu, Xiaohua; Pandharkar, Trupti ; Werbovetz, Karl Antimicrob Agents Chemother . 2012 , 56(3), 1182-1189.

4. Bahar, Mark; Deng, Ye; Zhu, Xiaohua; He, Shanshan; Pandharkar, Trupti ; Drew, Mark E.; Navarro-Vazquez, Armando; Anklin, Clemens; Gil,

Roberto R.; Doskotch, Raymond W.; et al Bioorg Med Chem Lett 2011 , 21(9), 2606-2610.

5. Wang, Michael Zhuo; Zhu, Xiaohua; Srivastava, Anuradha; Liu, Qiang; Sweat, J. Mark; Pandharkar, Trupti ; Stephens, Chad E.; Riccio, Ed; Parman, Toufan; Munde, Manoj; et al Antimicrob Agents Chemother . 2010 , 54(6), 2507-2516.

6. Bhasin, Deepak; Cisek, Katryna; Pandharkar, Trupti ; Regan, Nicholas; Li, Chenglong; Pandit, Bulbul; Lin, Jiayuh; Li, Pui-Kai Bioorg Med Chem Lett 2008 , 18(1), 391-395.

FIELDS OF STUDY

Major Field: Pharmacy

TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication ...... vi

Acknowledgments...... vii

Vita ...... ix

List of tables ...... xv

List of figures ...... xvi

Abbreviations ...... xix

Chapters:

1 Introduction 1.1 Leishmaniasis disease information ...... 1 1.1.1 Leishmaniasis: a neglected tropical disease ...... 1 1.1.2 Epidemiology ...... 3 1.1.3 Transmission and life cycle ...... 5 1.1.4 Clinical manifestations ...... 7 1.1.5 Current clinical treatments for leishmaniasis ...... 10 1.1.6 Neglected tropical diseases: drug discovery strategies, current drug targets and investigational drugs ...... 17 1.1.7 kDNA as anti-leishmanial drug target of aromatic diamidines ...... 22 1.2 Arylimidamides (AIAs) ...... 24 1.2.1 Discovery of arylimidamides as promising anti-leishmanial drug class . 24 1.2.2 Mechanism of action of diamidines and AIAs in protozoan parasites ..... 29 1.2.3 Objectives of the study...... 32

2. Mechanistic investigations of anti-leishmanial arylimidamides (AIAs): Comparative proteomic analysis 2.1 Introduction: Proteomic approach for mechanistic studies ...... 33 2.2 Materials and methods ...... 36 2.2.1 Parasites and culture conditions ...... 36 2.2.2 Assessment of cell viability and recovery after drug treatment ...... 36 2.2.3 Preparation of Leishmania cell extracts for 2D-DiGE analysis ...... 38 2.2.4 Sample labeling for DiGE ...... 39 2.2.5 Differential in gel electrophoresis ...... 40 2.2.5.1 Isoelectric focusing ...... 40 2.2.5.2 SDS PAGE ...... 41 2.2.6 Gel scanning and image analysis ...... 41 2.2.7 In gel digestion ...... 43 2.2.8 Protein identification ...... 43 2.2.8.1 Nano-LC/MS/MS ...... 43 2.2.8.2 Protein database search ...... 45 2.2.9 Mitochondrial membrane potential assay ...... 45 2.3 Results and Discussion ...... 46 2.3.1 Parasite viability after DB766 treatment ...... 46 2.3.2 Response of L.donovani to DB766 treatment based on 2D-DiGE analysis ...... 49 2.3.3 Effect of DB766 on mitochondrial membrane potential...... 56 2.4 Conclusions and future directions ...... 57

3 Mechanistic investigations of anti-leishmanial arylimidamides (AIAs): Transmission electron microscopy 3.1 Introduction: Transmission electron microscopy for mechanistic studies ...... 59 3.1.1 Rationale for protease and sterol metabolism hypotheses ...... 61 3.1.2 Parasitic proteases as drug targets...... 62 3.1.3 Sterol metabolism as drug target in Leishmania ...... 66 3.2 Materials and methods ...... 70 3.2.1 Transmission electron microscopy ...... 70 3.2.2 Leishmania lysate and OPB like serine protease assay ...... 71 3.2.3 Extraction and analysis of total Leishmania sterols by GC-MS ...... 72 3.3 Result and Discussion ...... 73 3.3.1 Ultrastructural alterations induced by DB766 in L.donovani axenic amastigotes ...... 73 3.3.2 Effect of DB766 on serine protease like OPB activity in L.donovani axenic amastigote lysate ...... 75 3.3.3 Effect of DB766 on Leishmania sterols ...... 77 3.4 Conclusions and future directions ...... 80

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4 Mechanistic investigations of anti-leishmanial arylimidamides (AIAs): Development and characterization of DB766 resistant Leishmania donovani 4.1 Introduction………………...... 82 4.2 Materials and methods ...... 86 4.2.1 Selection of a DB766 resistant Leishmania donovani cell line ...... 86 4.2.2 Transmission electron microscopy ...... 87 4.2.3 In vitro susceptibility studies ...... 87 4.2.4 Drug interaction assays: design, determination of FIC index and isobologram construction for analysis of nature of drug interactions ... 88 4.2.5 In vitro differentiation efficiency and growth curve ...... 89 4.2.6 Western Blotting …………… ...... 89 4.2.7 Real Time PCR………………...... 90 4.2.8 Culture conditions for CYP5122A1 half knockout Leishmania and in vitro susceptibility studies ...... 91 4.2.9 Hydroperoxide susceptibility assay ...... 92 4.2.10 Extraction and analysis of total sterols by GC-MS………………………92 4.3 Results and discussion ...... 93 4.3.1 Characterization of DB766 resistant Leishmania amastigotes ...... 93 4.3.2 Differentiation efficiency of L.donovani axenic amastigotes resistant to DB766...... 95 4.3.3 Drug susceptibilities of L.donovani axenic amastigotes resistant to DB766...... 97 4.3.4 Expression status of key sterol biosynthetic enzymes in L.donovani axenic amastigotes resistant to DB 766 ...... 100 4.3.5 Hydroperoxide susceptibility of L.donovani axenic Amastigotes resistant to DB766………………………………………...101 4.3.6 Sterol profile of DB766 sensitive and resistant Leishmania ……………102 4.3.7 Susceptibility of CYP5122A1 HKOs to DB766 and ketoconazole ...... 103 4.3.8 Drug interaction assays ...... 105 4.4 Conclusions ...... 108

5 DB766: Mechanistic conclusions, implications and future directions 5.1 Implications from proteomic and ultrastructural studies ...... 110 5.2 DB766 treatment induced alterations in the sterol composition and synergizes the anti-leishmanial action of posaconazole in Leishmania ...... 111 5.3 Implications from studies on DB766 resistant Leishmania ...... 112 5.3.1 Acquisition of resistance to DB766 alters the ultrastructure of Leishmania …………………………………………………………….. 112 6.3.2 Acquisition of resistance to DB766 alters drug susceptibility in Leishmania……………………………………………………………… …. 113 6.3.3 CYP5122A1, a CYP450 enzyme with a role in ergosterol metabolism, is a key determinant of susceptibility to DB766 antifungal azoles and oxidative stress in Leishmania …………………. .114 6.3.4 Acquisition of resistance to DB766 alters sterol composition without affecting ergosterol levels in Leishmania …………………….. .115

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6.3.5 Proposed model 1 for altered drug susceptibility and sterol metabolism in the light of reduced expression of CYP5122A1 in Leishmania ………………………………………………………….. .119 6.3.6 Proposed model 2 for altered drug susceptibility in the light Of reduced expression of CYP5122A1 in Leishmania …………………122 5.5 Conclusions……………………………………………………………………..123 5.6 Future directions ...... 124

References ...... 127

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LIST OF TABLES

Table Page

1.1 Main Leishmania species associated with different clinical outcomes in leishmaniasis ...... 8

1.2 Essential and validated drug targets with promising anti-leishmanial Leads ...... 19-21

2.1 Gel loading regimen for 2D DiGE ...... 40

2.2 Parasite viability after drug treatment ...... 48

2.3 Protein characterization and predicted localization data for protein isoforms identified in this study ...... 53-54

4.1 Susceptibility profiles of wild type and DB766 resistant Leishmania donovani axenic amastigotes 72h post drug treatments ...... 98

4.2 Sterol composition of WT and DB766 resistant Leishmania as determined by GC-MS ...... 103

4.3 Mean ΣFICs from two independent assays for studying interaction of DB766 with partner drugs ...... 105

LIST OF FIGURES

Figure Page

1.1 Geographical distribution of VL ...... 4

1.2 Geographical distribution of CL ...... 4

1.3 Life cycle of Leishmania ………………… ...... 6

1.4 Clinical manifestations of CL, MCL and VL ...... 9

1.5 Chemical structures of clinically approved anti-leishmanial drugs ...... 12

1.6 Different strategies for NTD drug discovery...... 17

1.7 A structure activity map of AIAs ...... 26

1.8 Structures of DB766, DB1960, DB1852 and DB1955…………………………..27

2.1 Work flow in a 2D- DiGE experiment… ...... 35

2.2 Representative 2D-DiGE gels showing L.donovani proteins ...... 51

2.3 Relative protein abundance measurements using 2D DiGE ...... 52

2.4 Basic steps in mitochondrial membrane potential dependent protein import in the mitochondrial matrix ...... 55

2.5 Effect of DB766 on Leishmania mitochondrial membrane potential ...... 56

3.1 Clans and families of L.major peptidases...... 63

3.2 Intracellular location of enzymes involved in early and late steps of sterol biosynthetic pathway in trypanosomatids ...... 67 xvi

3.3 Sterol biosynthetic pathway in trypanosomatids ...... 69

3.4 Electron micrographs of L. donovani axenic amastigotes ...... 73

3.5 Effects of DB766 on serine protease like oligopeptidase B activity in a Leishmania donovani axenic amastigote lysate and purified bovine β-trypsin .... 77

3.6 GC-MS traces of Leishmania sterol fractions ...... 79

4.1 Mode of action and resistance to pentavalent antimonials in Leishmania ...... 83

4.2 Modes of action and resistance to clinically approved anti-leishmanial drugs….84

4.3 Generation of a DB766 resistant L. donovani cell line ...... 94

4.4 Electron micrographs of wild type (A), DB766 resistant Leishmania donovani axenic amastigotes without DB766 pressure (B) and with DB766 pressure (c)……………………………………….94

4.5 ...... Growth curve of promastigotes adapted from wild type and DB766 resistant axenic amastigotes, over a period of five days in culture...... 96

4.6 Transformation efficiency of wild type (black histograms) and DB766 resistant axenic amastigotes (gray histograms) over a period of five days in culture as determined by hemocytometer based counting every 24h for 72h ...... 97

4.7 Quantitative real time PCR analysis of relative expression levels of CYP51, CYP5122A1, SCMT and CPR in DB766 sensitive and resistant L.donovani …………………………………………………………….100

4.8 Expression profiles of CYP5122A1 and CYP51 levels in wild type and 766R L. donovani axenic amastigotes………………………………………….101

4.9 Percentage viabilities of WT (black histograms) and DB766 resistant parasites (gray histograms) in response to treatment with 30 µM tBuOOH ...... 102

4.10 Susceptibility profile of wild type and CYP5122A1 HKO Leishmania donovani promastigotes to DB766 as determined by propidium iodide staining by flow cytometry ...... 104

4.11 Susceptibility profile of wild type and CYP5122A1 HKO Leishmania donovani promastigotes to Ketoconazole as determined by propidium iodide staining by flow cytometry ...... 104

4.12 Isobologram showing drug interaction against L.donovani axenic amastigotes

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at IC 50 concentration…. ………………………………………………………..106

5.1 Model for proposed routes to ergosterol biosynthesis in yeasts………………..118

5.2 Proposed model for role of CYP5122A1 in ergosterol metabolism in Leishmania and in ketoconazole susceptibility in DB766 resistant Leishmania …………………………………………………………….121

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LIST OF ABBREVIATIONS

Abbreviation Term

°C Degrees Celsius

3’-UTR 3’-untranslated region

AIA Arylimidamide

AQP-1 Aquaglyceroporin-1

BCA Bicinchonic acid

BPQ-OH Biphenylquinuclidine

CCCP Carbonyl cyanide m-clorophenylhydrazone

CD Circular Dichroism

CHAPS (3-[(3-chola-midopropyl) dimethylammonio]-1-

propanesulphonic acid)

CID Collision-induced dissociation

CL Cutaneous Leishmaniasis

CPA Cysteine protease A

CPB Cysteine protease B

CPR Cytochrome P450 reductase

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CRK-3 Cyclin dependent kinase

CYP51 Sterol 14 α-demethylase

DA Dalton

Ddi-1 DNA damage-inducible protein

DHFR-TS Dihydrofolate reductase-thymidine synthase

DiGE Difference in Gel Electrophoresis

DMSO Dimethyl sulfoxide

DNA 2΄-deoxyribonucleic acid

DNDi Drugs for Neglected Tropical Diseases Initiative

DTT Dithiothreitol

FICs Fractional Inhibitor Concentrations

FPPS Farnesyl Pyrophosphate synthase

FT/SQS Farnesyl transferase/ Squalene synthase

GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase

GC Gas Chromatography

GC-MS Gas Chromatography Mass Spectrometry

GCS Glutamylcysteine synthetase

GNNTD Global Network for Neglected Tropical Diseases

HKO Half knockout

HTS High Throughput Screening

IC 50 Inhibitory concentration 50%

IS Internal Standard

M Molar

MRPA Multidrug Resistance Protein A

Nano-LC/MS/MS Capillary-liquid chromatography-nanospray tandem

mass spectrometry

PBS Phosphate Buffered Saline

PKDL Post Kala Azar Dermal Leishmaniasis

PTR1 Pteridine reductase 1 kDNA Kinetoplastid DNA

MBCL Methylbenzethonium chloride

MCL Mucocutaneous Leishmaniasis

MVAK Mevalonate Kinase

NTD Neglected Tropical Diseases

OPB Oligopeptidase B

PgP P glycoprotein

PMT Photomultiplier Tube

PVDF Polyvinylidine fluoride

RIPA Radioimmunoprecipitation Assay

ROS Reactive oxygen species

SBI Sterol Biosynthesis Inhibitor

SCMT Sterol C-24 methyl transferase

SCMT-A Sterol C-24 methyl transferase-A

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

SEO Squalene epoxidase

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SMT Sterol-24-methyl transferase tBuOOH tert-butylhydroperoxide

TBST Tris Buffered Saline

TEM Transmission Electron Microscopy

TMRE Tetramethylrhodamine ethyl ester perchlorate

TMSI Trimethylsilyl imidazole

TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone

TryR Trypanothione reductase

VL Visceral Leishmaniasis

WHO World Health Organization

WRAIR Walter Reed Army Institute of Research

WT Wild Type

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CHAPTER 1

INTRODUCTION

1 .1 Leishmaniasis disease information

1.1.1 Leishmaniasis: a neglected tropical disease

“Neglected tropical diseases” (NTDs) comprise a large group of ancient infectious diseases that have afflicted human kind for centuries. Development of effective control tools through considerable research efforts lead to eradication of these diseases in the developed nations. However, these neglected diseases continue to significantly impact the health and socio-economic development of impoverished populations living in the developing countries. By the definition of the World Health Organization (WHO), NTDs are the diseases that almost exclusively impact poor people living in the low income countries. These infectious diseases are described as “neglected” due to several reasons:

1) The disease(s) are concealed at the community level as some of these can be deforming and disabling and hence associated with intense social stigma. At the national

1 level, these diseases are often poorly documented or rarely given a high priority by the finance or health ministries in the endemic countries. 2) Limited attention, up until recently, has been given to these diseases by the developed countries. 3) These disease are debilitating and affect millions of people but cause comparatively few deaths. This low mortality limits international attention and funds. 4) The development of effective drugs and vaccines for the “market that cannot pay” has negligible incentives for pharmaceutical companies (247).

However, in recent years there has been considerable improvement in the outlook for

NTDs. This is mainly due to increased awareness about the impact of NTDs on global health and economy. This increased awareness has led to important global health initiatives like the WHO Neglected Tropical Diseases Program, the Global Network for

Neglected Tropical Diseases (GNNTD), and the Neglected Tropical Diseases Initiatives

(NTDI). Partnerships between governmental organizations and foundations like the Bill and Melinda Gates Foundation, the Wellcome Trust, the World Bank and others has been a source of financial support for NTD R & D. Collaborative R & D efforts between non- profit organizations like the Drugs for Neglected Tropical Diseases Initiative (DNDi) and giant pharma companies might yield new treatments for NTDs in the near future (38, 127,

195).

The list of diseases designated NTDs by WHO, although not exhaustive, includes Buruli ulcer, Chagas diseases, dracunculiasis, human African trypanosomiasis, leishmaniasis,

2 leprosy, lymphatic filariasis, onchocerciasis, schistosomiasis, soil transmitted helminthiasis, yaws and other zoonotic diseases (248).

1.1.2. Epidemiology

Within the above mentioned list of NTDs, leishmaniasis is estimated to cause the ninth largest burden of disease (6). Leishmaniasis is a diverse and complex vector borne infection caused by over twenty different species of protozoan parasites of the genus

Leishmania (174). According to a recent report, leishmaniasis is endemic in 98 countries across five continents. While 90% of the visceral leishmaniasis (VL) cases occur in developing countries like Bangladesh, Brazil, India, Nepal and Sudan (Figure 1.1), 90% of the cases of cutaneous leishmaniasis (CL) occur in Afghanistan, Brazil, Iran, Peru,

Saudi Arabia and Syria (Figure 1.2). The official global incidences for VL and CL are over 58,000 and 220,000 cases each year respectively. The sparsely available mortality data suggests a case fatality of 10% i.e. 20,000 to 40,000 deaths due to leishmaniasis per year (6).

Figure 1.1 Geographical distribution of VL [Source: WHO website (244)]

Figure 1.2: Geographical distribution of CL

[Reprint with permission from Elsevier: The Lancet Infectious Diseases (194), copyright

(2007) Elsevier]

Leishmaniasis has a wider geographical distribution pattern than ever before as several endemic and previously non-endemic countries have registered a marked increase in the incidences of leishmaniasis. This increase could be attributed to several risk factors such as climatic changes and migration of non-immune populations from rural endemic areas to suburban and urban areas either due to occupational reasons, international tourism, military operations and political unrest (76). Other than deteriorating socio-economic factors, malnutrition also poses an additional risk factor within endemic countries as the disease affects the immune system (77). Since leishmaniasis is an opportunistic infection, emerging incidences of Leishmania /HIV co-infection is being considered as major risk factor for the increased incidence of the disease. Leishmania /HIV co-infection has already been reported in 35 countries around the world (78).

Together all these factors make leishmaniasis a complicated and difficult disease to treat.

Nonetheless better understanding of these risk factors is essential to develop better disease prevention, control and treatment strategies.

1.1.3 Transmission and life cycle

Leishmaniasis is diverse and complex vector borne infection caused by an obligate intracellular protozoan parasite of the genus Leishmania (family Trypanosomatidae, order

Kinetoplastida). Amongst the 20 species of Leishmania that have been identified, some of them cause infections in animals which then act as reservoirs. Over 15 species of

Leishmania are known to cause infections in humans and are categorized into two major

5 groups: 1) Old world leishmaniasis, caused by L. aethipica, L. tropica, L. major and the

L. donovani complex ( L. donovani and L. infantum ), and 2) new world leishmaniasis, caused by L. amazonensis, L. mexicana and the L. viannia complex ( L. braziliensis and L. guyanensis . While Phlebotomine species transmit Leishmania in Europe, Asia and

Africa, Lutzomyia transmits the parasite in the Americas (174).

Figure 1.3: Life cycle of Leishmania

[Source: CDC website (40)]

The digenetic life cycle of Leishmania alternates between the extracellular developmental

stage (the promastigote form) in the gut of the insect vector, a female phlebotomine

sandfly, and the intracellular development stage (the amastigote form) in the host

6 mammal (Figure 1.3). The life cycle begins as a parasitized sandfly draws a blood meal and in the process injects ~ 100-1000 promastigote forms of the parasite into the human host via the proboscis. These promastigotes are then quickly phagocytized by the macrophages, neutrophils and dendritic cells where they transform into amastigotes. The amastigotes are ovoid in shape with a short flagellum and undergo considerable metabolic changes to adapt to the hostile conditions within the host cell. Inside the macrophages, the amastigotes multiply and increase in numbers by binary fission until the cell finally bursts. These parasites then infect other macrophages and continue the cycle. The life cycle is complete when the sandfly vector ingests amastigote infected macrophages when they feed on a human host during a blood meal.

1.1.4 Clinical manifestations

The complex interaction between the genetic background of the infecting Leishmania spp and the immune status of the host determines the clinical manifestations of the disease.

The outcome is a broad spectrum of disease ranging from self-healing, localized skin lesions to diffuse involvement of the reticulo-endothelial system. In spite of the pleomorphic presentation of the disease, leishmaniases affecting humans can be subdivided in to three major clinical forms: cutaneous (CL), mucocutaneous (MCL) and visceral (VL). Within these three categories, a number of atypical and rare variants exist.

Sometimes more than one clinical syndrome can be produced by single Leishmania species and a single syndrome could be caused by multiple species (Table 1) (15)

CL MCL VL

Main clinical L. major, L. tropica, L. killicki, L. L. braziliensis, L. donovani, L. form aethiopica, L. mexicana, L. L. panamensis, infantum/ amazonensis, L. braziliensis, L. L. amazonensis, chagasi, L. guyanensis, L. infantum a, L. L. guyanensis tropica shawi a, L. naiffi a, L. lainsoni a, L. donovani a

Specific forms L. donovani : PKDL, of CL L. mexicana : ‘chiclero ulcer’, L. peruviana :‘uta’, L. guyanensis : ‘pian-bios’, L. mexicana a, L. aethiopica a, L. amazonensis a: DCL, L. tropica a, L. aethipica a, L. braziliensis b, L. panamensis b, L. guyanensis b, L. amazonensis b: recidivans and disseminated leishmaniasis Immunosup- Most species L. donovani a, L. L. donovani pressed infantum b,c , L. L. infantum c, L. patients major b,d , L. braziliensis b, L. (mainly HIV guyanensis b, L. mexicana b, L. positive) amazonensis b amazonensis b aRarely. bExceptionally. cIncluding ‘dermotropic zymodemes’. dLocal extension to mucosa.

Table 1.1: Main Leishmania species associated with different clinical outcomes in leishmaniasis.

[Reprint with permission from John Wiley and Sons: Clinical Microbiology and Infection

(15), copyright (2011) John Wiley and Sons]

The cutaneous form of leishmaniasis is more widespread and can present itself as a

variety of chronic ulcerative self-healing skin lesions or non-ulcerative disseminated skin

nodules (Figure 1.4, panel A). The pleomorphic nature of these lesions makes diagnosis

of CL complicated and delays the initiation of treatment. Skin lesions associated with CL

can persist for months before healing spontaneously and often leave hypopigmented

8 ineradicable scars that could have dramatic social consequences. Depending on the infecting Leishmania species and the host immunity, CL may evolve to develop satellite lesions, in rare cases causing enlargement of the original lesions to form recidivans leishmaniasis and in extreme cases disseminate to form DCL. The diffuse non-ulcerative skin nodules associated with DCL fail to heal spontaneously, reappear as satellite lesions on face and extremities, often leave unattractive scars and sometimes even cause death

(15).

In MCL, the parasite causes extensive destruction of mucosal linings of the oral, nasal and pharyngeal cavities leading to unsightly disfigurement of the face that is often accompanied by intense social stigma (Figure 1.4, panel C). Mortality due to MCL is very low but the resulting morbidity is substantial in terms of the social consequences.

Figure 1.4 Clinical manifestations of CL (panel A), MCL (panel B) and VL (panel C) [Source: WHO website (243, 245, 246)]

VL is a characterized by a systemic parasitic infection that spreads through the lymphatic system to organs like the liver and spleen (Figure 1.4, panel C). The onset of symptoms include fever, malaise, loss of body weight, hyperpigmentation of the skin (hence the term ‘kala azar’ for Old World VL), hepato-splenomegaly and or lymphadenopathies and pancytopenia. VL represents the most severe form of leishmaniasis and can be fatal if left untreated. With an estimated fatality rate of 20,000-40,000 deaths per year, it stands second after malaria in terms of fatalities caused by parasitic disease (15, 74). A number of people in India and Africa, in spite of successful response to treatment, develop post kala azar dermal leishmaniasis (PKDL). PKDL usually develops within 1-2 years after apparent clinical cure and can persist for as long as 20 years. The condition is characterized by the presence of hypo/hyper pigmented macular, papular or nodular rash on the face, trunk and extremities. PKDL develops in about 50-60% and 5-10% of VL patients in Sudan and India respectively (15).

Although most cases of Leishmania -HIV co-infection have been reported in concert with

VL, several countries have reported their co-existence with CL, DCL and MCL. The

clinical presentation of leishmaniasis during Leishmania -HIV infection is diverse,

ranging from being asymptomatic to the presence of highly pleomorphic lesions (15).

1.1.5 Current clinical treatments for leishmaniasis

Although there has been considerable progress in developing vaccines for the

immunotherapy of leishmaniasis, none of these vaccine candidates are fully effective

(68). In the absence of an effective vaccine, chemotherapy remains the prime strategy for management of the disease. Clinically approved drugs for the management of VL and or

CL (Figure 1.5) include pentavalent antimonials in the form of sodium stibogluconate

(Pentostam®) and meglumine antimoniate (Glucantime®), lipid formulations of

Amphotericin B (Fungizone®, Ambisome®, Amphocil®, Abelcet®), miltefosine, paromomycin, and pentamidine. Miltefosine was the first oral drug approved for treating

VL in India (217). Paromomycin was recently registered for managing VL in India (218) and is also available as a 12% topical formulation for CL (9, 58, 83). Pentamidine is approved for certain cases of CL in South America only (163). Clinically approved treatment options for CL as opposed to VL are limited not only due to differences at the species level but also in required pharmacokinetic properties of the drugs (distribution to viscera in VL vs the dermis in CL) (56).

Figure 1.5 Chemical structures of clinically approved anti-leishmanial drugs

Pentavalent antimonials have remained as the first line of treatment for all forms of leishmaniasis for more than 60 years. These drugs require parenteral administration over extended periods of time (from a few weeks to a few months), making the treatment both painful and expensive. Further, the use of antimonials is now limited due to the extensive development of drug resistant strains in India (65% failure and/or relapse rates) (196).

Another well recognized drawback is that the biologically active species and molecular and cellular mechanism of action of these drugs is poorly understood. Serious adverse reactions like cardiotoxicity, hepatic and renal dysfunction, shock and (rarely) sudden death have been documented with the use of these drugs (66, 155, 169, 241).

Pentamidine isethionate, an aromatic diamidine, was used as a second line of treatment for VL in India and is also approved for the treatment of some types of CL cases in South

America (163, 165). However, its costs, diminishing efficacy over a period of time and serious toxicity issues such as irreversible insulin-dependent diabetes, cardiotoxicity and nephrotoxicity has led to its discontinuation for treatment of VL in India (58, 241).

Amphotericin B (3), a polyene antifungal antibiotic, is also widely used in the treatment of VL. Given the loss of effectiveness of pentavalent antimonials due to wide spread drug resistance in India, amphotericin B is now a first line drug for management of VL in this region. It is also approved for treating CL, MCL and PKDL. While Fungizone, the micellar formulation of amphotericin B is highly effective (cure rate > 95%), requirement of prolonged course of intravenous administration (30-40 days) and drug and infusion related adverse effects limits its use. Abelcet and AmBisome, the lipid based formulations offer the advantage of high efficacy, significantly reduced course of treatment (3-5 days) and less adverse effects compared to Fungizone (222, 223).

However, requirement for i.v. administration that necessitates hospitalization makes the treatment costs prohibitive (241). Further limited temperature stability of the formulations (the storage condition recommended by the manufacturer is 25 °C) pose a barrier to the treatment from reaching to the patient population in the tropical endemic areas. Cases of VL that are clinically unresponsive to amphotericin B due to development of drug resistant strains are beginning to appear (186).

The outstanding anti-leishmanial activity of the anticancer alkylphospholipid derivative miltefosine led to its clinical approval as the first oral drug for treating VL in India. The drug is highly effective in antimony unresponsive cases of VL. Further, miltefosine is proven to be effective and approved for the treatment of certain cases of CL (212, 213,

219). Issues of concern with the use of this drug include 1) its teratogenic potential and long half-life that forbid its use in women of child bearing age, who may become pregnant, 2) poor patient compliance due to the prolonged course of treatment (28 days),

3) over-the-counter availability and misuse leading to an increased risk of the development of resistance. Resistance to miltefosine develops quickly in vitro (57, 175).

Although clinical resistance due to point mutations in miltefosine specific targets is not yet verified, clinically unresponsive cases of VL following relapses have appeared for miltefosine. As miltefosine is also known to eliminate parasites in VL by modulating host immune response, involvement of host factors such as host immunity in the development of tolerance to the drug has been suggested as the reason for the observed unresponsiveness to the miltefosine treatment (25, 182).

In a randomized controlled phase III study paromomycin (15 mg/kg for 21 days by intramuscular route) demonstrated efficacy for treatment of VL in India by producing a cure rate of 94% (218). A parenteral formulation of paromomycin is now registered for use against VL only in India. However the same regimen failed to demonstrate clinical efficacy (cure rate < 50%) in a multi-center, open-label, randomized study in Sudan

(101). Although the cure rate increased to 85% with longer duration (15 mg/kg for 28 days) and higher dose regimen (20 mg/kg for 21 days) of paromomycin in a phase II

14 study in Sudan (156), the use of paromomycin as a monotherapy is precluded for treatment of Sudanese VL. Despite its availability the clinical efficacy of several paromomycin formulations for treatment of CL has been variable. While a topical formulation of 15% paromomycin with 12% methylbenzethonium chloride (MBCL) in white paraffin is effective in treating CL (83), increasing reports of skin irritations due

MBCL has limited the usefulness of this preparation (7, 8, 83, 84, 122) for treatment of

CL. The second topical formulation containing 15% paromomycin and 10% urea in white paraffin was non-irritating. Given the problems in the design of studies for self-healing diseases like CL, the clinical cure rates with the ointment were only slightly better than placebo in Iran and Tunisia (9, 21, 108). A recent, third generation hydrophilic formulation containing 15% paromomycin and 0.5% gentamycin demonstrated higher efficacy with a clinical cure rate of 94%, compared to 71% in the placebo control group and was tolerated well (20). Of all the treatments available for VL, paromomycin is the cheapest and is being considered as an alternative to antimonials and expensive amphotericin B parenteral formulations (164).

Sitamaquine, an oral 8-aminoquinoline, demonstrated outstanding anti-leishmanial activity in preclinical studies (120, 255). However, it failed to show cure rates > 90% in the subsequent extensive phase II trials for VL in India and Africa (112, 238) and hence is no longer in development. Oral antifungal azoles (fluconazole, ketoconazole, itraconazole and posaconazole) have demonstrated variable effectiveness in clinical trials depending on the Leishmania species involved and the geographical location. While clinical trials demonstrated the efficacy of fluconazole for CL due to L. major and L.

15 braziliensis (85, 214), ketoconazole was effective against CL caused by L. mexicana and

L. panamensis (160, 199). Allopurinol, either used alone or in combination with pentavalent antimonials or pentamidine, demonstrated clinical effectiveness in treating

South American CL and Indian VL (111, 200, 225). To circumvent the issues associated with the toxicity and cost of approved treatments and the emergence of drug resistant strains, DNDi and other organizations are actively pursuing projects to evaluate the preclinical safety and efficacy of potential drug combinations. Although several other combination studies have been tried and more are underway, none of them have been clinically approved for treating VL or CL thus far. Immunotherapy including vaccines and immunomudulators for the prevention and control of leishmaniasis has also been the subject of extensive clinical research. Topical imiquimod, a imidazoquinoline TLR7 agonist and US-FDA approved immune response modifier, in combination with pentavalent antimony showed a significantly higher cure rate in Peruvian CL than pentavalent antimony alone (146). Recent reports on phase I clinical trials with LEISH-

F1+MPL-SE vaccine candidate have demonstrated its safety and immunogenicity against

CL, MCL and VL in Brazil, Peru and India respectively (41, 129, 159).

While significant progress has been made in terms of the recent approval of miltefosine and paromomycin for treating leishmaniasis, all the existing drugs have some limitations including cost, patient compliance, safety, efficacy, toxicity and the potential of emerging drug resistant strains. Clearly the need for the discovery and development of new anti- leishmanial agents is urgent.

1.1.6 Neglected tropical diseases: drug discovery strategies, current drug targets and investigational drugs

Despite the challenges associated with the drug discovery for NTDs in general, the research support from philanthropic groups (Wellcome Trust, the Bill and Melinda Gates

Foundation etc.) and work conducted by non-profit organizations (DNDi, WRAIR,

CPDD) and public-private partnerships (with pharma partners such as Novartis and Pfizer etc.) have rejuvenated neglected diseases research (56). Figure 1.6 shows different strategies for NTD drug discovery.

Figure 1.6 Different strategies for NTD drug discovery

[Reprint with permission from Nature Publishing Group: Nature Reviews Drug

Discovery (162), copyright (2006), Nature Publishing Group]

The three major strategies for tropical diseases drug discovery including leishmaniasis are 1) ‘Label extension/therapeutic switching’- which is extending indications of existing treatment for human/animal disease conditions to tropical diseases, exemplified by therapeutic approval of the antifungal agent amphotericin B for leishmaniasis, 2) ‘piggy back strategy/drug-target repurposing’- exemplified by the discovery of miltefosine, an anticancer agent with potent anti-leishmanial activity, and 3) ‘ de novo ’ drug discovery,

which involves identification of synthetic or natural product derived leads. This strategy

uses the knowledge of potential targets for rational drug design and or high throughput

screening (162). Data from the completed genome of L. major, L. braziliensis and L.

infantum , functional genomic studies (target essentiality and validation based on gene

knockout/knockdown/overexpression studies) and protein structural data provide an

increased understanding of the metabolic pathways and enzyme targets. The TDR drug

target database (http://tdrtargets.org), an open access tropical disease pathogens database,

has been created to integrate all this diverse information and manually curate inhibitors

and targets. Availability of this information to researchers is expected to facilitate NTD

drug discovery through identification and prioritization of potential drug targets (59).

Once the drug target is identified, several combinations and iterations of strategies such

as virtual screening methodologies, structure based drug design and target and or whole

organism based high throughput screening (HTS) of chemical libraries can be applied for

lead identification and optimization. A search based on essentiality and validation data

(obtained using orthologs in other species) and druggability led to a list of 44 candidate

Leishmania drug targets in the TDR drug target database. Examples of ‘essential’ and validated drug targets that have led to identification of some promising anti-leishmanial

18 leads include tubulins, proteases (cysteine proteinase A or CPA), protein kinases (cyclin- dependent cdc2-related serine/threonine protein kinase or CRK3), enzymes involved in sterol and isoprenoid metabolism (sterol 14 α demethylase or CYP51, farnesyl pyrophosphate synthase or FPPS, sterol C24 methyl transferase or SCMT, farnesyl transferase/squalene synthase or FT/SQS), enzymes involved in anti-oxidant metabolism

(trypanothione reductase or TryR), folate/pteridine metabolism (dihydrofolate reductase- thymidylate synthase or DHFR-TS and pteridine reductase 1 or PTR1) and the glycolytic pathway (glyceraldehyde 3-phosphate dehydrogenase or GAPDH) (Table 1.2).

Drug Metabolic Validation Druggability Inhibitors target pathway CYP51 Ergosterol Chemical 0.8 Antifungal azoles- biosynthesis inhibition by fluconazole, azoles in vitro(19) ketoconazole, and in vivo - CL itraconazole, and VL(3) posaconazole

FPPS Isoprenoid Chemical 0.8 Risedronate, biosynthesis inhibition of target Alendronate in cell free system, inhibition of growth in vitro and in vivo, overexpression confers resistance to the drug (133, 201, 254) SCMT Ergosterol Chemical 0.6 Azasterols biosynthesis inhibition in cell free system, inhibition of growth in vitro GAPDH Glycolytic Chemical 0.8 Adenosine analogs pathway inhibition in cell free system Continued

Table 1.2 Essential and validated drug targets with promising anti-leishmanial leads 19 19 Table 1.2 continued TryR Thiol Genetic disruption 0.9 Pentavalent antimonials metabolism (81), chemical (62), Arsenicals, inhibition in cell Glutathione analogs free system, (63), inhibition of quaternary growth in vitro arylalkylammonium-2- qmino-4-chlorophenyl sulfides and N-acyl analogs of 2- amino-4-chlorophenyl sulfide (172), phenothiazine analogs (22), 9,9-dimethyl xanthene tricyclics (43). DHFR- Folate & Genetic disruption 1 2,4- TS Pteridine (60), chemical diaminoquinazolines, metabolism inhibition in cell 5 substituted 2,4- free system and diaminopyrimidines inhibition of (211), growth in vitro 2,4-diaminoquinazolines (117), PTR1 Folate & Genetic disruption 0.7 2,4-dihydropyrimidone Pteridine (158), chemical analogs (210), 2,4- metabolism inhibition in cell diaminopteridines and free system and quinoxalines (39), inhibition of Quinaxolines (139), growth in vitro and piperidine-pteridine in vivo (115) derivatives (52), Diverse set nonfolate structures (88) CPA Genetic disruption 0.6 Cystatin (69), Aziridine- (75), chemical 2,3-dicarboxylates inhibition in cell (180), epoxysuccinates free system and (51) inhibition of growth in vitro and in vivo

FT/ Sterol Chemical 0.8 Allylamines- terbinafine SQS metabolism inhibition in cell (256), quinuclidine and protein free system and derivatives (228), farnesyla- growth inhibition ER119884 and ER5700 tion in vitro (87) CRK-3 Genetic disruption 0.9 2,6-disubstituted purines (104), chemical and 3,7-disubstituted Continued 20

Table 1.2 continued

inhibition in cell pyrazolo [4,3d] free system and pyrimidines (47), inhibition of Indirubins (99) growth in vitro and in vivo Tubulin N/A chemical 1 3,5-Dinitro inhibition in a cell sulfanilamides analogs free system (26) DNA Diamidine analogs of pentamidine (249)

In addition to the target based approaches and HTS of compound libraries, traditional screening of plant derived natural products has yielded novel and potent anti-leishmanial leads. The following phytochemicals, belonging to different structural classes, have demonstrated potent anti-leishmanial activity in vitro and in vivo: 1) licochalcone A, a chalcone from Glycerrhiza spp (efficacy in mouse CL and mouse and hamster VL model)

(42), 2) amarogentin, a secoiridoid glycoside from S. chirata (efficacy in hamster VL

model) (192), 3) 2-substituted quinolines from G. longiflora (efficacy in mouse CL

model) (94), 4) clerodane diterpenes from P. longifolia (oral efficacy in hamster VL

model) (147), 5) Momordicatin, a 4-(o-carboethoxyphenyl)-butanol from M. charantia

(efficacy in hamster VL model) (100), 6) furoquinoline alkaloid and coumarins from H.

apiculata (efficacy in mouse CL model) (89), 7) peganine hydrochloride, harmaline and

harmine from P. harmala (efficacy in mouse VL and CL model) (118, 188) and 8)

coumarins from C. brasiliense (efficacy in mouse CL model) (224).

1.1.7 kDNA as anti-leishmanial drug target of aromatic diamidines

The kinetoplast is a concave, elongated disc shaped organelle formed by a highly unusual

DNA network within the extended, convoluted mitochondrion of all the flagellated protozoans belonging to the order Kinetoplastida (209). kDNA is an attractive drug target because it possesses several unusual features, including 1) a unique catenated presentation of kDNA maxicircles and minicircles that encode rRNA, mitochondrial proteins and guide RNAs for mRNA editing respectively, 2) curved double helical structure and relatively abundant A-T rich sequences in the kDNA minicircles compared to nuclear DNA, 3) affinity of DNA binding cationic drugs to these sequences and 4) absence of cationic histone proteins that would otherwise hinder the binding of cationic drugs (30, 209). Cationic DNA intercalating dyes such as ethidium bromide and acridines are known to induce loss of kDNA structure (dyskinetoplasty) and block growth in trypanosomatids by inhibiting kDNA dependent enzymes and replication (30, 209). kDNA is thus a validated target for anti-kinetoplastid drug discovery. Anti-trypanosomal and anti-leishmanial activities of cationic diamidines such as pentamidine, propamidine, and diminazine aceturate have been attributed in part to their affinity for A-T rich sequences within the minor groove of kDNA (82). Altered topology of kDNA following electrostatic interaction between the negatively charged nucleic acid back bone and positively charged diamidines is believed to interfere with the normal topoisomerase II function resulting in replication errors, formation of kDNA-toposiomerase II cleavable complex (topoisomerase poisoning) and finally destruction of the kDNA network (205).

Since kDNA is known to encode several mitochondrial proteins associated with cellular

22 respiration, loss of mitochondrial genes due to kDNA damage is thought to results in ultimate collapse of mitochondrial structure and function (71). Alteration in the mitochondrial membrane potential and ultrastructure (swelling of the mitochondrion, disintegration and loss of the kDNA network) due to pentamidine treatment in

Leishmania supports the above proposed mechanism of action of pentamidine and other diamidines (55, 249).

Pentamidine displays good in vitro activity against extracellular pathogens like African trypanosomes and is clinically approved for treating HAT and CL in South America.

However, several factors like poor bioavailability, efficacy and toxicity both in vitro and in vivo have limited the use of pentamidine as an effective anti-microbial agent, not only against African trypanosomes but also against intracellular pathogens like Leishmania

and T. cruzi (239). To overcome these drawbacks the CPDD has synthesized and evaluated series of diamidine analogs for their anti-parasitic activity against several pathogens including Leishmania. Although many of these diamidines displayed promising in vitro activity against Leishmania axenic amastigotes (10-12, 107, 136), the few that were tested against intracellular Leishmania were found to be inactive (27). In vitro (intracellular Leishmania ) and in vivo efficacy evaluation for most of these diamidines as anti-leishmanial candidates has not been documented. However, previous reports of the limited in vivo efficacy of pentamidine and other diamidines preclude their broad clinical utility for management of CL and VL (102, 131, 215). The dicationic nature of these molecules limits their permeability across biological membranes, posing a major challenge for developing such agents as orally effective drugs (240).

1.2 Arylimidamides

1.2.1 Discovery of arylimidamides as promising anti-leishmanial drug class

Structural modification to mask the dicationic amidine function of diamidines, by attachment of the ‘imino function’ of the amidine to an ‘anilino’ nitrogen, resulted in discovery of new class of diamidines termed ‘arylimidamides (AIAs)’, previously also known as ‘reversed amidines’. In 2003, Stephens et al. showed that AIAs have extraordinary anti-parasitic activity (submicromolar IC 50 s), especially against intracellular pathogens like T. cruzi and Leishmania (216) . According to Wang et al. , the

superior activity of AIAs can be attributed to their significantly improved physico-

chemical properties compared to that of diamidines. AIAs, with their lower pKas (~7 vs

11.6 of pentamidine) and higher lipophillicity (4.12 vs <-2.0 for pentamidine), have a

better chance of reaching the intracellular pathogens by crossing host cell and

phagolysosomal membranes (237). Thus far several bis-AIAs (compounds containing

two terminal heteroaromatic rings attached to the diphenyl furan linker via imidamide

groups) bearing different structural motifs have been synthesized and evaluated against

intracellular Leishmania. The initial series of eight symmetrical bis-AIAs synthesized by

Stephens et al. that showed promising activity against intracellular L. donovani (IC 50 range 0.15-3.6 µM) had different substitutions on the phenyl rings of the diphenylfuran linker (216). In 2011, Collar et al. reported the biological activity of 55 symmetrical bis-

AIAs with different substitutions on and or modifications in 1) the phenyl rings of the

diphenyl furan linker, 2) the terminal heteroaromatic rings, and 3) the furan linker against

L. amazonensis intracellular amastigotes. Compounds with 2-pyridyl terminal groups and large hydrophobic substituents (cyclopentyloxy or trifluroethoxy) on the diphenyl furan linker group had potent activity (IC 50 = 1.4 µM and 1.1 µM respectively, against intracellular L. donovani ). However, sterically bulky substituents (benzyloxy) reduced the potency (IC 50 = 4.5 µM against intracellular L. donovani ). With respect to terminal

groups, compounds with 2-pyridyl termini were superior to their 2-pyrimidyl, 2-

pyrazinyl, 4-imidazolyl, and 3-pyridazinyl counterparts. However, compound with 2-

methyl-5-thiazolyl terminal group retained potent activity. Also a compound with 2-

pyridyl terminus, isopropoxy substituent and a thiophene linker retained potent activity

(50). Recently Banerjee et al. reported the effect of different linker groups on the potency

of bis-AIAs against intracellular Leishmania. Within this subclass replacement of the

central furan with a 1,4-phenylenediamine linker resulted in a dramatic loss of activity

(IC 50 range- 5 to > 10 µM against intracellular L. amazonensis ), while compounds with linkers such as 4,4’-biphenyl, 4,4’-oxydianiline and 4,4’-oxybis(methylene)diamine linkers retained activity (IC 50 < 1 µM against intracellular L. donovani ) (13). Four

‘unsymmetrical bis-AIAs’ with an isopropoxy group on one of the phenyl rings ortho to

the furan and different substitutions ortho to the furan on the other phenyl ring of the

diphenylfuran linker were also synthesized and evaluated against Leishmania intracellular amastigotes. While all four unsymmetrical bis-AIAs displayed potent activity with sub-micromolar IC 50 s, the compound containing an isopropoxy substituent

on one of the phenyl rings and a hydrogen ortho to the furan on the other phenyl ring was

the most potent in this series (IC 50 = 5.3 nM and 93 nM against intracellular L. donovani and L. amazonensis respectively) (193). A structure activity map (Figure 1.7)

25 summarizing the effect of different substituents on biological activity against intracellular

Leishmania was recently reported by Reid et al (193) .

Figure 1.7 A structure activity map of AIAs

[Reprint with permission from Elsevier: Bioorganic and Medicinal Chemistry Letters

(193), copyright (2012), Elsevier]

With respect to the substituents on the phenyl ring of the diphenylfuran linker, halogen, alkyl, and alkoxy substituted bis-AIAs had improved activity compared to their unsubstituted diphenylfuran counterparts. Increasing the size of substitution improved the activity. However, while derivatives with substituents as big as the cyclopentyloxy group retained potent activity (mid nanomolar IC 50 ), derivatives with bulkier substituents such

as benzyloxy groups displayed 3- and 6-fold loss of potency compared to the

corresponding isopropyloxy and isopentyloxy congeners. Bis-AIAs with unsymmetrical

substitutions retain mid-nanomolar potency similar to their symmetrical counterparts.

(193).

Bis-AIAs with high in vitro potency and selectivity were further screened for their in vivo toxicity. Based on the in vivo toxicity profile of bis-AIAs, the following structure toxicity relationship can be summarized. Although a considerable number of bis-AIAs showed potent in vitro activity against intracellular Leishmania, only symmetrical bis-AIAs with larger alkoxy substituents (isopropoxy in DB766 and cyclopentyloxy in DB1852) on the phenyl ring of the diphenylfuran are relatively non-toxic to the mice. Although DB745, the ethoxy substituted symmetrical bis-AIA derivative, had in vitro and in vivo efficacy

similar to DB766, it was significantly more toxic in vivo. All four unsymmetrical AIAs were highly toxic to mice (193).

Figure 1.8 Structures of DB766, DB1960, DB1852 and DB1955

Of all the structural variants of AIAs synthesized and evaluated thus far, the most promising pre-clinical drug candidates in terms of the in vivo anti-leishmanial efficacy are 1) DB766 and DB1960, dihydrochloride and dimesylate salts of 2,5-bis[2-(2-

27 propoxy)-4-(2-pyridylimino)aminophenyl]furan respectively 2) DB1852 and DB1955, dihydrochloride and dimesylate salts of 2,5-bis[2-(2-cyclopentyloxy)-4-(2- pyridylimino)aminophenyl]furan respectively (Figure 1.8).

In 2010, Wang et al. showed that DB766 had outstanding and selective anti-leishmanial

activity in vitro (IC 50 values of 36, 87, and 14 nM against intracellular amastigotes of L. donovani , L. amazonensis , and L. major , respectively and selectivity index > 30) which is comparable to that of amphotericin B, a clinically approved drug for treatment of CL and

VL. Oral administration of DB766 at 100 mg/kg/day × 5 reduced the parasite burden in the liver by 71% and 89% in mouse and hamster VL models. DB766 was non-mutagenic based on Ames tests results and at the given oral dose of 100 mg/kg/day × 5 did not alter the serum chemistry values in treated mice. DB1852, although not as potent in vitro as

DB766, was equally efficacious in reducing the liver parasite burden in the mouse VL model (237). However, limited aqueous solubility of DB766 precluded its testing at doses higher than 100 mg/kg and necessitated synthesis and evaluation of DB1960 and

DB1955, the more soluble mesylate salts of DB766 and DB1852, respectively. Published studies from our lab by Zhu et al. showed that DB1960 and DB1955 are 5.3-fold and 3.1 fold more soluble than DB766 and DB1852, respectively. This enhanced solubility allowed testing of doses up to 500 mg/kg for repeat dose toxicological studies. These studies indicated that while DB1960 was more toxic than DB1955, high doses of both mesylate salts resulted in kidney, liver and hematological toxicity and low doses resulted in irreversible microscopic changes in the GI tract in mice. Given these toxicities, the estimated maximum tolerated oral dose for both DB1955 and DB1960 are < 100

28 mg/kg/day (258). Lack of a sufficient therapeutic window for the bis-AIAs led to discontinuation of preclinical development of these molecules as antileishmanials. Our efforts to improve the efficacy and reduce the toxicity of bis-AIAs have met with limited success. We were interested in determining the mechanism of action of the bis-AIAs with hopes that mechanistic information would give clues to the in vitro potency of these molecules and aid future antileishmanial drug discovery efforts.

1.2.2 Mechanism of action of diamidines and AIAs in protozoan parasites

Given the structural similarities between AIAs and diamidine analogs of pentamidine, it is reasonable to assume that they share the same anti-parasitic target/mechanism of action. Several lines of evidence support the long standing mechanistic hypotheses that the anti-parasitic activity of diamidines results from binding to the A-T rich sequences in the minor groove of kDNA, similar to that of pentamidine. Results from fluorescence, transmission electron microscopy and flow cytometry studies show that inherently fluorescent diamidines, such as furamidine and related analogs progressively accumulate in the kinetoplast of American ( T. cruzi ) and African trypanosomes ( T. brucei ), and that

treatment of T. cruzi with diamidines results in the disintegration of the kDNA network,

collapse of the mitochondrial membrane potential (∆Ψ m) and dramatic changes in the mitochondrial morphology followed by cell death (18, 64, 208, 249). Additional evidence for similarities in the target/mechanism of pentamidine and diamidines comes from the observation made with pentamidine resistant L. donovani promastigotes. This resistance was associated with reduced ∆Ψ m and reduced mitochondrial uptake of pentamidine and

29 diamidines (151). Finally, the DNA binding studies using conserved synthetic minicircle sequences and purified whole kDNA preparation from T. cruzi confirm that binding with

a diamidine results in topological changes in the kDNA network (67, 249). However, a

recent study by Daliry et al. confirmed previous observations made in T. cruzi and T.

brucei that there is no correlation between affinity for A-T rich sequences, binding

induced structural changes in the kDNA network (increased Tm, change in CD spectra)

and anti-T. cruzi activity of select diamidines and AIAs. It has been suggested that diamidines and AIAs can induce profound alterations in the kDNA topology, an effect more relevant to the biological activity than the simple DNA binding ability. Altered kDNA topology could contribute to parasite death via impaired access of different proteins to kDNA and disruption of protein-kDNA interactions, leading to replication errors, kDNA instability and destruction (67). While part of the anti-parasitic activity of diamidines appears to be related to their selective effects on kDNA, the existence of other subcellular targets in the mechanism of action of diamidines cannot be ruled out. In this regard, diamidines have also been shown to accumulate within punctate, DNA free acidic organelles, which are most likely acidocalcisomes, in Trypanosoma spp . Accumulation within this sub-cellular compartment has been suggested to play a role in the mechanism of action and/or sequestration of diamidines in trypanosomes (249). Some of the other anti-trypanosomatid targets/mechanisms reported for diamidines include interference with polyamine metabolism (33), nuclear DNA topoisomerase II (53), DNA polymerase

(113) and protease inhibition (149). While superior physicochemical properties may account for the improved potency of AIAs compared to diamidines against intracellular parasites (237), it is possible that a distinct sub-cellular target/unique mechanism of

30 action exist. There are few mechanism of action studies regarding AIAs in protozoan parasites. Studies on N. caninum and T. gondii suggested that treatment of these apicoplastids with AIA exclusively resulted in ultrastructural damage to intracellular parasites (increased vacuolization, appearance of electron dense bodies in the cytoplasm etc.) by involving the host cell in the process of parasite killing (increased abundance of lipid droplets surrounding the parasitophorous vacuole membrane, prolonged parasite growth inhibition in AIA pretreated host cells) (124). Studies on T. cruzi trypomastigotes and amastigotes treated with AIAs showed defects typically observed with diamidines: decreased ∆Ψ m, ultrastructural alterations in the nuclei, enlargement of the mitochondrion, and disintegration of kDNA. However, effects atypical for diamidines were also observed in AIA-treated T. cruzi : ultrastructural alterations in microtubule

structure and organization and the presence of abundant vesicles in the flagellar pocket

indicative of increased exocytic activity (17, 208). Similar effects were also reported in

trypanosomatids treated with other metabolic inhibitors and thus are suggestive of alternate anti-parasitic target(s) for AIAs. As discussed previously, AIAs have also shown promising anti-leishmanial activity in vitro and in vivo (216, 237, 259). However, studies characterizing the cellular effects of AIAs in Leishmania are lacking. Without knowledge of the antileishmanial target(s) of AIAs, empirical medicinal chemistry based lead optimization efforts to improve the efficacy and reduce the toxicity of AIAs have met with limited success. With the hope that the identification of anti-leishmanial target(s) and a greater understanding of the mechanism of action of AIAs will guide future drug discovery efforts, we have performed studies to identify the target and understand anti-leishmanial mechanism of action of AIAs.

1.2.3. Objectives of the study

The task of identifying the target and understanding the mechanism of action of a small molecule cannot be achieved by using a single method. While no standard set of approaches exist, knowledge of the pharmacology of related drugs and their derivatives from literature precedents if available and the preliminary data obtained from complementary approaches (phenotypic data, proteomics etc.) can be useful in formulating and testing hypothesis concerning the mechanism of action of a small molecule of interest. Recognizing this fact we employed three approaches to identify the target and understand the anti-leishmanial mechanism of action of AIAs: 1) A 2D-DiGE-

MS assisted comparative proteomics analysis to study changes in the Leishmania proteome post-treatment with the lead AIA-DB766. 2) Transmission electron microscopy to study the ultrastructural alterations caused by DB766 treatment in Leishmania. 3)

Generation and characterization of a Leishmania cell line that is over 10-fold resistant to

DB766 through stepwise increases in the concentration of the compound.

CHAPTER 2

Mechanistic investigations of anti-leishmanial arylimidamides (AIAs):

Comparative proteomic analysis

2.1 Introduction: Proteomic approach for mechanistic studies

Proteomics involves quantitative comparison and functional characterization of differentially expressed proteins by the genome at a given time under given conditions

(36, 161). In the post-genomic era with the availability of information about different

‘omes’ (genome, proteome, metabolome etc.), the classical approaches to target identification (biochemical and/or genetic methods, characterization of drug-resistant cells) are now being complemented by modern ‘omic’ techniques like comparative genomics and proteomics. The large volume of information obtained through ‘omics’ technology can be useful in formulating hypotheses concerning drug mechanism and aid in the design of biochemical and genetic studies to evaluate the hypotheses (242).

Proteomics is especially useful in studies concerning trypanosomatids as the genomes of

33 these organisms are constitutively expressed and gene and protein expression are regulated by post-transcriptional (stability and rate of mRNA translation) and post- translational (post translational modifications) mechanisms (46). Proteomics has contributed significantly to our understanding of parasite biology, host-parasite interactions and mechanisms of drug resistance in Plasmodium (26) , Trypanosoma and

Leishmania (61) . Proteomic studies employing global profiling (whole cell wide proteome analysis) and chemoproteomics based targeted profiling strategies (analysis of the sub-proteome or protein target(s) captured by affinity or activity based probes) have contributed to a greater understanding of or previously unknown mechanisms of action of anti-malarial drugs like chloroquine, artemisinin (184), and doxycycline (29) and investigational agents like napthoimidazoles in T. cruzi (144) and 2,4-diaminopyrimidine based inhibitors of cyclin dependent kinases in T. brucei, L. donovani and L. major (145).

Given the conceptual simplicity and technical feasibility of the global proteome profiling strategy we used a 2D-gel based platform, difference in gel electrophoresis (DiGE) and mass spectrometry (MS) in an attempt to identify and quantify differentially expressed proteins in DB766 treated L. donovani axenic amastigotes . The 2D-DiGE technique allows detection and quantitation of the relative abundance of individual proteins between paired samples from different biological groups. The merit of the technique lies in the ability to run and analyze up to three different samples, labeled with three specific fluorescent dyes (Cy2, Cy3 and Cy5), simultaneously within the same gel, thus eliminating gel to gel variability (121). Figure 2.1 illustrates the experimental work flow in 2D-DiGE.

Figure 2.1 Work flow in a 2D- DiGE experiment

(Figure adapted from http://www.appliedbiomics.com/proteomics_2d_dige.html)

2.2 Materials and methods

The CellTiter reagent was obtained from Promega (Madison, WI). Miltefosine was purchased from Cayman Chemical Company (Ann Arbor, MI). DB1111 and DB766 were synthesized according to previously reported methods (106, 216). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

2.2.1 Parasites and culture conditions

Leishmania donovani promastigotes (WHO designation MHOM/SD/62/1S-CL2 D) were

adapted to axenic amastigote forms by culturing the former at 37 °C in a humidified 5%

CO 2 atmosphere and in a slightly modified version of the amastigote medium described previously by Joshi et al. (114). The axenic amastigote medium is composed of fetal bovine serum (FBS) at a final concentration of 20%, 15 mM KCl, 115 mM KH 2PO 4, 10

mM K 2HPO 4, 0.5 mM MgSO 4, 24 mM NaHCO 3, an 0.8 × concentration of RPMI-1640

vitamins and amino acids, 2.0 mM L-glutamine, 22 mM D-glucose, 50 units/mL

penicillin, 50 µg/mL streptomycin, 0.1 mM adenosine, 1 µg/mL folate, and 25 mM MES adjusted to pH 5.5.

2.2.2 Assessment of cell viability and recovery after drug treatment

Cell viability was assessed using three methods. Briefly, 1 × 10 7 and 2 × 10 7 parasites/mL

of L. donovani axenic amastigotes in a total volume of 500 µL were treated with or

36 without different concentrations of DB766 (0.3, 0.4, 0.5, 1.0 µM) in a 24 well plate for 12 h at 37 °C. At the end of the incubation period the cell viability was determined by three different methods. 1) Tetrazolium dye . The tetrazolium dye based CellTiter reagent

(Promega, Madison, WI) was employed as described previously (72). 2) Propidium iodide (PI) staining flow cytometry . Briefly, 10 6 parasites/mL from untreated and DB766

treated cell cultures were harvested by centrifugation at 3000 × g for 10 min followed by

resuspension in fresh axenic amastigote medium. Propidium iodide was added to a final

concentration of 10 µg/mL and incubated for 5 min before analyzing the fluorescence on

the PerCP-Cy5.5 channel of a BD FACS Canto II flow cytometer (NJ, USA). 3)

Hemocytometer based counting . Briefly, the cell suspensions of samples were diluted 10

fold with PBS. A 10 µL volume of the diluted suspension was loaded on the counting

chamber of a Neubauer hemocytometer and cell density was calculated using the standard

procedure. Percentage viability by each method was calculated with respect to the

untreated controls. Cell morphology was also assessed by light microscopy at 40×

magnification. For assessing the recovery of the parasites after drug treatment, 5 × 10 6 parasites were harvested after 12h of exposure to drug and washed once with fresh drug free medium by centrifugation at 3000 × g for 10 min. The cells were then re-suspended

in 500 µL of fresh drug free medium in a 24-well plate and allowed to incubate for an

additional 24 h at 37°C (24 h drug washout period). At the end of the incubation, cell

viability was assessed by hemocytometer based counting.

2.2.3 Preparation of Leishmania cell extracts for 2D-DiGE analysis

Leishmania cell extracts were prepared according to established protocols developed by

Burchmore et al. (personal communication). Briefly, based on the results from the experiment described above, 1 × 10 7 parasites/mL were exposed to DMSO vehicle or 0.4

µM DB766 for 12h in quadruplicate. At the end of 12h, 1 × 10 9 parasites were harvested

from four different cultures of untreated DMSO control or DB766 treated samples and

washed 3 × with cold PBS (pH 7.4) by centrifugation at 3000 × g for 10 min at 4 °C. The

cell pellets were then resuspended by pipetting several times in 1 mL DiGE lysis buffer

[7 M urea, 2 M thiourea, 25 mM Tris base, 4% (w/v) CHAPS (3-[(3-chola-midopropyl) dimethylammonio]-1-propanesulphonic acid) (PlusOne reagents, GE Healthcare), 100 µL of 10× protease inhibitor cocktail (Roche) and 10 µL of 100× phosphatase inhibitor cocktail (Sigma)]. Samples were sonicated (MicrosonTM ultrasonic cell disruptor,

Misonix Inc.) using ten 1-2 s pulses (power output set to 10) with 1 min cooling on ice in between. Lysis was allowed to proceed by incubating the samples for another 10 min at room temperature with intermittent mixing. The samples were then centrifuged at 10,000

× g for 15 min at 4°C to remove the insoluble matter. To precipitate the proteins, cold acetone (4 × volume of the sample or 4 mL) was added to the clarified supernatant; the tubes were vortexed and incubated at -20 °C for 1 h. Protein pellets were collected by centrifugation at 3000 × g for 15 min at 4 °C. The protein pellets were washed 2 × with cold 80% acetone, air dried briefly and then re-solubilized in 200 µL of DiGE lysis buffer. Protein concentration was estimated in triplicate per sample by the Bradford assay

(Biorad) using bovine serum albumin as a standard. Samples were then adjusted to a concentration of 5 mg/mL with DiGE lysis buffer.

2.2.4 Sample labeling for DiGE

DiGE experiments, beginning with sample labeling, were carried out by Jocelyn Hach and Kari-Green Church using OSU Shared Proteomic Resources.

Soluble proteins (60 µg), from control and DB766 treated parasite samples at a concentration of 1 mg/mL, were used for DiGE labeling. Samples were stored in the dark to avoid destruction of the dye. 18 µg of each sample was pooled to create an internal standard (IS). The remaining 42 µg from each sample and the internal standard were labeled with Cydye DiGE flour minimal dyes (GE healthcare) as follows: pooled internal standard was labeled with Cy2; two of each control and DB766 treated samples were labeled with Cy3, and another two of each control and DB766 treated samples were labeled with Cy5. Cydyes were added to each sample in a ratio of 8 pM/1 µg protein and incubated at 4 °C for 30 min. The labeling reaction was quenched by addition of 1 µL of

10 mM lysine at 4 °C for 10 min. A sample of unlabeled pooled standards (500 µg) was prepared for the preparative gel.

2.2.5 Differential in gel electrophoresis

2.2.5.1 Isoelectric focusing

Gel ID Cy3 Cy5 Cy2 64678 30 µg Control 1 30 µg Treated 1 30 µg IS 65487 30 µg Control 2 30 µg Treated 2 30 µg IS 64683 30 µg Treated 3 30 µg Control 3 30 µg IS 64684 30 µg Treated 4 30 µg Control 4 30 µg IS

Table 2.1 Gel loading regimen for 2D DiGE

Labeled samples were combined as shown in Table 1, vortexed, and diluted with rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS), 1% pH 4-7 IPG buffer (GE

Healthcare), 50 mM DTT) followed by addition of 1% saturated bromophenol blue solution. Samples were centrifuged for 20 mins at 4°C at 16,000 × g. A 24 cm

immobiline dry strip pH 4-7 IEF strip (GE Healthcare) was rehydrated overnight with

450 µL of each sample and mineral oil. The IEF strips were then focused on an

isoelectric focusing apparatus (IPGphorII, GE Healthcare) in a ceramic manifold at 20

°C. IEF was performed at an initial current of 75 µA per strip. The voltage was then

progressively increased to achieve isoelectric focusing (500V for 1 h, gradual increase to

1000V over 1 h, a gradual increase to 10,000V over 3 h, and finally a hold at 10,000V for

3.25 h). Immediately following this protocol, the IEF strips were placed in a plastic sheet,

wrapped in aluminum foil and stored at -80 °C until further processing.

2.2.5.2 SDS PAGE

IEF strips were equilibrated at room temperature in 5ml of equilibration buffer A (50 mM

Tris pH 8.8, 6M urea, 30% glycerol, 2% SDS, 65 mM DTT, 1% saturated bromophenol blue solution) for 15 min, followed by 5 mL of equilibration buffer B (50 mM Tris pH

8.8, 6M urea, 30% glycerol, 2% SDS, 135 mM iodoacetamide, 1% saturated bromophenol blue solution) for 15 min. The reduced and alkylated strips were rinsed briefly with 2 × SDS-PAGE running buffer (50 mM Tris, 384 mM glycine, 0.2% SDS) and placed on top of 12% SDS-PAGE gels (20 × 24 cm). The IEF strips were sealed in place using 0.5% agarose in 2 × SDS-PAGE running buffer containing 1% saturated bromophenol blue solution. Gels were run in a DaltII electrophoresis system (GE

Healthcare) at an initial voltage of 2 watts per gel for 45 mins followed by 15 watts per gel until the dye front reached the bottom (~4 h).

2.2.6 Gel scanning and image analysis

Gels were rinsed with water and immediately scanned in a Typhoon 9400 variable mode scanner (GE Healthcare) at recommended laser and filter settings for each CyDye fluorophor. To maximize the sensitivity and avoid image saturation the PMT voltages were optimized for each channel. The gels were scanned for Cy5, followed by Cy3 and

Cy2 labels at 100 µM resolution. Gel images were cropped and saved for analysis. The preparative gel was stained with Sypro Orange (Sigma) dye (diluted 1:10,000 in 7%

41 aqueous acetic acid) for 2 h after fixing for 2 h in 10% aqueous methanol and 7% acetic acid.

For image analysis, the gel images were loaded into DeCyder 2D software (version 6, GE

Healthcare) and analyzed individually. All saturated spots, dust spots, and noise were removed before gel normalization. The gel with the most abundant spots was labeled as the master gel to which all other gel images were matched.

Gel alignment, spot matching, quantitation and normalization of spot volume and statistical analysis of variation in spot volume ratio between different gels were done in a

Decyder BVA module. For statistical analysis, Student's t test was used to select the protein spots that changed significantly in abundance in response to DB766 treatment.

The spots showing a statistically significant difference in abundance ( P-value < 0.05) were then selected for identification by MS analysis.

Protein spots selected for identification were marked on the gel images and the ‘pick list’ was saved in the Decyder BVA module. These protein spots in the ‘pick list’ were matched and their co-ordinates were applied to the gel image of the Sypro Orange stained preparative gel. The pick list was then exported from Decyder to the Ettan Spot Handling

Workstation.

2.2.8 In gel digestion

The Ettan Spot Handling Workstation was used to core protein spots of interest from the imported ‘pick list’ and placed in a 96 well plate (following recommendations from the

Ettan Spot Handling Workstation 2.1 User Manual from Amersham Biosciences). The in gel digestion was preformed manually. Gel pieces were washed in 100 µL of solvent mixture comprised of 50% methanol and 5% acetic acid for 1 hour. The wash step was repeated twice. The gel pieces were washed with acetonitrile for 5 min. Upon removal of acetonitrile, the gels pieces were placed in a vacufuge for 2 min until they were completely dry. Gels pieces were digested with sequencing grade trypsin from Promega

(Madison WI). Gel pieces were rehydrated in 50 µL of 5 µg/mL sequencing grade modified trypsin prepared in 50 mM ammonium bicarbonate for 4 h. The peptides were extracted 3 times from the polyacrylamide gel with 50 µL of 50% acetonitrile and 5% formic acid. The extracts were pooled and mixed with 50 µL acetonitrile for 15 min. The extracts were then dried for 30 min in a vacufuge and removed immediately to prevent complete drying.

2.2.8 Protein identification

2.2.8.1 Nano-LC/MS/MS

Capillary-liquid chromatography-nanospray tandem mass spectrometry (Nano-

LC/MS/MS) was performed on a Thermo Finnigan LTQ mass spectrometer equipped

43 with a nanospray source operated in positive ion mode. The LC system was an

UltiMate™ Plus system from LC-Packings A Dionex Co (Sunnyvale, CA) with a Famos autosampler and Switchos column switcher. Solvent A was water containing 50 mM acetic acid and solvent B was acetonitrile. A 5 µL aliquot of each sample was first injected on to the trapping column (LC-Packings A Dionex Co, Sunnyvale, CA), and washed with 50 mM acetic acid. The injector port was switched to inject and the peptides were eluted from the trap onto the column. A 5 cm × 75 µM ID ProteoPep II C18 column

(New Objective, Inc. Woburn, MA) packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the LTQ system using a gradient of 2-80% B over 50 min, with a flow rate of 300 nL/min. The total run time was 60 min. To obtain MS/MS spectra, briefly, a nanospray source operated with a spray voltage of 3 KV and a capillary temperature of 200 °C was used.

The scan sequence of the mass spectrometer was based on the TopTen™ method; the analysis was programmed for a full scan recorded between 350 – 2000 Da , and a MS/MS

scan was employed to generate product ion spectra. The amino acid sequence of the

peptide was derived from consecutive instrument scans of the ten most abundant peaks in

the spectrum. The CID fragmentation energy was set to 35%. Dynamic exclusion was enabled with a repeat count of 30 s, exclusion duration of 350 s, a low mass width of 0.5 and high mass width of 1.50 Da.

2.2.8.2 Protein database search

Sequence information from the MS/MS data was processed by converting the raw data files into a merged file (.mgf) using MGF creator (merge.pl, a Perl script). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.2.1 (Boston,

MA). The database was searched against the full SwissProt database version 54.1. The mass accuracy of the precursor ions were set to 2.0 Da given that the data was acquired on an ion trap mass analyzer and the fragment mass accuracy was set to 0.5 Da.

Modifications (variable) considered during database searching were methionine oxidation and carbamidomethylation of cysteine. Two missed cleavages for the enzyme were also permitted. Peptides with a score less than 20 were filtered. Protein identifications were checked manually and proteins with a Mascot score of 50 or higher with a minimum of two unique peptides from one protein having a ‘-b’ or ‘-y’ ion sequence tag of five

residues or better were accepted.

2.2.9 Mitochondrial membrane potential assay

Tetramethylrhodamine ethyl ester perchlorate (TMRE, Invitrogen) was used for measurement of mitochondrial membrane potential by flow cytometry. A 4 mg/mL stock solution was prepared by dissolving TMRE in DMSO and stored at -20 °C. Leishmania donovani axenic amastigotes, at a seeding density of 1 × 10 7 cells/mL were exposed to

0.4 µM of DB766 or DMSO vehicle for 12 h. For determination of mitochondrial

membrane potential at the end of 12 h drug treatment, 1 × 10 6 parasites/mL from each

45 sample were harvested by centrifugation at 3000 × g for 10 min at 4 °C and washed once with PBS. The parasites were then resuspended at 1 × 10 6 parasites/mL in fresh warm medium containing 100 nM TMRE and incubated at 37 °C for 30 min. As a positive control, 200 µM of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to cells already labeled with TMRE during the 30 min incubation period and incubated for

15 min. The fluorescence from TMRE was recorded on the PE channel of a BD FACS

Canto II flow cytometer (NJ, USA).

2.3 Results and discussion

2.3.1. Parasite viability after DB766 treatment

Selection of the proper concentration of a compound of interest can have a significant impact on the outcome and analysis of a proteomics experiment. While treatment with a sub-lethal concentration of the compound may not be sufficient to induce significant qualitative and quantitative changes in the proteome, treatment conditions that are too stringent risk over-representation of non-specific proteins, mostly associated with cell death events in the proteome and thus can complicate the data analysis. To ensure that the changes in the Leishmania proteome represent a DB766 specific response rather than

non-specific effects associated with cell death, a treatment regimen based on the

morphology (viewed under 40× objective of bright field microscope), cell viability

(hemocytometer based counting and PI staining by flow cytometry) and the ability of

treated parasites to recover after a 24 h drug washout period was selected

(hemocytometer based counting). Cell viability assays indicated that exposure of L. donovani axenic amastigotes at an initial seeding density of 1 × 10 7 parasites/mL to 0.4

µM DB766 for 12h reduced the growth by ~50% and had very little effect on the

morphology of the cells (Table 2.2). The treated cells fully recovered after removal of the

compound from the culture during the 24 h washout period (cell density at the end of 24

h) (Table 2.2) indicating that DB766 treatment affected the cell growth slightly and did

not lead to irreversible effects and cell death. Hence this treatment regimen was selected

for assessing differential effects produced by DB766 on Leishmania proteome.

Seeding density 1 × 10 7/ml 2 × 10 7/ml Cell viability (% of DB766 (µM) DB766 (µM) control) 0 0.3 0.4 0.5 1.0 0 0.3 0.4 0.5 1.0 Cell counting 100 89.2 54.1 ND ND 100 97.7 84.4 55.6 ND MTS viability 100 96.8 60.3 45.2 21.7 100 96.3 72.1 46.5 15.8 PI viability 100 78.2 51.6 51.6 33 100 80.7 79.3 63.4 45.2 Morphology (40×) ++++ ++++ ++ + ND ++++ ++++ +++ + ND

Recovery at 24 h wash out 100 110 97.3 55.6 ND 100 108.3 107.6 95.3 ND (cell counting) 48 Morphology descriptors: ++++ is normal morphology; ++ is few dead cells; + is lot of dead cells; ND is not determined due to extensive cell damage

Table 2.2 Parasite growth after the drug treatment.

2.3.2 Response of L. donovani to DB766 treatment based on 2D-DiGE analysis

The DiGE approach used in this study (Figure 2.2 and 2.3) allowed reproducible resolution of about ~ 2000 protein spots (pI range 4-7), which is consistent with the earlier proteomic studies reported for Leishmania spp (80). DB766 treatment resulted in a significant ( P < 0.05) and reproducible (n = 4) difference in expression of proteins (spot ratios ≥ 1.50) corresponding to 50 spots on four protein gels; proteins corresponding to

16 spots and 34 spots were up-regulated and down-regulated respectively. These data are consistent with the earlier observations of low number of differentially modulated proteins after exposure of protozoan parasites like Plasmodium spp (28, 183) and T. cruzi

(143) and even mammalian cancer cell lines (14) to drugs. Of the 50 differentially modulated spots, proteins corresponding to 31 spots could not be identified due to technical difficulties associated with isolation and identification of less abundant proteins. Proteins corresponding to the remaining 19 spots were identified by nano-

LC/MS/MS. A total of 25 distinct proteins, with specific accession numbers, were identified of which 10 were up-regulated and 15 were down-regulated. While heat shock proteins (HSPs) like HSP-83 and HSP-60 were identified in six and five spots respectively, α- and β-tubulins appeared in four and two spots respectively. Tubulins and

HSPs are the two most abundant proteins and are consistently over-represented across the proteomes of eukaryotes and protozoan parasites. While the presence of tubulin and

HSP83 in multiple spots indicates potential post translational modifications and processing, consistent with earlier observations in apicomplexans (92) and kinetoplastids

(93), it might represent non-specific common features associated with treatment with a

49 toxic compound. Table 2.3 lists the identified proteins along with their accession numbers, biological function, predicted localization, and fold change values. From the biological function and predicted localization it is clear that most of the differentially modulated proteins like RNA helicase, mitochondrial tryparedoxin peroxidase

(mTXNPx), HSP60, ATP synthase and ATPase are nuclear encoded mitochondrial proteins. While proteins like RNA helicase, mTXNPx, ATP synthase and ATPase had reduced expression, four proteins corresponding to HSP60 were overexpressed, three of which were identified as mitochondrial precursors.

Figure 2.2 Representative 2D-DiGE gels showing L. donovani proteins. (a) Cy-2 labeled pooled protein standards (internal standard). (b) Overlay of images from Cy3 (DB766 treated) and Cy5 (untreated) labeled proteins from untreated and DB766 treated L.donovani axenic amastigotes. The inset C shows representative spot images, 3D-landscapes, and quantitative data for mTXNPx that exhibits a 1. 5 fold decrease in abundance in DB766 treated Leishmania.

Figure 2.3 Relative protein abundance measurements using 2D DiGE to assess protein expression in DB766 treated and untreated L. donovani . Spot images, 3D-landscape representation and summary of quantitative data for the four distinct proteins identified as HSP60, mitochondrial precursor, HSP60, RNA helicase and mitochondrial tryparedoxin peroxidase.

Master # T-test Fold GI accession # MASCOT Protein Predicted change score localization 261 0.016 -2.92 gi|146097493 401 HSP83 [ Leishmania infantum ] 262 0.018 -2.5 gi|146097493 286 HSP83 [ Leishmania infantum ] 264 0.0034 -2.13 gi|146097493 242 HSP83 [ Leishmania infantum ] gi|145552563 60 hypothetical protein [ Paramecium tetraurelia strain d4-2] 369 0.016 -1.47 gi|136429 230 gi|157867985 111 HSP [ Leishmania major strain Friedlin] 599 0.019 -1.91 gi|146097493 253 HSP83 [ Leishmania infantum ] 665 0.0056 2.36 gi|322490787 123 HSP60, mt precursor [ Leishmania mexicana ] gi|123666 91 HSP83 [ Typanosoma brucei brucei ] 670 0.027 1.91 gi|322490787 121 HSP60, mt precursor [ Leishmania mexicana ] Mitochondrial gi|28400787 85 cofactor-independent phosphoglycerate mutase

53 [Leishmania mexicana ] 675 0.0017 2.32 gi|322490788 274 HSP60, mt precursor [ Leishmania mexicana ] Mitochondrial 871 0.0021 2.03 gi|3025866 66 chaperonin 60 [ Leishmania braziliensis ] Mitochondrial 973 0.033 -1.71 gi|606648 422 α tubulin [ Leishmania donovani ] gi|71661631 195 ATPase β subunit [ Trypanosoma cruzi strain CL Mitochondrial Brener] gi|322490787 126 HSP60, mt precursor [ Leishmania mexicana ] Mitochondrial gi|23957265 123 α -tubulin [ Metacylis angulata ] 1174 0.039 -1.64 gi|606648 316 α tubulin [ Leishmania donovani ] 1189 0.013 -1.5 gi|146086104 116 RNA helicase [ Leishmania infantum JPCM5] 1200 0.031 -1.6 gi|606648 959 α tubulin [ Leishmania donovani ] gi|91983206 118 α tubulin [ Leishmania mexicana ] gi|114421 106 ATP synthase subunit beta, mt precursor Mitochondrial [Nicotiana plumbaginifolia ] gi|157868378 82 peptidylprolyl isomerase-like protein [ Leishmania major ] Continued 53 Table 2.3 Protein characterization and predicted localization data for protein isoforms identified in this study Table 2.3 continued

1309 0.018 -1.64 gi|606648 577 α tubulin [ Leishmania donovani ] gi|146081643 444 enolase [ Leishmania infantum JPCM5] 1373 0.0038 1.64 gi|146078079 213 β tubulin [ Leishmania infantum JPCM5] gi|13569565 201 β tubulin [ Leishmania mexicana ] gi|146088643 128 protein phosphatase [ Leishmania infantum JPCM5] gi|606648 64 α tubulin [ Leishmania donovani ] 1456 0.038 -1.65 gi|146086185 487 β tubulin [ Leishmania infantum JPCM5] gi|146078079 472 β tubulin [ Leishmania infantum JPCM5] gi|606648 106 α tubulin [ Leishmania donovani ] gi|22297081 85 β tubulin Cyp-1a [ Cyathostomum pateratum ] 2132 0.048 -1.52 gi|61619796 303 mt tryparedoxin peroxidase [ Leishmania Mitochondrial amazonensis ] 2207 0.017 -1.58 gi|146102004 128 hypothetical protein [ Leishmania infantum ]

Based on this observation, we hypothesized that processing of mitochondrial targeted proteins is disrupted in DB766 treated Leishmania . Mitochondrial processing of pre- proteins could be compromised at the level of mitochondrial protein import which in turn depends on ∆Ψm (Figure 2.4). Disruption of ∆Ψm dependent protein import would lead

to accumulation and subsequent degradation of unprocessed pre-proteins. We thus sought

to evaluate the effect of DB766 on ∆ψ m.

Figure 2.4 Basic steps in mitochondrial membrane potential dependent mitochondrial protein import together with the proposed mechanism for the reduced expression of mitochondrial proteins based on loss of mitochondrial membrane potential induced by DB766. Loss of ∆Ψ m would disrupt mitochondrial protein import leading to accumulation and degradation of unimported pre- proteins.

[Reprint with permission from Annual Reviews Inc: Annual Reviews of Cell and

Developmental Biology (179), Copyright (1997), Annual Reviews Inc. ]

2.3.3 Effect of DB766 on mitochondrial membrane potential

Flow cytometry based membrane potential assays indicate that DB766 treatment reduced the ∆Ψm to ~60% (Figure 2.5), which is consistent with our hypothesis and explains the

differential modulation of mitochondrial proteins in the proteome of DB766 treated

Leishmania . Disruption of ∆Ψm and down-regulation of important mitochondrial proteins would impair the mitochondrial function that can be contributing factors to devastating effects induced by DB766 in Leishmania .

120

100

80 * 60

20 Fluorescence (% of Control) *

0 Control 200 µM CCCP 0.4 µM DB766 Treatments

Figure 2.5 Effect of DB766 on Leishmania mitochondrial membrane potential. Values are expressed as percentage of promastigotes relative to the total cell density. Results indicate mean ± SE from at least three individual measurements (* P < 0.005).

2.4 Conclusions and future directions

Important information regarding the protein changes associated with DB766 treatment was obtained using 2D-DiGE analysis that ultimately led to evaluation and confirmation of mitochondrial involvement in response to DB766 in Leishmania . Although the mitochondrion in Leishmania appears to be affected by the AIA-DB766, consistent with the impact of diamidines on the kinetoplastid parasites, it is important to note that 1)

DB766 induced altered expression of mitochondrial proteins and loss of ∆Ψm could be a secondary effect downstream of primary target(s) and 2) the observed changes in the proteome are associated only with reversible effects on cell growth under the selected sub-lethal treatment conditions. Further our DiGE study encountered several limitations inherent to global proteomic profiling of the effects of a toxic small molecule such as the over-representation of more abundant proteins (tubulins and HSPs) and under- representation of membrane proteins, limiting the coverage and analysis of less abundant proteins (14). To minimize this ‘abundance bias’ sub-cellular fractionation and or affinity enrichment strategies that allow a more focused analysis of drug specific changes in expression and post translational modifications are suggested for future studies. A prefractionation protocol using digitonin extraction, by successfully removing tubulin and cytoskeleton associated abundant proteins, increased the representation of less abundant proteins and allowed identification of novel life cycle stage specific proteins in L. infantum (93) .

As an alternative to gel-based global protein profiling strategies, chemical proteomics strategies for the identification of drug target(s) are becoming increasingly popular as they use an ‘affinity or activity probe’ specifically engineered to capture target protein(s) or sub-proteome comprising a class of structurally or functionally related proteins (14).

Identification of CRK3 as a potential drug target of 2,4-diaminopyrimidine analogs in trypanosomatids (145) and cysteine peptidases (rhodesain and TbCatB) and other novel proteins as drug targets of vinyl sulfone analogs of K11777 (cysteine peptidase inhibitor) in T. brucei (252) exemplifies the affinity and activity based chemical proteomics

approach for anti-parasitic target discovery respectively. Such a chemical proteomic

strategy with AIA based affinity or activity probe is recommended as an alternate

strategy for future studies. Further, DB766 resistant Leishmania were found to be cross resistant to other AIAs (discussed later in section 4.3, chapter 4), indicating that they most likely have common target(s) and act through a common mechanism of action.

Hence there is high probability that AIA based probes would bind the same target proteins as the parent AIAs, thus facilitating identification of AIA target proteins in

Leishmania .

While proteomic studies indicated mitochondrial involvement in response to DB766 treatment in Leishmania , it did not give any specific clues with respect the anti- leishmanial target(s) and mechanism of action of AIAs. In absence of the specific target information and considering the fact that mitochondrial involvement could be a secondary event downstream of a primary target, a more detailed investigation employing other approaches was undertaken.

Chapter 3

Mechanistic investigations of anti-leishmanial arylimidamides (AIAs):

Transmission electron microscopy

3.1 Introduction: Transmission electron microscopy for mechanistic studies

Transmission electron microscopy (TEM) has been a popular approach to study the ultrastructural alterations induced by different drugs acting on different metabolic pathways such as sterol and lipid metabolism in parasite protozoa. As the metabolic pathways are often compartmentalized within specific sub-cellular organelles (e.g. mitochondrion), small molecules, primarily by inhibiting target metabolic enzyme(s), often interfere with the structure and function of the corresponding subcellular organelles and also affect related secondary target organelles in parasitic protozoa (231). It is therefore logical that structurally diverse drugs display similar mechanisms of action by affecting common enzymes or metabolic pathways and hence result in similar ultrastructural alterations to the primary and secondary subcellular targets. For example, the sterol biosynthesis pathway is a well known, essential and validated anti-parasitic drug target in trypanosomatids. Structurally diverse small molecule inhibitors of different

59 enzymes in the sterol biosynthetic pathway deplete the endogenous sterol pool and induce common ultrastructural alterations such as swelling of the mitochondrion, damage to the flagellum, presence of vesicular structures in the flagellar pocket, multivesicular bodies, myelin like figures and autophagosomal structures in the cytoplasm, increased cytoplasmic vacuolization and abundant electron dense bodies in trypanosomatids (197).

Thus ‘profile matching’, i.e. assessment of the ultrastructural effects of drugs and/or drug candidates and their comparison to ultrastructural effects produced by reference compounds with a known target/mechanism of action in the common target cells could provide important clues for understanding the mechanism of action of new compounds of interest. Although the exact phenotypic descriptors employed for profile matching in our study (shape and size of subcellular organelles, cell processes affected, mode of cell death) are rather empirical, as a proof of concept a more intensive high content high throughput phenotype based screening strategy using multiparameter cell imaging technology has been successfully applied to identify known and unknown anti-cancer drug targets. The approach is based on the same rationale that a compound induces specific subcellular changes mediated by unique target(s). In this study, a database of subcellular morphological changes induced by 100 different compounds in HeLa cells was created. Matching morphological profiles induced by treatment with different compounds revealed that drugs with common targets induced similar profiles while drugs with broader pharmacological effects (protein biosynthesis inhibitors) resulted in diverse profiles. This approach also led to identification and subsequent validation of the target for a compound with a previously unknown mechanism of action (177). TEM, although low in throughput, yields high content phenotypic information. The popularity of this

60 approach in studying the effects of drug candidates against parasitic protozoa has led to the availability of a large amount of phenotypic data (ultrastructural effects produced by different metabolic inhibitors in trypanosomatids including Leishmania ) and was recently reviewed by Vannier-Santos et al. (231) and Fernandes Rodrigues et al. (198). The data included in these reports was used as a reference to match the ultrastructural profile induced by DB766 in Leishmania and facilitate identification of potential drug target(s) for further validation studies.

3.1.1 Rationale for protease and sterol metabolism hypotheses

As discussed later in section 3.3.1, the ultrastructural profile matching approach employed in this study revealed that the ultrastructural profile induced by DB766 was similar to that produced by sterol biosynthesis and protease inhibitors in Leishmania .

In addition to the ultrastructural similarities induced by serine protease (207) and HIV protease inhibitors (202) in Leishmania (damage to the flagellar membrane, vesicular structures in the flagellar pocket, cytoplasmic vacuolization and abundant electron dense bodies), there are several literary precedents for serine protease inhibitory activity for diamidines including pentamidine. Being that DB766 is a diamidine analog of pentamidine, it is thus reasonable to hypothesize that serine protease inhibitory activity contributes to the observed ultrastructural effects induced by DB766 in Leishmania. The inhibitory activity of benzamidine and bis-benzamidines against several mammalian serine proteases is known including trypsin, coagulation factor Xa, thrombin, plasmin,

61 urokinase, matriptase and mast cell trypatase (37, 97, 176, 185, 227). The structural similarity among benzamidine, the bis-benzamidines berenil and pentamidine, and the natural substrate arginine has been proposed as the basis for their trypsin inhibitory activity. A recent crystallographic study of bovine β-trypsin in complex with ligands like

arginine, berenil and pentamdine has provided further structural evidence for binding of

these anti-trypanosomal drugs to serine proteases (176). Yet another study proposed

trypanosomal oligopeptidase B, a serine protease, as a target for trypanocidal drugs like

pentamidine and berenil based on inhibition of the said enzyme at therapeutic

concentrations of diamidines (149). Taken together, these data provide rationale for the

hypothesis that the observed ultrastructural effects of DB766 might be related to serine

protease inhibitory potential in Leishmania (see below).

3.1.2 Parasitic proteases as drug targets

Several key virulence factors responsible for the pathogenesis observed as a result of parasitic diseases have been identified with recent advancements in genomic analysis of some of these organisms. Proteases are one of the most widely studied key virulence factors in parasites. Parasitic proteases play significant roles in establishing, sustaining and exacerbating the infection via aiding the penetration of the host tissue barrier and degrading host proteins for nutrition and immune evasion (138). Biochemical studies and genome sequencing efforts have revealed the presence of several families and clans of aspartyl, threonine, cysteine, metallo and serine proteases in Leishmania spp (206)

(Figure 3.1).

Figure 3.1 Clans and families of L. major peptidases.

[Reprint with permission from Elsevier: International Journal for Parasitology (24),

While the Leishmania major genome indicates the presence of two aspartyl peptidases –

presenilin and an intramembrane signal peptide peptidase (24), a recent study identified

and characterized a retroviral aspartyl protease domain containing DNA damage-

inducible protein (Ddi-1) in L. major (178). The presence of a protein with a retroviral

aspartyl protease fold explains the sensitivity and ultrastructural alterations induced by

HIV protease inhibitors in Leishmania spp (202).

Of all the proteases, cysteine proteases are the most extensively studied virulence factors

in Leishmania because of their role in host-parasite interaction. Several cathepsin-L like

cysteine proteases of clans A and B and cathepsin-B like peptidase of clan C have been

characterized in Leishmania spp (24). Genetic disruption studies with Clan B cysteine

protease (CPB) in Leishmania indicate that CPB is an essential and validated drug target

in trypanosomatids (5, 150). GP63, also called major surface protease (MSP) or

leishmanolysin, is a metalloprotease and is also the most abundantly expressed

glycoprotein on the surface of the trypanosomatids. Multiple studies in Leishmania have

identified GP63 as a key virulence factor involved in disease pathogenesis (206). Several

studies have reported and characterized serine protease activities in Leishmania spp (44,

65, 202). An intracellularly located 115 kDa secretory serine protease with the capability

to degrade host extracellular matrix proteins such as fibronectin and collagen was

recently reported in Leishmania culture supernatants (44). A clan SB, family S8 serine

protease, the subtilisin (SUB) serine protease with a unique catalytic triad was recently

identified and functionally characterized in Leishmania spp. Studies with SUB gene

knockout (-/-) parasites revealed the role of this protease in the maturation of terminal

64 enzymes of the trypanothione reductase system, an antioxidant defense system in trypansomatids including Leishmania . SUB deficient Leishmania promastigotes, with their compromised anti-oxidant defense system and reduced ability to withstand oxidative stress had significantly reduced virulence both in vitro and in vivo (220). Subtilisin like peptidases of apicomplexan parasites play a role in proteolytic processing of proteins and host invasion and thus have been identified as potential drug targets in P. falciparum and

T. gondii (138, 250). Treatment with the serine protease inhibitors L-1-tosylamido-2- phenylethyl chloromethyl ketone (TPCK), benzamidine and Kunitz-type inhibitor (ShPI

1) derived from a sea anemone reduced the viability and induced ultrastructural damage reflective of disruption of the endocytic pathway in Leishmania (207). Oligopeptidase B

(OPB), a clan SC, family 9A serine oligopeptidase which is different from trypsin like serine protease, was recently identified and functionally characterized in Leishmania spp.

A role for OPB in regulating expression of parasite enolase, a virulence factor, was established based on studies with OPB gene knockout (-/-) Leishmania . Further, infection of macrophages with OPB (-/-) Leishmania induced a dramatic protective response from the macrophages (upregulation of proteins involved in cytokines and interferon response) as opposed to the wild type parasites, thus establishing the role of OPB in evading the immune response (221). While retention of the virulence by OPB (-/-) Leishmania indicates that the enzyme might not be a prevalent virulence factor, the presence of an additional OPB like enzyme (OPB2) has been suggested to compensate for the loss of

OPB in OPB deficient Leishmania mutants (154). A trypanosome ortholog of Leishmania

OPB, a previously identified important virulence factor (32), was shown to be inhibited

65 by pentamidine at therapeutic concentrations and was proposed as a target in the mechanism of action of pentamidine and related diamidines (149).

3.1.3 Sterol metabolism as a drug target in Leishmania

Sterols, the ubiquitous isoprenoid components of biological membranes, perform

structural and regulatory functions in prokaryotic and eukaryotic cells. Unlike

mammalian cells, C-28 ergostane based sterols form the major components of the

membranes of kinetoplastids and fungi. Ergosta-5,7,24(28)-trien-3β-ol (5-

dehydroepisterol), ergosta-5,7,22-trien-3β-ol (ergosterol) and ergosta-7,24(28)-trien-3β-

ol (episterol) are the most abundant sterols in Leishmania . In Leishmania promastigotes

C-29 stigmastane based sterols comprise up to 5% of total sterols and this amount

increases up to 20% in amastigotes of certain Leishmania species (197).

The sterol biosynthetic pathway involves at least 20 metabolic steps (Figure 3.3), some of

which are catalyzed by enzymes unique to fungi and trypanosomatids. The carbon

skeleton of ergostane based sterols, with the exception of the C-24 methyl group in the

side chain, is derived either from acetyl-CoA from glucose, fatty acid or amino acid

intermediary metabolism (197) or from HMG-CoA obtained via methylglutaconyl CoA

from leucine degradation (98). Studies on intracellular localization of the isoprenoid and

sterol biosynthetic pathways show that enzymes in these pathways are distributed within

different sub-cellular compartments: mitochondrion, glycosomes, cytosol and

endoplasmic reticulum in trypanosomatids (Figure 3.2). These studies indicate that

66 enzymes involved in mevalonate synthesis are located in the mitochondrion, mevalonate kinase that phosphorylates mevalonate to isopentenyl diphosphate and dimethylallyl diphosphate is located in the glycosomes, farnesyl pyrophosphate synthase (FPPS) is in the cytosol and sterol C24 methyl transferase (SCMT), an enzyme in the late steps of the pathway is located mainly within the endoplasmic reticulum (see Figure 3.2) (34).

Figure 3.2 Intracellular location of enzymes involved in early and late steps of the sterol biosynthetic pathway in trypanosomatids

[Reprint with permission from Elsevier: International Journal of Parasitology (34),

While enzymes in the early steps involving synthesis of lanosterol are common to all

eukaryotes, the pathway diverges beyond lanosterol and becomes exclusive to mammals

or fungi and trypanosomatids. Close similarity to the sterol biosynthetic pathway in fungi

and the unique ability to synthesize ergosterol and related 24-alkylated sterols makes the

67 sterol biosynthesis pathway an attractive drug target in trypanosomatids including

Leishmania (197).

Depending on the enzyme being inhibited in the sterol biosynthesis pathway, sterol biosynthesis inhibitors (SBI) can be categorized in to four groups: 1) bisphosphonates that inhibit FPPS 2) zargozic acid and biphenylquinuclidine (BPQ-OH) derivatives that inhibit squalene synthase (SQS) 3) allylamines that inhibit squalene epoxidase (SEO) 4) imidazole and triazole based antifungal agents that inhibit sterol 14 α-demthylase

(CYP51) and 5) azasterol derivatives that inhibit SCMT. Irrespective of the enzymes

being inhibited in the pathway, treatment of trypanosomatids with either class of SBIs

resulted in compromised cell viability that correlated with depletion of the endogenous

pools of C-24 alkylated sterols and accumulation of aberrant sterol intermediates. Sterols

are important structural components of cell and biological membranes of the

mitochondrion, Golgi complex, nucleus, endoplasmic reticulum and flagellum. The

ultrastructural damage to these organelles is thus consistent with the loss of structural

integrity of the biological membranes caused by altered sterol composition following SBI

treatment (197, 231). Further, these effects have been shown to induce formation of

acidocalcisomes and initiate the autophagic process in trypanosomatids (232). Given the

unique sterol requirements of the trypanosomatids, the existence of de novo sterol

biosynthesis pathway similar to fungi in these organisms, and the sensitivity of such

parasites to antifungal sterol biosynthesis inhibitors, sterol metabolism is an attractive

target for the pursuit of new anti-parasitic drugs in trypanosomatids.

Figure 3.3 The sterol biosynthetic pathway in trypanosomatids

[Reprint with permission from Elsevier: Molecular and Biochemical Parasitology (197), copyright (2003), Elsevier]

3.2 Materials and methods

3.2.1 Transmission electron microscopy

5 × 10 6 cells/mL of axenic amastigotes were incubated for 24 or 48 h in amastigote medium in the presence of DB766, DB1111, or DMSO vehicle, then were harvested and washed with cold phosphate buffered saline (PBS) by centrifugation at 3000 × g for 10

min at 4 °C. The cells were then fixed for 3 h in a buffer containing 2% glutaraldehyde,

4% paraformaldehyde, and 0.1 M sucrose in 0.1 M phosphate buffer, pH 7.4. After

fixation and washing four times with 0.1 M sucrose in 0.1 M phosphate buffer, pH 7.4,

the cells were post fixed for 1 h in 1% osmium tetroxide in phosphate buffer. The cells

were rinsed twice before setting in 2% agarose and then chilled on ice for 10 min.

Ultrathin agarose sections were stained with 2% uranyl acetate in lead citrate followed by

dehydration of the cells with successive gradients of 50-100% ethanol. Residual ethanol

was removed from the samples by propylene oxide treatment (initially alone followed by

propylene oxide-Spurr resin treatment at 1:1 and 1:2 ratios for 1 h and 12 h, respectively).

Stained sections were then embedded by overnight polymerization in Spurr resin and

viewed using a FEI Tecnai G2 Spirit Transmission Electron Microscope (Hillsboro,

Oregon USA).

3.2.2 Leishmania lysate and OPB like serine protease assay

Leishmania lysates were prepared and OPB like serine protease activity was measured according to a previously reported method (221). Stationary phase cultures of L. donovani axenic amastigotes were harvested and washed 3 × with cold PBS (pH 7.4) by

centrifugation at 3000 × g for 10 min at 4 °C. The cell pellet was lysed in a lysis buffer containing 50 mM Tris, pH 8.0 and 0.25% Triton X-100 by pipetting and vortexing.

Lysis was allowed to proceed for 1 h at 4 °C with intermittent mixing. Insoluble material in the lysate was removed by centrifugation at 10,000 × g for 10 min at 4 °C. The

clarified supernatant was sterilized by passing through 0.2 µM syringe filter, aliquoted

and stored at -20 °C. The substrate Z-GGR-AMC (Bachem Inc) was used to measure

serine protease activity. Stock solutions of 10 mM Z-GGR-AMC in DMSO, 20 mM

PEFABLOC [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) from

Merck Chemicals] in water, 1 mM pepstatin A (Sigma) in ethanol, 50 mM disodium

EDTA (Sigma), 10 mM pentamidine (Sigma) and 10 mM DB766 in water were prepared,

aliquoted and stored at -20 °C. A 12 µL volume of bovine β trypsin (30 µg/mL) or sterile

Leishmania donovani axenic amastigote lysate was pre-incubated with and without

different inhibitors (2 mM AEBSF, 1 µM pepstatin A, 5 mM EDTA, 100 µM

pentamidine and 100 µM DB766 ) for up to 1 h in 50 mM Tris.HCl buffer, pH 8.0 (total

volume per well 120 µL). A 100 µL aliquot of this mixture was added to 20 µM of

substrate in 50 mM Tris.HCl buffer, pH 8.0. The progressive release of the fluorochrome

AMC was monitored at 25 °C with a FlexStation microplate spectrofluorimeter

(Molecular Devices, Sunnyvale, CA) using excitation and emission wavelengths of 355

71 and 460 nm, respectively. SoftMax Pro v 5.4 software was used to calculate the V max values.

3.2.3 Extraction and analysis of total Leishmania sterols by GC-MS

This study was performed by Joy Laydbak and Frederick Buckner, University of

Washington, Seattle.

Sterols from L. donovani axenic amastigotes were extracted according to an established protocol developed by Buckner et al. (personal communication). Briefly, 5 × 10 6 cells/mL of late log phase Leishmania donovani axenic amastigotes were incubated in the presence of IC 50 concentration of DB766 or DMSO vehicle, harvested, washed twice

with cold PBS, and frozen at -80 °C until required. For sterol extraction, duplicate

samples of cell pellets were resuspended in 5 mL chloroform-methanol (2:1), vortexed

and centrifuged at 4000 × g for 10 min. The resulting supernatant was concentrated under

a stream of nitrogen (N2) and extracted twice with 2 mL petroleum ether. The petroleum ether extract was dried under N 2 and derivatized with an excess of trimethylsilyl imidazole (TMSI) + pyridine reagent at 70 °C for 30 min. Samples were then directly injected into a Hewlett-Packard 6890 series II gas chromatograph coupled to an

HP5973A mass spectrometer. Upon injection, the column temperature was kept at 150 °C for 1 min, and then the following temperature gradient was employed: 150 °C to 200 °C at 2 °C/min, 200 °C to 250 °C at 1 °C/min, and finally 250 °C to 300 °C at 10 °C/min.

The helium carrier gas flow was kept constant at 1.1 mL/min, and the injector and detector temperatures were 250 °C and 280 °C, respectively.

3.3 Results and discussion

3.3.1 Ultrastructural alterations induced by DB766 in L. donovani axenic amastigotes

Figure 3.4. Electron micrographs of L. donovani axenic amastigotes incubated in the presence of (A) 0.1% DMSO, (B) 0.68 µM DB1111 for 24h, (C-F) 0.15 µM DB766 for 48h. N = nucleus; M = mitochondrion; K = kinetoplast; F = flagellum; FP = flagellar pocket; V = vesicular bodies. Bars, 0.5 µm (A, C, D, F), 2 µm (B and E).

We and others have previously demonstrated trypanosomatid kDNA and the mitochondrion as main subcellular targets of pentamidine and other diamidines (90, 106,

249). Hence we compared the ultrastructural effects of AIAs with the diamidine DB1111 in Leishmania donovani axenic amastigotes. While DB1111 caused dilation of the L.

donovani mitochondrion as observed previously (106), DB766 treatment did not result in

any ultrastructural change in the mitochondrial morphology of these parasites (Figure

3.4). Thus the subcellular target/mechanism of action of the AIA DB766 appears to be

distinct from that of diamidines. Instead, we noted dramatic ultrastructural alterations in

other organelles, including increased vesicular structures in the flagellar pocket, damage

to the flagellar membrane, increased cytoplasmic vacuolization and abundant electron

dense bodies. Based on the ultrastructural profile matching approach (discussed in section

3.1), we noted that the ultrastructural effects of DB766 resembled those produced by

sterol biosynthesis inhibitors (197, 231) and serine protease inhibitors in Leishmania

(207) .

As discussed earlier in section 3.1.1, the ultrastructural effects produced by DB766 and

the published precedents for serine protease inhibitory activity by diamidines including

pentamidine prompted us to test the hypotheses that AIAs act via oligopeptidase B

inhibition and/or disturbances in sterol metabolism in Leishmania . To investigate

Leishmania oligopeptidase B as a potential target for AIAs, we evaluated and compared

74 the inhibition of OPB like activity in Leishmania lysates and purified bovine β trypsin by

DB766 and pentamidine using flurogenic OPB substrate based enzyme assays. To

investigate sterol metabolism as potential target for AIAs, changes in the Leishmania

sterol profile induced by DB766 were evaluated by GC-MS analysis.

3.3.2 Effect of DB766 on serine protease like OPB activity in L. donovani axenic

amastigote lysates

The presence of specific protease inhibitor sensitive aspartyl, cysteine and serine protease

activities has been demonstrated and characterized using Leishmania whole cell extracts

or culture supernatants (44, 51, 230). For proteases that are expressed at high levels, a

whole cell lysate thus seems to be a reasonable starting point for preliminary detection

and biochemical characterization of enzymatic activities. The synthetic flurogenic peptide

Z-GGR-AMC used previously for the characterization of OPB activity in Leishmania

lysates (221) was used to assess OPB like serine protease activity in the Leishmania

lysate in this study. Purified bovine β trypsin was used as a positive control for serine

protease activity in these studies. Since the whole cell extract is a cocktail of proteins

including a variety of proteases, the assay design necessitated verification of the

proteolytic activity being tested. The proteolytic activity in the whole cell lysate was

characterized by testing serine protease specific PEFABLOC (AEBSF), aspartyl protease

specific pepstatin and metallopeptidase specific EDTA as inhibitors. The enzymatic

activity in the lysate and of trypsin was completely abolished by PEFABLOC (1 mM)

thus confirming the presence of a high serine protease activity in the lysate. Further,

75 pepstatin (1 µM) and EDTA (5 mM) had no effect on this enzymatic activity indicating that the major proteolytic activity in the assay was not due to the presence of aspartyl protease or metallopeptidase (Figure 3.5). Despite the fact that cysteine peptidase account for a high level of protease activity in trypanosomatids including Leishmania (31), standard cysteine protease inhibitors (e.g. E64), due to their unavailability at the time, were not included in the these assays. However, previous findings indicated that

PEFABLOC inhibitable OPB activity dominates the Leishmania lysate at pH 8.0 over that of cysteine peptidases which require acidic pH (5.5) for optimum activity (221).

Given this observation, while the possibility of the presence of cysteine peptidases in the whole cell lysate and their interference with the assay results cannot be excluded, we believe that the major proteolytic activity under the given assay conditions is due to serine proteases. While both pentamidine (100 µM) and DB766 (100 µM) abolished trypsin activity by ~ 98%, they produced only marginal inhibition of OPB like serine protease activity (~24% and 21% respectively) in the Leishmania lysate. It thus appears that Leishmania OPB, unlike trypanosomal OPB, is not likely to be a potential target for diamidines and AIAs.

120 Lysate βββ 100 Bovine trypsin

% Inhibition % of OPB like activity 0

-20 No inhibitor AEBSF Pentamidine DB766 PepstatinA EDTA 100 µM 100 µM

Figure 3.5 Effects of DB766, pentamidine, and known inhibitors on serine protease like oligopeptidase B activity in a Leishmania donovani axenic amastigote lysate and on purified bovine β-trypsin. Bars represent the means ± standard error of at least three independent measurements.

3.3.3 Effect of DB766 on Leishmania sterols

GC-MS analysis of Leishmania sterols indicated that exposure to posaconazole, a potent

CYP51 inhibitor that disrupts sterol metabolism in Leishmania , resulted in a typical

dramatic decrease in the peak size of ergosterol in Leishmania . Exposure to the IC 50 concentration of DB766 for 48h resulted in relative increase in the peak size of lanosterol, indicative of accumulation of this sterol, with respect to peak size of ergosterol in DB766 treated Leishmania as compared to the untreated controls (Figure

3.6). This result is consistent with our hypothesis that DB766 alters sterol metabolism in

Leishmania. While this effect appears to be consistent with sterol 14 α-demethylase

(CYP51) inhibition in Leishmania, it is not as dramatic as that produced by posaconazole

in Leishmania . Two possible explanations for this observation are that 1) the dramatic

alterations in the sterol profile might be a potency related or dose dependent effect

suggesting that DB766 might be a weaker inhibitor of CYP51 than posaconazole or 2)

DB766 might alter the sterol profile in Leishmania by inhibiting alternate enzymes in the

sterol biosynthesis pathway or by modulating proteins/enzymes in other parallel

pathways, such as lipid metabolism, involved in sterol homeostasis. Treatment with

miltefosine, a known inhibitor of phospholipid metabolism, also leads to alterations in

sterol biosynthesis in Leishmania (190) .

A B

C D

79 Relative abundance Relative

Retention time (min)

Figure 3.6 GC-MS traces of Leishmania sterol fractions. Differences in the sterol profile in untreated (A) and

posaconazole treated (B) Leishmania promastigotes and untreated (C) and DB766 treated (D) Leishmania donovani

axenic amastigotes. 1: cholesterol; 2: ergosterol; 3: ergosta-5,7,22,24(28)-tetraen- βββ-ol; 4: ergosta-5,7,24(28)-triene- βββ-ol;

5: Ergosta-7,24(28)-dien-3βββ-ol; 6: Stigmasterol; 7: Stigmasta-7,24(28)-dien-3βββ-ol; 8: lanosterol

3.4 Conclusions and future directions

The data from the serine protease assay suggests that Leishmania OPB, unlike

trypanosomal OPB, is unlikely to be potential target for diamidines and AIAs, which is

consistent with the structural and functional differences between the two enzymes (70,

140, 154). However, the results of the assay should be interpreted with the following

considerations: 1) While interference with the assay by cysteine peptidase activity is

expected to be minimal, this remains to be confirmed in our case. Additional assays with

the whole cell lysate in the presence of selective cysteine peptidase inhibitors such as E64

are recommended in the future. 2) The marginal OPB inhibitory activity observed with

DB766 and pentamidine might be due to binding of the drugs by non-target proteins in

the lysate resulting in dilution of the drug available for inhibition of protease. Follow up

studies with purified enzyme preparations to confirm the preliminary results obtained

with Leishmania lysates is recommended in the future. 2) Serine protease activity,

measured using whole cell preparations is not exclusively due to OPB. Serine proteases

other than OPB, capable of hydrolyzing the Z-GGR-AMC substrate could contribute to

the observed total serine protease activity in the lysate. In this regard, it is important to

note that OPB belongs to family of prolyl oligopeptidases (POP) and is not a classical

trypsin like serine protease (148), which might explain the inability of pentamidine and

DB766 to inhibit Leishmania OPB. Other than the POP class, the L. major genome encodes several other serine peptidases (24). As stated earlier, the presence of at least two different serine protease activities has already been reported in Leishmania spp (44, 45).

Given the significant inhibition of trypsin like serine proteases by diamidines and

80 pentamidine, ‘trypsin like’ serine proteases should be further explored as targets of AIAs in the future. Nonetheless, while pentamidine inhibited trypanosomal OPB, neither

DB766 nor pentamidine showed oligopeptidase B inhibitory activity above 20% in the

Leishmania lysates, even at the concentration of 100 µM. Hence this potential target was not pursued further.

GC-MS analysis of sterols (Figure 3.6) supports our hypothesis that DB766 disrupts sterol biosynthesis in Leishmania . The exact molecular basis for this alteration remains to be uncovered, however, and will be the subject of the investigations described in the following chapter.

Chapter 4

Mechanistic investigations of anti-leishmanial arylimidamides (AIAs):

Development and characterization of DB766 resistant Leishmania donovani

4.1 Introduction

Given the limited arsenal of clinically available anti-leishmanial drugs and the emergence of drug resistant strains, a better understanding of mechanisms of drug resistance becomes imperative. Our current understanding of mechanisms of drug resistance in

Leishmania and other related protozoa are either based on studies with clinical isolates or laboratory generated drug resistant strains (171). These studies indicate that protozoan parasites may evade the toxic effects of drugs by 1) thwarting their uptake 2) overexpressing efflux pumps, 3) modifying the drug, 4) suppressing their activation, 5) sequestering the drug, 6) increasing target expression, or 7) altering the target site (166,

171). Despite the diversity of mechanisms, both in vitro and in the clinic, drug resistance most frequently includes amplifications of drug efflux pumps and changes in target genes

(171). Figures 4.1 and 4.2 indicate the mode of action and resistance to pentavalent

82 antimonials and various other anti-leishmanial drugs.

Figure 4.1 Mode of action and resistance to pentavalent antimonials in Leishmania. Pentavalent antimony (SbV) can enter the macrophages or the parasite within the phagolysosome, as such or after being reduced to trivalent form in the cytosol (or within the phagolysosome). While transporters for the uptake of SbV are unknown, aquaglyceroporin AQP1 facilitates uptake of SbIII by the amastigotes. Within the parasite, SbV can be converted to SbIII by either thiols, or possibly by the novel ACR2 or TDR1 reductases. SbIII probably interacts with some cellular targets but can also form conjugates with various thiols including cysteine, glutathione and trypanothione. Enzymatic involvement in these conjugation reactions has not been verified yet. Antimony-resistant strains often have increased levels of trypanothione, presumably for increasing conjugation and disposition of the conjugated metal. The metal-thiol conjugate can be either sequestered into an organelle by the ABC transporter MRPA or extruded out of the cell by another ABC transporter containing efflux system.

[Reprint with permission from Elsevier: Drug Resistance Updates (167), copyright

(2004), Elsevier]

Figure 4.2 Modes of action and resistance to clinically approved anti-leishmanial drugs. Potential mechanisms of resistance for miltefosine, pentamidine and AmB are indicated by broken circles. (A) Aminophospholipid P-type ATPase (oval) transporter facilitates uptake of miltefosine. Drug resistance is due to decreased accumulation of miltefosine and is presumably related to the point mutations in this transporter. Potential extrusion of miltefosine, outside of the cell, can occur by an ABC transporter (175). (B) Transporter responsible for pentamidine uptake (triangle) is yet to be identified in Leishmania . Mitochondria of sensitive but not resistant cells accumulate pentamidine (16). Drug extrusion by an ABC transporter (circle) can occur in Leishmania (48). Amphotericin B interacts with ergostane- based membrane sterols in Leishmania . Ergosta-5,7,24(24’)-trien-3β-ol (structure on the right) is one of the most abundant sterol in Leishmania (197). Resistance to amphotericin B is associated with functional loss of SCMT followed by concomitant buildup of ergosterol precursors such as cholesta-5,7,24-trien-3-ol (structure on the left) for which the drug has lower affinities (137, 181).

[Reprint with permission from Elsevier: Drug Resistance Updates (167), copyright

(2004), Elsevier]

Genome sequencing and transcriptomic and proteomic approaches have led to the identification of several genes involved in the development of resistance and have furthered our understanding of mechanisms of drug resistance in protozoan parasites including Leishmania . Knowledge of both mechanisms of resistance and mechanisms of action will continue to provide information permitting the more rational uses of drugs, will allow evaluation of new combination strategies to minimize the development of resistance, and may increase the effectiveness of treatment and the life span of currently available anti-parasitic drugs. Identification of efflux pumps and alterations in trypanothione metabolism as mediators of resistance to pentavalent antimonials has led to the evaluation of anti-leishmanial combination strategies involving resistance reversal agents (verapamil, a P glycoprotein efflux pump inhibitor) and a trypanothione biosynthesis inhibitor (buthionine sulfoximine or BSO) both in vitro and in vivo (166).

Identification of resistance markers might also facilitate monitoring of drug resistance in the field. For example, a set of proteins such as a miltefosine transporter (P-type

ATPase), pyridoxal kinase, LdRos3 multidrug resistance protein A (MRPA), γ-

glutamylcysteine synthetase (GCS) and aquaglyceroporin-1 (AQP-1) are being evaluated

as potential biomarkers for miltefosine (49) and antimony resistance (123) in Leishmania .

Understanding mechanisms of drug resistance has also permitted the identification of

novel intracellular targets and/or mechanism of drug action that could be exploited for the

rational development of novel inhibitors (167). For example, the increased dependence of

antimony resistant Leishmania on trypanothione dependent anti-oxidant defense has led

to interest in developing inhibitors of parasite enzymes involved in thiol metabolism

(105).

Drug resistance that specifically results from mutations or alteration in the gene copy number provide a more tractable and reliable approach and can in fact serve as a ‘gold standard’ as opposed to traditional genetics and affinity based approaches for identification and validation of drug targets (236). This approach has led to the identification and/or confirmation of physiological targets for anticancer and anti- parasitic agents (54, 91, 236). This approach typically involves 1) generation of resistant mutants from their drug sensitive counterparts by continuous exposure to increasing drug concentrations or insertional mutagenesis followed by selection of clones resistant to lethal concentration of drugs, 2) comparison of drug sensitive and resistant clones using phenotypic, genomic and proteomic approaches and the identification of distinct phenotypes along with differentially expressed genes/proteins, and 3) gene overexpression, silencing and/or often heterologous expression in a suitable model organism for functional analysis of differentially expressed genes/proteins (153). In this study, phenotypic comparison between DB766 sensitive and resistant organisms along with genetic validation permitted the identification of a DB766 target in Leishmania .

4.2 Materials and methods

4.2.1 Selection of a DB766 resistant Leishmania donovani cell line

Axenically grown Leishmania donovani amastigotes were subjected to increasing DB766 pressure starting at a concentration of 0.05 µM and rising to 8 µM. A stepwise increase in the DB766 concentration was applied only when pressured cultures showed a growth rate

86 equivalent to that of untreated cultures. Parasites displaying a stable resistance phenotype were maintained under constant compound pressure throughout our experimental procedures.

4.2.2 Transmission electron microscopy

Axenic amastigotes (either wild type or resistant parasites), in the absence and presence of drug pressure were harvested and washed with cold PBS by centrifugation at 3000 × g for 10 min at 4 °C. The cells were fixed and processed for transmission electron

microscopy according to the protocol described in section 3.2.1.

4.2.3 In vitro susceptibility studies

The in vitro susceptibility of the DB766 resistant cell line was evaluated after allowing

the cells to grow in the culture in the absence of DB766 for at least 3 days. Briefly, 10 6 parasites/mL of DB766 sensitive or DB766 resistant axenic amastigotes in a total volume of 60 µL were treated with a 2-fold dilution series of each compound in a 96 well plate at

37 °C for 72 h. At the end of the treatment, cell viability was determined by using the tetrazolium dye based CellTiter reagent (Promega, Madison, WI) as described previously

REF. IC 50 values were calculated using SoftMax Pro software (Amersham Biosciences,

Piscataway, NJ). Each compound was tested in at least three independent experiments.

4.2.4 Drug interaction assays: design, determination of FIC index and isobologram analysis to determine the nature of drug interaction

Drug interaction assays were performed according to the procedure described by Siefert et al. (203). Briefly, the highest concentrations of the drugs used in these assays were 25

µM posaconazole, 50 µM miltefosine, 200 µM terbinafine, 2.5 µM amphotericin B and

25 µM DB766. A series of ten concentrations were prepared for each combination by making 2-fold dilution series of ‘fixed ratio solutions’ of posaconazole and DB766 in combination with various ‘partner drugs’ in ratios of 5:0; 4:1; 3:2; 2:3; 1:4 and 0:5. As for susceptibility assays, 10 6 parasites/mL of DB766 sensitive wild type axenic amastigotes were exposed to serial dilutions of drug combinations in different ratios.

Each point was tested in duplicate. Endpoints were determined as described previously in drug susceptibility assays. This setup allowed determination of IC 50 values for each drug alone from ‘fixed ratio solutions’ of 5:0 and 0:5 as well as IC 50 s of drug combinations

from ‘fixed ratio solutions’ of 4:1; 3:2; 2:3; 1:4.

Fractional inhibitory concentrations (FICs) and sum FICs ( ΣFIC) for DB766 and

posaconazole in combination with each other or other partner drugs were determined as

follows:

FIC (DB766) = IC 50 of DB766 in combination IC 50 of DB766 alone

ΣFIC = FIC (DB766) + FIC (partner drug)

FICs were used for constructing the isobolograms. Mean ΣFIC allowed for the analysis of the nature of drug interaction. The interaction is classified as ‘synergistic’ if ΣFIC ≤ 0.5; indifferent if 0.5 ≤ ΣFIC < 4 and antagonistic if ΣFIC > 4.

4.2.5 In vitro differentiation efficiency and growth curve

Briefly, the transformation of axenic amastigote forms to promastigotes was initiated by inoculating 5 × 10 6 axenic amastigotes/mL in 4 mL in RPMI 1640 medium containing

20% FBS, 50 units/mL penicillin and 50 µg/mL streptomycin at pH 6.88 at 23 °C. The

cell density and number of promastigote-like slender forms were determined by

hemocytometer based counting every 24 h for 72 h.

4.2.6 Western blotting

Cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (Pierce) and

protein determinations were performed using the bicinchonic acid (BCA) protein assay

kit (Pierce). Equal amounts of proteins were electrophoresed on a 10% polyacrylamide

gel by standard denaturing SDS PAGE electrophoresis using precast gels from Biorad.

For Western blotting, the proteins were transferred to a polyvinylidine fluoride (PVDF)

membrane (GE Lifesciences) at a constant voltage of 80 kV for 2h. 5% Non-fat milk in

0.1% Tween in Tris buffered saline (TBST) was used for blocking and probing the

membrane with a 1:20,000 dilution of anti-CYP5122A1 antibody (provided by Dr.

Chandrima Shaha, National Institute of Immunology, New Delhi, India), a 1:500 dilution

89 of anti-CYP51 antibody (provided by Dr. Frederick S. Buckner, University of

Washington, Seattle, WA, USA) and a 1:1000 dilution anti-α-enolase antibody (provided

by Dr. Paul Michels, Catholic University of Louvain, Brussels, Belgium) as loading

control. To visualize the bands, enhanced chemiluminescence was performed according

to the manufacturer’s instructions (Cell Signaling Technologies, Danvers, MA).

4.2.7 Real time PCR

Total RNA was extracted from wild type and DB766 resistant Leishmania using the

RNeasy isolation kit (Qiagen, Valenecia, CA) according to the manufacturer’s protocol.

Reverse transcription was performed using the Superscript III reverse transcriptase kit

(Life Technologies, Grand Islands, NY) according to the manufacturer’s directions. The

synthesized cDNA were amplified for α-tubulin, sterol 14 α-demethylase (CYP51),

CYP5122A1, sterol C-24 methyl transferase-A (SCMT-A) and cytochrome P450

reductase (CPR ) genes. The sequence of forward and reverse primers for SCMT-A was

based on the 3’-untranslated region (3’-UTR). Real time PCR was performed in the

StepOnePlus TM system (Applied Biosystems) using SYBR Green (Invitrogen) chemistry.

Following the initial denaturation step at 95 °C for 10 min, target genes were amplified

by 40 cycles of denaturation at 95 °C for 15 s followed by annealing and extension at 60

°C for 1 min. The fold change in the expression levels were determined by the ∆∆ Ct method (128). The target/reference ratio for each sample was normalized to the target/reference ratio of calibrator. Here, the target/reference ratio for DB766 sensitive wild type Leishmania was used as a calibrator and α-tubulin was used as a reference. The

90 primers used for quantitative real time PCR were as follows: for α-tubulin, 5’-

ATGTCGTGCCGAAGGATGTC-3’ (F) and 5’-TGAATTGTCCGCTTCGTCTTG-3’

(R); for CYP51, 5’-TGCTCATGCGCAAGGTATTG-3’ (F) and 5’-

GCACGACGTACTTGCCCAC-3’ (R); for CYP5122A1, 5’-

CCGTGGTCGAAAATGTCGA-3’ (F) and 5’-TTTTGTGGCAGGTTCTTCTCG-3’ (R); for SCMT-A, 5’-CATCTTCCCTCCCTTTCCTC-3’ (F) and 5’-

CCGCATGAACAACAGAGAGA-3’ (R); for CPR, 5’-CGCTGTTTCACCCCATCA A-

3’ (F) and 5’-TGTTCGCTTCCTCTGCAG TCT-3’ (R).

4.2.8 Culture conditions for CYP5122A1 half knockout Leishmania and in vitro

susceptibility studies

Wild type and CYP5122A1 half knockout promastigotes (HKOs) of Leishmania

donovani (UR6) were used for DB766 and ketoconazole susceptibility assays. Data with

CYP5122A1 HKOs, used in the following studies, were generated by Radhika Mathur

and Chandrima Shaha, National Institute of Immunology, India according to previously

established procedures (109, 141, 152, 234).

To evaluate the difference in susceptibility to DB766 in WT and CYP5122A1 HKO L.

donovani , 10 6 promastigote/mL were incubated with or without DB766 (50-750 nM) and ketoconazole (10 or 30 µM) at 23 °C for 24 h. To assess the viability of these organisms, promastigotes were harvested by centrifugation at 1100 × g for 5 min followed by

91 resuspension in PBS. Propidium iodide was added at a final concentration of 2 µg/mL and incubated for 5 min before analyzing the fluorescence on FL2 channel of a BD FACS caliber flow cytometer.

4.2.9 Hydroperoxide susceptibility assay

5 × 10 5/mL wild type or DB766 resistant L. donovani axenic amastigotes in the axenic

amastigote medium described previously were exposed to 30 µM tert-butylhydroperoxide

(tBuOOH). Cell densities were determined at the end of 72 h of incubation by

hemocytometer based counting. Percentage viability was calculated with respect to

untreated controls.

4.2.10 Extraction and analysis of total sterols by GC-MS

These studies were performed by Joy Laydbak and Frederick Buckner, University of

Washington, Seattle

Briefly, 2 × 10 8 late log phase wild type or DB766 resistant Leishmania donovani axenic amastigotes were incubated in the presence of DB766 or DMSO vehicle, harvested, washed twice with cold PBS, and frozen at -80 °C until required. Total sterols were extracted and analyzed according to a previously established protocol described in detail in section 3.2.3.

4.3. Results and discussion

4.3.1 Characterization of DB766 resistant Leishmania amastigotes

As indicated in Figure 4.3, the development of resistance to DB766 occurred with

difficulty in culture. The time required to induce ~10-fold resistance to DB766 (as

assessed by comparing IC 50 values of resistant versus wild type parasites) was about 18

months. Further, this resistance was maintained for five months in the absence of DB766

pressure, showing a stable chemoresistant phenotype. Transmission electron micrographs

(Figure 4.4) of DB766 resistant parasites both in the absence and presence of DB766

pressure showed abundant “electron dense bodies” as compared to wild type counter

parts.

Figure 4.3. Generation of a DB766 resistant L. donovani cell line . Leishmania donovani axenic amastigotes were cultured under increasing DB766 pressure starting at a concentration of 0.05 µM and rising to a final concentration of 8 µM. As indicated in the figure, the development of resistance to DB766 occurs with difficulty in culture.

Figure 4.4 Electron micrographs of (A) wild type, (B) DB766 resistant Leishmania donovani axenic amastigotes without DB766 pressure, and (C) DB766 resistant Leishmania donovani axenic amastigotes with DB766 pressure. N = nucleus; M = mitochondrion; K = kinetoplast. The asterisks indicate electron dense bodies. Note the normal morphology in DB766 sensitive parasites (A) and increase in electron density and abundance of electron dense bodies both in the absence and presence of DB766 pressure in DB766 resistant parasites (B and C). Bars, 0.5 µm (A and B), 2 µm (C).

4.3.2 Differentiation efficiency of L. donovani axenic amastigotes resistant to DB766

The wild type and DB766 resistant axenic amastigotes had comparable growth rates

(doubling time ~ 12h). To test their ability to differentiate into promastigotes, axenically

grown amastigotes were cultured in promastigote medium in the absence of DB766 at 23

°C. The growth rate (doubling time ~ 24 h) and maximum cell density of promastigotes

adapted from DB766 resistant axenic amastigotes was significantly lower than the WT

cells (doubling time ~ 12 h) (Figure 4.5), and these cells were much smaller and less

motile than their WT counterparts. Also about 80% of the population of WT axenic

amastigotes transformed into promastigotes within 48 h; the transformation was complete

at 72 h. Under the same experimental conditions, the transformation of DB766 resistant

amastigotes was incomplete, with these cultures containing only about 60%

promastigotes at the end of 72 h (Figure 4.6).

Figure 4.5 Growth curve of promastigotes adapted from wild type and DB766 resistant axenic amastigotes over a period of five days in culture. 5 × 10 6 axenic amastigote forms/mL were cultured in promastigote medium in the absence of DB766 as described before. The number of promastigote-like slender forms derived from DB766 sensitive axenic amastigotes (filled circles) and DB766 resistant axenic amastigotes (empty circles) were determined by hemocytometer based counting every 24 h for 72 h. Results indicate mean ± SE from at least three individual measurements

** ** *

Figure 4.6 Transformation efficiency of wild type (black bars) and DB766 resistant axenic amastigotes (gray bars) over a period of five days in culture as determined by hemocytometer based counting every 24 h for 72 h. Values are expressed as the percentage of promastigotes relative to the total cell density. Results indicate mean ± SE from at least three individual measurements (* P < 0.01, ** P < 0.005).

4.3.3 Drug susceptibilities of L. donovani axenic amastigotes resistant to DB766

Besides overexpression of efflux pumps, drug resistance can arise due to compensatory changes in the target or metabolic pathways involving the target, often conferring cross resistance or hypersensitivity to other drugs acting on the same or parallel pathways.

Comparing susceptibilities of drug sensitive and resistant cell lines can thus be useful in understanding the mechanism of action/resistance of new small molecules that are structurally unrelated to chemotherapeutic agents with known targets/mechanisms of

97 action (257). The susceptibility profile of the DB766 resistant cell line to other structurally related and unrelated drugs is summarized in Table 4.1.

Compound Fold difference IC 50 ±±± SD (µM) DB766 sensitive DB766 resistant Pentamidine 1.3 ± 0.2 1.2 ± 0.0 DB766 0.66 ± 0.15 7.7 ± 1.4 + 11.7* DB745 0.677 ± 0.22 5.53 ± 1.29 + 8.16* DB1852 1.305 ± 0.32 6.47 ± 0.04 + 4.96* Verapamil > 100 > 100 DB766 + 50 µM Verapamil 1.1 ± 0.7 11 ± 2 + 9.6* Nelfinavir 22 ± 5 17 ± 1 Amphotericin B 0.15 ± 0.04 0.14 ± 0.04 Miltefosine 2.7 ± 0.1 1.2 ± 0.3 - 2.25* Ketoconazole 45 ± 1 0.016 ± 0.005 -2830** Fluconazole 140 ± 50 120 ± 30 Posaconazole 12 ± 0.5 0.0010 ± 0.0005 - 12, 200** Terbinafine 99 ± 25 77 ± 5 *P < 0.005, ** P < 0.0005

Table 4.1 Susceptibility profiles of wild type and DB766 resistant Leishmania donovani axenic amastigotes 72 h post drug treatments. Results indicate mean ± SD from at least three individual measurements.

There was no significant difference (P > 0.05) between resistant and wild-type axenic amastigotes in susceptibility to pentamidine, amphotericin B, or fluconazole. Resistance to DB766 was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance associated with overexpression of P glycoprotein (PgP) type efflux pumps. Further, DB766 resistant parasites are cross resistant to the other bis-AIAs

DB745 and DB1852. Interestingly, DB766 resistant parasites were twice as sensitive to miltefosine, more than 2000-fold more sensitive to ketoconazole and over 12000-fold more sensitive to posaconazole than the wild type parasites.

Miltefosine and antifungal azoles are known to alter lipid and sterol metabolism in

Leishmania (125, 189, 190). It is conceivable that reduced expression of key sterol

biosynthetic enzymes like CYP51 (sterol 14-α demethylase, an antifungal azole target)

would hypersensitize these cells to the lethal effects of azoles, consistent with the earlier

observations of enhanced sensitivity to antifungal azoles in squalene epoxidase,

cytochrome P450 reductase (CPR) and sterol 14 α-demethylase (CYP51) knockout fungi

(96, 132, 142, 157, 173, 226, 233, 251). Also, amphotericin B resistant Candida albicans and Leishmania mexicana and Leishmania donovani lacking functional sterol 24C- methyl transferase (SCMT) were shown to be hypersensitive to anti-fungal azoles (110,

181, 187). Recently Verma et al identified a novel cytochrome P450 protein,

CYP5122A1, essential for survival, virulence and drug response in Leishmania . The facts that CYP5122A1 half knockouts (knockout of a single allele of CYP5122A1 in

Leishmania , HKO) had 3.5 fold less ergosterol and were twofold more sensitive to miltefosine compared to their WT counterparts provides strong evidence that

CYP5122A1 plays an important role in ergosterol metabolism in Leishmania (234).

Based on the above observations, we hypothesized that modulation of CYP51 and/or other sterol metabolizing enzymes such as CYP5122A1, SCMT and CPR and/or alterations in sterol metabolism in Leishmania occurred as a consequence of acquired resistance to DB766. Hence studies to investigate the contribution of these enzymes to the mechanism of action and resistance to DB766 in Leishmania axenic amastigotes were undertaken.

4.3.4 Expression status of key sterol biosynthetic enzymes in L. donovani axenic

amastigotes resistant to DB766

While there was no significant difference in the transcript level of CYP5122A1 in WT

versus DB766 resistant Leishmania as assessed by real-time qPCR, the latter had reduced

abundance of CYP51, SCMT and CPR transcripts (~ -1 to -1.5 fold) as compared to the

former (Figure 4.7). At the protein level, while there is statistically significant increase in

expression of CYP51 between wild type and resistant organisms, expression of the

CYP5122A1 protein is significantly reduced in the resistant parasites (Figure 4.8).

2 DB766 sensitive Leishmania DB766 resistant Leishmania

Ct) 1 ∆∆ ∆∆ ∆∆ ∆∆ *

-1 Normalized value Normalized ( * ** -2 CYP51 CYP5122A1 SCMT CPR

Gene

Figure 4.7 Quantitative real time PCR analysis of relative expression levels of CYP51, CYP5122A1, SCMT and CPR in DB766 sensitive and resistant L. donovani. Data are target/reference ratio for each sample, normalized by target/reference ratio of the calibrator. Here, the target/reference ratio of DB766 sensitive parasites is used as a calibrator and α-tubulin as the reference. Results indicate mean ± SE from at least three individual measurements (* P ≤ 0.05, ** P < 0.001).

100

Figure 4.8 Expression profiles of CYP5122A1 and CYP51 levels in wild type and 766R L. donovani axenic amastigotes. A western blot is shown for 10 µg of total protein from wild type and DB766 resistant Leishmania donovani axenic amastigotes that were probed with anti-CYP51 (upper panel) and anti-CYP5122A1 antibodies (middle panel). α-Enolase was used as loading controls. The figure is representative of at least three independent measurements.

4.3.5 Hydroperoxide susceptibility of L.donovani axenic amastigotes resistant to

DB766

CYP5122A1 HKO Leishmania had increased susceptibility to H 2O2 induced oxidative damage in vitro (234). Since DB766 resistant Leishmania had significantly reduced expression of CYP5122A1 protein we sought to investigate whether there were differences in susceptibility to oxidative damage in WT vs DB766 resistant Leishmania.

The viability of WT or DB766 resistant axenic amastigotes exposed to 30 µM for 72 h was determined by hemocytometer based cell counting. As expected, exposure to tBuOOH significantly reduced the cell viability of DB766 resistant parasites (20%, P <

0.0005) as opposed to their wild type counterparts (Figure 4.9)

101

Figure 4.9 Percentage viabilities of WT (black bars) and DB766 resistant axenic amastigotes (gray bars) in response to treatment with 30 µM tBuOOH. Cell viability was measured by hemocytometer based counting at the end of 72 h. Values are expressed as the percentage viability relative to the appropriate untreated controls. Results indicate mean ± SE from three individual measurements (*P < 0.005).

4.3.6 Sterol profile of DB766 sensitive and DB766 resistant Leishmania

Since CYP5122A1 HKO Leishmania had a lower level of ergosterol compared to wild type parasites (234), we investigated the effect of reduced expression of CYP5122A1 on sterol metabolism in DB766 resistant organisms. GC-MS analysis shows that while there is no change in ergosterol levels (Table 4.2), there is a distinct perturbation with respect to other sterol intermediates, such as decrease in levels of 5-dehydroepisterol (47% less) with concomitant accumulation of episterol (230% more), decrease in the levels of lanosterol (42% less) in DB766 resistant Leishmania as compared to their WT counterparts .

102

Peak # 1 2 3 4 5 8 Wild type 26.4 31.8 4.7 27.7 8.1 1.2 766R 25.1 33.1 2.5 20.0 18.7 0.7

Table 4.2 Sterol composition of WT and DB766 resistant Leishmania (766R) as determined by GC-MS. 1: Cholesterol; 2: Ergosterol; 3: Ergosta 5,7,22,24-tetraen- 3β-ol; 4: 5-dehydroepisterol; 5: Episterol; 8: Lanosterol

4.3.7 Susceptibility of CYP5122A1 HKOs to DB766 and ketoconazole

To provide definitive evidence that a reduction in CYP5122A1 expression alone causes reduced susceptibility to DB766, we tested the susceptibility of DB766 and ketoconazole in CYP5122A1 HKO promastigotes. As shown in Figures 4.10 and 4.11, exposure to different concentrations of DB766 (50-750 nM) resulted in statistically significant less cell death (less PI positive cells) indicating that CYP5122A1 HKOs are significantly less susceptible to DB766 than their wild type counterparts. However exposure to ketoconazole (10 and 30 µM) resulted in statistically significant more cell death (less PI positive cells) indicating that these organisms are more susceptible to ketoconazole than their WT counterparts. These data underscores the importance of CYP5122A1 in susceptibility and resistance to DB766 in Leishmania .

103

Figure 4.10 Susceptibility profiles of wild type and CYP5122A1 HKO Leishmania donovani promastigotes to DB766 as determined by PI staining and by flow cytometry. The values represent percentage cell death relative to untreated controls. Results indicate the mean ± SE of three independent measurements (# P ≤ 0.05, * P < 0.001).

Figure 4.11 Susceptibility profiles of wild type and CYP5122A1 HKO Leishmania donovani promastigotes to ketoconazole as determined by propidium iodide staining by flow cytometry. The values represent percentage cell death relative to untreated controls. Results indicate mean ± SE of at least three independent measurements.

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4.3.8 Drug interaction assays

Drug combination Expt IC 50 IC 50 ΣΣΣFIC ± SD Nature of # (Main (partner drug drug) a drug) b interaction Posaconazole/Miltefosine 1 12.5 1.75 0.958 ± 0.2 Indifferent 2 12.3 2.11 0.855 ± 0.09 Posaconazole/Terbinafine 1 12.6 56.4 0.780 ± 0.30 Indifferent 2 12.3 95.5 0.854 ± 0.19 Posaconazole/AmB 1 12.8 0.18 0.694 ± 0.46 Indifferent/ 2 11.5 0.13 0.563 ± 0.41 Synergistic DB766/Posaconazole 1 0.458 9.78 0.611 ± 0.22 Synergistic 2 0.487 12.5 0.407 ± 0.04 DB766/Miltefosine 1 0.394 2.79 1.332 ± 0.22 Indifferent 2 0.553 2.87 1.308 ± 0.08 DB766/AmB 1 0.477 0.158 1.277 ± 0.12 Indifferent 2 0.422 0.312 1.539 ± 0.03

Table 4.3 Mean ΣΣΣFICs from two independent assays for studying interaction of DB766 with partner drugs. a IC 50 of the ‘main drug’ (Posaconazole/DB766) alone that was used to calculate FICs b IC 50 of the ‘partner drug’ alone that was used to calculate FICs

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A B C 106 D E F

Figure 4.12 Isobolograms showing in vitro drug interactions between (A) Posaconazole/Miltefosine, (B) Posaconazole/Terbinafine, (C) Posaconazole/Amphotericin B, (D) DB766/Posaconazole, (E) DB766/Miltefosine, and (F) DB766/ Amphotericin B, against L. donovani axenic amastigotes at IC 50 level.

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Given the effects of DB766 on sterol metabolism in Leishmania and hypersensitivity of

DB766 resistant Leishmania (both DB766 resistant cell line and CYP5122A1 HKO

Leishmania ) to ketoconazole and or miltefosine, we assessed the mechanistic nature of in vitro interaction between DB766 and posaconazole, miltefosine and amphotericin B

(AmB) by using the modified version of the ‘fixed ratio isobologram method’ as described previously by Croft et al (203). The overall mean ΣFICs for all the combinations, in both experiments, ranged from 0.4 to 1.54 (Table 4.3). Antifungal azoles, miltefosine and AmB have mechanistic similarities as they all affect parasite sterols or sterol associated functions and hence are anticipated to be synergistic. Also based on previous reports of synergistic in vivo interaction between antifungal azoles and miltefosine and antagonistic in vitro interaction between antifungal azoles and AmB in

Leishmania (191, 204) , we used posaconazole-miltefosine, posaconazole-terbinafine and posaconazole-AmB combinations as internal controls in our assay. However, in our assay, posaconazole-miltefosine (mean ΣFIC = 0.906) and posaconazole-terbinafine

(mean ΣFIC = 0.817) interactions proved to be ‘indifferent’, while posaconazole-AmB interaction was somewhat ‘synergistic’ ( ΣFIC = 0.628). DB766-miltefosine (mean ΣFIC

= 1.315) and DB766-AmB (mean ΣFIC = 1.408) interactions were ‘indifferent’. Based on the mean ΣFIC value of 0.509 and the concave isobologram (Figure 4.12), DB766- posaconazole interaction was classified as ‘synergistic’. DB766-posaconazole synergism indicates mechanistic overlaps between these potent anti-leishmanial agents and is consistent with our hypothesis that like posaconazole, DB766 alters sterol metabolism in

Leishmania .

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4.4 Conclusions

In an attempt to obtain further mechanistic information concerning the lead AIA DB766,

we developed and characterized L. donovani axenic amastigotes that are over 10-fold resistant to this compound. Our electron microscopy studies revealed a unique difference in the ultrastructure of WT and DB766 resistant parasites compared to wild type both in the presence and in the absence of DB766 pressure (Figure 4.4): the abundance of electron dense bodies in the resistant organisms, a feature consistent with what was observed in DB766 treated Leishmania (Section 3, Figure 3.4).

The in vitro susceptibility assays with the DB766 resistant cell line show that 1) there is no significant difference in susceptibility to pentamidine in WT and DB766 resistant

Leishmania , reinforcing the notion that the cellular target of AIAs in Leishmania is distinct from that of diamidines, 2) resistance to DB766 is not reversed by verapamil, indicating that the overexpression of P-gp type efflux pumps is unlikely to be responsible for resistance, 3) DB766 resistant parasites are cross resistant to other AIAs indicating that all the AIAs share common target(s), 4) DB766 resistant parasites are twice as sensitive to miltefosine and over 1000-fold more sensitive to ketoconazole and posaconazole than the wild type parasites, and 5) DB766 synergizes the anti-leishmanial efficacy of posaconazole indicating a mechanistic overlap.

The effect of DB766 on Leishmania sterol metabolism together with the dramatically

enhanced sensitivity of DB766 resistant parasites to antifungal azoles prompted us to

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investigate the hypothesis that acquisition of resistance to DB766 was the cause/effect of

downregulation of one or more of the key sterol biosynthetic enzymes in Leishmania -

CYP51, SCMT, CPR and or CYP5122A1 . DB766 resistant parasites displayed

dramatically reduced expression of CYP5122A1 protein, consistent with this hypothesis.

These resistant organisms also displayed characteristics in common with CYP5122A1

HKO Leishmania : a significantly reduced growth rate compared to their wild type

counterparts (Figure 4.5), failure to differentiate completely to promastigotes (Figure

4.6), and significantly greater susceptibility to oxidative damage induced by tBuOOH

than their wild type counterparts (Figure 4.9). However, our GC-MS analysis shows that

unlike CYP5122A1 HKO Leishmania (234), DB766 resistant Leishmania show no

change in ergosterol levels compared to their WT counterparts (Table 4.2) . Nonetheless, the resistance of CYP5122A1 HKOs to the lethal effects of DB766 and their hypersensitivity to ketoconazole (Figures 4.8 and 4.9) establishes a definitive role of

CYP5122A1 in the anti-leishmanial mechanism of action of DB766 and antifungal azoles.

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CHAPTER 5

DB766: Mechanistic implications, conclusions and future directions

5.1 Implications from proteomic and ultrastructural studies

In the present investigation, while the comparative proteomic study suggests involvement of the mitochondrion, the ultrastructural studies show that the mitochondrion is not the primary target and that the mechanism of action of DB766 is distinct from that of diamidines in Leishmania . DB766 induced changes in the proteome are consistent with the loss of mitochondrial membrane potential and are consistent with a secondary role for the mitochondrion in response to the lethal effects of DB766 in

Leishmania. The ultrastructural alterations induced by DB766 in Leishmania , such as the appearance of abundant electron dense bodies, increased cytoplasmic vacuolization and abnormalities in the flagellar pocket, matches the ultrastructural profile of sterol biosynthesis inhibitors (198) and protease inhibitors in Leishmania (202, 207). Based on the ultrastructural similarities, we evaluated hypotheses that protease inhibition and/or

110 alterations in sterol metabolism contribute to the anti-leishmanial effects of AIAs.

However, the ultrastructural profile matching based protease inhibition hypothesis was refuted by the lack of Leishmania OPB inhibitory activity of DB766 and hence OPB was not explored further as a potential antileishmanial target of AIAs.

5.2 DB766 treatment alters sterol composition and synergizes the anti-leishmanial action of posaconazole in Leishmania

The ultrastructural profile matching approach led us to investigate sterol metabolism in

DB766 treated Leishmania. The GC-MS analysis of Leishmania sterols revealed that

DB766 treatment, under the given conditions, resulted in relative accumulation of lanosterol (a 14 α-methyl sterol). This effect, although less prominent, is consistent with posaconazole (a potent CYP51 inhibitor that disrupts sterol metabolism in fungi and trypanosomatids) induced changes in the sterol composition in Leishmania and suggest inhibition of sterol 14 α-demethylase or other enzymes in the sterol biosynthetic pathway in Leishmania . Further, the effect of DB766 on Leishmania sterol metabolism provides a logical explanation for the observed synergism between DB766 and posaconazole due to mechanistic overlaps and thus opens up possibilities for developing novel combination strategies for the treatment of leishmaniasis.

The ultrastructural effects induced by DB766 can thus be attributed to the observed alterations in sterol composition in L. donovani in this study and are thus similar to ultrastructural effects induced by other sterol biosynthesis inhibitors in Leishmania.

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Sterols are important structural components of physiological membranes. Disruption of sterol metabolism, as a primary event, has been shown to have downstream effects on the structure and function of several subcellular organelles including the parasite mitochondrion (231). We therefore believe that the loss of mitochondrial membrane potential and the accompanying changes in the proteome occur downstream of the disruption of sterol metabolism in DB766 treated Leishmania .

5.3 Implications from studies on DB766 resistant Leishmania

5.3.1 Acquisition of resistance to DB766 alters the ultrastructure of Leishmania

Our electron microscopy studies revealed a difference in the ultrastructure of WT and

DB766 resistant parasites both in the presence and in the absence of DB766 pressure

(Figure 4.4): the abundance of electron dense bodies in the latter, a feature consistent with what was observed in DB766 treated Leishmania (Figure 3.4). Interestingly, a similar feature has also been reported and speculated to be organelles called acidocalcisomes in electron micrographs of Leishmania exposed to sterol biosynthesis inhibitors (198), protease inhibitors (202, 207), diamidines (135), N-alkyl and N-aryl- bisphosphonates (229) and 8-aminoquinolines (35, 130) in trypanosomatids. Although the identity of these electron dense bodies as acidocalcisomes remains to be verified in

DB766 treated/resistant Leishmania, the AIA DB766 appears to be a good candidate for accumulation in acidocalcisomes due to its weakly basic nature (pKa ~ 7) and lipophilicity (237). While acidocalcisomes have been proposed as antiprotozoal drug

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targets (79), the lack of correlation between acidocalcisomal accumulation and

sensitivity to these drugs in trypanosomatids (130, 134) makes it tempting to speculate

that sequestration of drugs in these organelles might instead serve as a mechanism to

reduce the toxic effects of drugs and mediate resistance (168).

5.3.2 Acquisition of resistance to DB766 alters drug susceptibility in Leishmania

The DB766 resistant L. donovani axenic amastigotes described in this study are cross resistant to other AIAs, consistent with the hypothesis that DB766 and the other AIAs mediate lethal effects through common target(s). While the inability of verapamil to reverse this resistance argues against a role for efflux pumps in the acquisition of resistance, the heightened susceptibility of DB766 resistant parasites to miltefosine (2.5 fold more sensitive) and antifungal azoles like ketoconazole and posaconazole (over 1000 fold more sensitive) indicates compensatory alterations in the common target or metabolic pathways related to the target. This conclusion is based on the previous observation of the hypersensitivity of amphotericin B resistant Leishmania (over 10 fold more sensitive) and Candida to ketoconazole (over 8 fold more sensitive) (181, 226).

Compensatory alterations following acquisition of drug resistance can thus confer cross- resistance or hypersensitivity to structurally diverse drugs acting on the same or parallel pathways.

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5.3.3 CYP5122A1, a CYP450 enzyme with a role in ergosterol metabolism, is a key

determinant of susceptibility to DB766, antifungal azoles and oxidative stress in

Leishmania

Reduced expression of key sterol biosynthetic enzymes like CYP51, SCMT and CPR has

previously been shown to enhance the susceptibility to antifungal azoles and/or

miltefosine in fungi and Leishmania (96, 132, 142, 157, 173, 226, 233, 251). Western

blot analysis of key enzymes in Leishmania sterol metabolism indicates that while

DB766 resistant parasites had significantly increased expression of CYP51 expression of

CYP5122A1, a recently identified novel cytochrome P450 in Leishmania , was dramatically reduced in these resistant organisms as (Figure 4.8), compared to their wild type counterparts. Knockout of a single allele of CYP5122A1 in Leishmania (half knockout, HKO) resulted in a 3.5-fold decrease in ergosterol levels. The resultant

CYP5122A1 HKO parasites had significant growth defects, impaired metacyclogenesis and reduced infectivity in vitro and in vivo. The observed growth defects in CYP5122A1

HKO parasites were partially rectified upon supplementation with exogenous ergosterol.

Genetic complementation of CYP5122A1 HKO parasites with episomally expressed

CYP5122A1 partially rescued the growth phenotype, by increasing the ergosterol levels in these parasites (234). These observations with both DB766 resistant Leishmania and the CYP5122A1 HKO strain underscore the importance of CYP5122A1 in ergosterol metabolism in Leishmania . Further, drug susceptibility assays with CYP5122A1 HKO parasites indicate that they are less susceptible to DB766 (Figure 4.10) and more susceptible to ketoconazole (Figure 4.11), which is consistent with our observations

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concerning CYP5122A1 deficient DB766 resistant parasites. Taken together, these data

highlight the critical role played by CYP5122A1 in determining the susceptibility of

Leishmania parasites to DB766 and antifungal azoles. Besides similarities between

CYP5122A1 deficient DB766 resistant Leishmania and CYP5122A1 HKO Leishmania in their susceptibilities to DB766 and azoles, these two parasite lines also displayed important growth and transformation defects (Figure 4.5 and 4.6), and significantly greater susceptibility to oxidative damage induced by tBuOOH than their wild type counterparts (Figure 4.9).

5.3.4 Acquisition of resistance to DB766 alters sterol composition without affecting ergosterol levels in Leishmania

Since CYP5122A1 HKO Leishmania had a lower level of ergosterol compared to wild type parasites, we investigated the effect of reduced expression of CYP5122A1 on sterol metabolism in DB766 resistant organisms. GC-MS analysis of Leishmania sterol shows that unlike CYP5122A1 HKO Leishmania that exhibited 3.5-fold less ergosterol than the corresponding wild type cell line , DB766 resistant Leishmania had distinct alterations in sterol composition without affecting ergosterol levels compared to their WT counterparts

(Table 4.2) . The lack of obvious alteration in ergosterol composition in the DB766 resistant cell line suggests functional compensation by overexpressed CYP51 in DB766 resistant parasites. Also it might be due to differences in effects associated with acute knockdown in CYP5122A1 HKO and gradual downregulation of CYP5122A1 during adaptation to increasing concentrations of DB766 over a 19 month period in DB766

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resistant Leishmania . Thus, while CYP5122A1 knockdown resulted in a 3.5-fold decrease in ergosterol, its loss during acquisition of resistance to DB766 might have been compensated by the re-routing of sterol intermediates in Leishmania . Although not yet

directly demonstrated in trypanosomatids, multiple biosynthetic routes to ergosterol have

already been reported in Saccharomyces (95) and Aspergillus species (4), enabling these

organisms to synthesize ergosterol even when several sterol biosynthetic enzymes are

suppressed. One of these studies described the analysis of the sterol composition of erg 3

mutants of an Aspergillus strain defective in sterol C5 desaturase activity (4). This study

indicated that despite increased accumulation of C5-saturated sterol intermediates (12.4%

over 4.8% in wild type), these mutants had ergosterol levels (75%) comparable to their

wild type counterparts (76%) (4). Based on the 1) structural and functional evidences

from L. infantum CYP51, 2) ability of trypanosomatids to synthesize 14 α demethylated sterols and 3) ability of trypanosomatids to sustain growth despite treatment with sterol biosynthesis inhibitors, Hargrove et al. (103) and Goad et al. (19) suggest that sterol biosynthesis in Leishmania diverges into several branches beyond squalene, so as to allow variation in the order of subsequent biochemical reactions (Figure 5.1). Such variation allows the parasite multiple options to adjust the sterol flux to permit survival, especially in the event of inhibition of key sterol biosynthetic enzymes by small molecule inhibitors. Our observation that DB766 resistant parasites can synthesize wild type levels of ergosterol in spite of reduced levels of the intermediates lanosterol [ 8], 5- dehydroepisterol [ 4] and ergosta-5,7,22,24-tetraen-3β-ol [ 3] (Table 4.2) is consistent with

the studies cited above describing multiple routes to ergosterol biosynthesis in

trypanosomatids and fungi. A similar observation was also made by Rakotomanga et al. ,

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where miltefosine treatment resulted in a 43% reduction in 24-alkylated sterol intermediates in L. donovani promastigotes without affecting ergosterol levels compared to the untreated controls (189).

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Figure 5.1 Model for proposed routes to ergosterol biosynthesis in yeast. 1: lanosterol; 2: ergosterol; 3: zymosterol; 4: 4,4-dimethylzymosterol; 5: 4 α- methylzymosterol; 6: cholesta-7,24-dien-3β-ol; 7: cholesta-5,24-dien-3β-ol; 8: cholesta-5,7,24-trien-3β-ol; 9: 4 α-methyl-ergosta-8,24-dien-3β-ol; 10: fecosterol; 11: episterol; 12: 5,6-dihydroergosterol; 13: ergosta-5,7,22,24-tetraen-3β-ol; 14: ergosta- 8,22-dien-3β-ol; 15: ergosta-8,22,24-trien-3β-ol; 16: ergosta-8-ene-3β-ol; 17: ergosta- 22,24-dien-3β-ol; 18: ergosta-7-en-3β-ol; 19: ergosta-5,7,24-trien-3β-ol; 20: ergosta- 5,7-dien-3β-ol

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[Reprint with permission from the American Chemical Society: Journal of the American

Chemical Society (95), copyright (1973), American Chemical Society ]

5.3.5 Proposed model 1 for altered drug susceptibility and sterol metabolism in the light of reduced expression of CYP5122A1 in DB766 resistant Leishmania

Based on the knockdown of CYP5122A1 in the CYP5122A1 HKO cell line (234), it appears that both CYP5122A1 and CYP51 perform vital roles in ergosterol biosynthesis in Leishmania donovani . The altered sterol profile in the DB766 resistant organisms described in this study suggests that reduced expression of CYP5122A1 may cause the cells to depend on CYP51 for ergosterol production. DB766 resistant parasites, which lack CYP5122A1, are hypersensitive to CYP51 inhibition by ketoconazole and posaconazole (Table 4.1), and CYP5122A1 HKOs are less susceptible to DB766 and more susceptible to ketoconazole than their WT counterparts (Figures 4.10 and 4.11).

Although the physiological function of CYP5122A1 at this point is unknown, based on 1) its important contribution to the ergosterol biosynthesis, 2) evidence that it could be a potential target of DB766 and other AIAs and 3) its potential for inhibition by DB766 leading to a change in sterol composition indicative of inhibition of sterol 14 α-

demethylase activity, we propose that CYP5122A1 provides additional sterol 14 α- demethylase activity in Leishmania , despite the fact that it has low sequence similarity to

Leishmania CYP51 (~20%). By sequence similarity to mammalian CYP4 and fungal

CYP52, CYP5122A1 appears to belong to a family of CYP450s with fatty acid hydroxylase activity. From an evolutionary standpoint, CYP51 has been suggested as a

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common ancestor whose duplication and divergence might have led to evolution of other

CYP450 enzymes with sterol and secondary metabolite syntheses and xenobiotic

metabolism capacities (116). It is thus possible that CYP5122A1 may also have evolved

from CYP51. While acquiring new function(s), it may have retained its sterol 14 α- demethylase activity. Considering our data and the facts discussed above, we propose that both CYP51 and CYP5122A1 carry out sterol 14 α-demethylation by acting on different substrates within the post-squalene pathway of sterol biosynthesis in Leishmania . We

propose divergence of the sterol biosynthetic pathway downstream of squalene into two

distinct branches: the CYP51 dependent and the CYP5122A1 dependent routes that can

provide required levels of ergosterol and potentially distinct pools of ergosterol related

sterols to the trypanosomatids. If the activity of one of these enzymes is reduced either by

genetic knockout of one allele or by the action of a small molecule inhibitor (azoles in the

case of CYP51 and DB766 in the case of CYP5122A1), the normal sterol pool will be

affected. Inhibition of either route to sterol biosynthesis or perturbation of the typical

sterol pool may lead to toxic effects on Leishmania . As illustrated in Figure 5.2, the re-

routing of ergosterol biosynthesis through the CYP51 dependent pathway in DB766

resistant L. donovani could result in the required levels of ergosterol even in the absence

of CYP5122A1, but at the expense of a normal pool of sterol intermediates (Table 4.2)

and possibly ergosterol related sterols unique to each route. Further, absence of the

CYP5122A1 dependent pathway and presence of only the CYP51 dependent pathway for

ergosterol biosynthesis could make DB766 resistant Leishmania hypersusceptible to

inhibition by azole drugs like ketoconazole and posaconazole.

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Figure 5.2 Proposed model for the role of CYP5122A1 in ergosterol metabolism in Leishmania and in ketoconazole susceptibility in DB766 resistant Leishmania . The route indicated by broken arrows is a hypothetical CYP5122A1 dependent pathway and the route shown by solid black arrows is a hypothetical CYP51 dependent pathway. The choice of C4-dimethyl or C4-desmethyl sterols as the preferred substrate of CYP5122A1 is based on the C4-monomethyl sterols as preferred substrates of L.infantum CYP51 (103) . Absence of CYP5122A1 in DB766 resistant Leishmania could redirect (blue thick arrow) ergosterol biosynthesis via the CYP51 dependent pathway, making these organisms hypersusceptible to the lethal effects of azoles. The situation is likely to be more complex than depicted in the model.

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5.3.6 Proposed model 2 for altered drug susceptibility in the light of reduced

expression of CYP5122A1 in DB766 resistant Leishmania

An alternative role for CYP5122A1 in regulating the sensitivity of L. donovani to both

DB766 and azoles is possible. Cytochrome P450s (CYP450s), with their ability to

catalyze monooxygenation of a plethora of compounds, can modulate drug susceptibility through chemical modification for drug detoxification or activation (126). The presence of phenobarbital inducible CYP450 activity, similar to mammalian CYP1A1, has been previously reported in kinetoplastid microsomal fractions (1, 2, 23). CYP5122A1 shares sequence similarity with mammalian CYP4A10 (> 25%) and fungal CYP52 (~ 25%), both of which are fatty acid hydroxylases (119) that play a role in drug metabolism.

Detoxification by CYP450s has been suggested as a potential mechanism by which parasites exhibit drug resistance (2). In fact, overexpression of CaALK8, a member of the

CYP52 family, conferred multidrug resistance to fluconazole, itraconazole and 4- nitroquinoline oxide in a C. albicans mutant bearing multiple disruptions in several ABC efflux pumps (170). The capacity of CYP61, a sterol C22-desaturase with an indispensible role in ergosterol biosynthesis, for xenobiotic metabolism in

Saccharomyces species has been reported previously (116). While CYP61 is believed to

have evolved from CYP51, it has been suggested that this enzyme retained its role in ergosterol biosynthesis and also developed detoxification capabilities (116). Given its role in drug response and its sequence similarity to CYP410 and CYP52, it is possible that CYP5122A1 may also play a role in xenobiotic metabolism in Leishmania . Thus,

CYP5122A1 could be required for metabolic activation of DB766 to a more active

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species leading to the observed leishmanicidal effects. Under DB766 pressure,

downregulating CYP5122A1 (to minimize metabolic activation) may be beneficial for

survival, which is observed in insects resistant to permethrin or organophosphate based

insecticides (253). If CYP5122A1 plays a role in drug metabolism, increased sensitivity

to miltefosine and azoles in CYP5122A1 deficient HKOs and DB766 resistant

Leishmania raises the possibility that CYP5122A1 metabolically inactivates these antileishmanial drugs. CYP5122A1 may thus play a dual role in ergosterol and xenobiotic metabolism, providing an alternative explanation for the observed resistance to DB766 and hypersensitivity to miltefosine and azoles in Leishmania donovani in this

study.

Nonetheless, we favor the hypothesis that CYP5122A1 is a molecular target rather than a

metabolic activator of DB766 based on its perturbation of sterol metabolism in L.

donovani (Figures 3.4 and 3.6). Further work is required to distinguish these hypotheses.

5.5 Conclusions

Effects of DB766 on the ultrastructure, sterol metabolism and synergism with

posaconazole in Leishmania support our hypothesis that DB766 targets sterol metabolism

in these organisms. Given the important role of the CYP5122A1 protein in survival,

virulence, drug response and ergosterol metabolism in Leishmania , its dramatic

downregulation in our DB766 resistant parasites at least partially explains their resistance

to DB766 and hypersensitivity to antifungal azoles. These studies suggest CYP5122A1 as

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a potential target of AIAs, inhibition of which can result in disturbances in sterol

metabolism , and downregulation of which offers a survival advantage during the

acquisition of resistance to AIAs in Leishmania . This further highlights the importance of

sterol metabolism in the action of antileishmanial drugs and drug candidates. A better

understanding of this process could inform the discovery of new antikinetoplastid agents.

5.6 Future directions

To validate CYP5122A1 as an anti-leishmanial target of DB766, genetic complementation of DB766 resistant Leishmania with episomally expressed CYP5122A1 and susceptibility studies with such complemented parasites will be performed in our laboratory. To better understand the role of CYP5122A1 in ergosterol metabolism and in determining the susceptibility to DB766 and ketoconazole, crystallographic and enzyme activity studies for structural and functional characterization of CYP5122A1 are recommended in the future. Knowledge of CYP5122A1 structure and function will permit a rational approach for 1) further lead optimization of the AIA class and 2) further exploration (target driven HTS based approaches) for identification and development of novel small molecule inhibitors of this protein as new anti-leishmanial drug candidates.

We have examined key enzymes of the ergosterol biosynthetic pathway in Leishmania including CYP51, SCMT-A, and CPR. Deficiency in these enzymes has been previously documented to enhance the sensitivity to azoles in fungi and Leishmania (96, 132, 142,

157, 173, 226, 233, 251). The reduction in the level of SCMT-A and CPR mRNA

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transcripts in DB766 resistant Leishmania fits our original hypothesis and might explain the enhanced sensitivity of these organisms to miltefosine and antifungal azoles and distinct perturbations in their sterol composition. However, it is important to note that these reduction are only modest (1-1.5 fold) compared to wild type counterparts. Given the post-transcriptional and post-translational regulation of gene expression in

Leishmania , reduced expression of these and other sterol biosynthetic genes remains to be confirmed at the protein level in DB766 resistant Leishmania . Follow-up studies should include measurement of the levels of these proteins in the DB766 resistant cell line.

Further, we have not examined gene products other than enzymes of the ergosterol biosynthetic pathway that have previously been established to play a direct or indirect role in the response to azoles in fungi. A recent genome-wide screening study of a yeast deletion mutant library identified ~100 genes whose deletion enhanced susceptibility to azoles in Schizosaccharomyces pombe . This list included genes in the ergosterol biosynthetic pathway, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction etc. The majority of these genes were involved in membrane trafficking and were subunits of adaptor protein-1 and 3 (86). Similar observations were made with adaptor protein-1 deletion mutants of Leishmania . Deletion of subunits of adaptor protein-1 altered sterol homeostasis by disrupting endosome to lysosome trafficking of sterols and as a result enhanced the sensitivity to ketoconazole in

Leishmania (235). Thus, potential modulation of genes other than those of the sterol biosynthetic pathway could also contribute to azole hypersensitivity and resistance to

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DB766 in Leishmania . To identify other potential targets, genome wide studies employing either genome sequencing, transcriptomics or proteomics to identify mutations conferring resistance, followed by validation of appropriate targets, are recommended for

DB766 resistant Leishmania.

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