OLD TARGETS AND NEW BEGINNINGS: A MULTIFACETED APPROACH TO COMBATING , A NEGLECTED TROPICAL DISEASE

DISSERTATION

Presented in Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy

from the Graduate School of The Ohio State University

By

Adam Joseph Yakovich, B.S.

*****

The Ohio State University 2007

Dissertation Committee:

Karl A Werbovetz, Ph.D., Advisor Approved by

Pui-Kai Li, Ph.D.

Werner Tjarks, Ph.D. ______

Ching-Shih Chen, Ph.D Advisor

Graduate Program In Pharmacy

ABSTRACT

Leishmaniasis, a broad spectrum of disease which is caused by the protozoan parasite , currently affects 12 million people in 88 countries worldwide. There are over 2 million of new cases of leishmaniasis occurring annually. Clinical manifestations of leishmaniasis range from potentially disfiguring to the most severe manifestation, , which attacks the reticuloendothelial system and has a fatality rate near 100% if left untreated. All currently available therapies all suffer from drawbacks including expense, route of administration and developing resistance.

In the laboratory of Dr. Karl Werbovetz our primary goal is the identification and development of an inexpensive, orally available antileishmanial chemotherapeutic agent. Previous efforts in the lab have identified a series of dinitroaniline compounds which have promising in vitro activity in inhibiting the growth of Leishmania parasites. It has since been discovered that these compounds exert their antileishmanial effects by binding to and inhibiting polymerization.

Remarkably, although mammalian and Leishmania are ~84 % identical, the dinitroaniline compounds show no effect on mammalian tubulin at concentrations greater than 10-fold the IC 50 value determined for inhibiting Leishmania tubulin

ii polymerization. These results indicate that Kinetoplastid tubulin may present a useful chemotherapeutic target.

Ongoing drug development efforts with the dinitroaniline compounds will require that the next generation analogues be analyzed in vitro against not only the parasite but also purified parasite tubulin. Previously, tubulin has been purified from the pathogenic Leishmania amazonensis . There are several drawbacks with utilizing

L. amazonensis as a tubulin source, including, low growth densities, expense of growth medium and risk of to laboratory personnel. Leishmania tarentolae utilizes the gecko lizard as a , and does not infect humans. Furthermore, L. tarentolae can be grown to high densities and can be cultured in inexpensive medium. To assess the suitability of L. tarentolae tubulin as a viable alternative to the corresponding from L. amazonensis for compound screening, both the α- and β-tubulin genes were sequenced for comparisons with pathogenic species. Both

α- and β-tubulin protein sequences are at least 98% identical to sequences from closely related Leishmania species. Additionally, two in vitro experiments were conducted to examine the degree of dinitroaniline binding site congruency between

L. amazonensis and L. tarentolae tubulin. IC 50 values, in terms of inhibiting tubulin polymerization, for our lead compound GB-II-5 were 6.7 µM and 6.8 µM for L. amazonensis and L. tarentolae tubulin, respectively. Dissociation constants (K d), determined by fluorescence quenching, were used to compare binding affinities of

GB-II-5 for tubulin from both L. amazonensis and L. tarentolae . The K ds for GB-II-5 for L. amazonensis and L. tarentolae were determined to be 1.7 µM and 2.4 µM,

iii respectively. These data taken together indicate that tubulin from L. tarentolae is a suitable alternative to tubulin from pathogenic species for drug evaluation.

The K ds and IC 50 values determined for numerous GB-II-5 analogues synthesized in our lab against Leishmania tubulin has allowed us to utilize molecular modeling and molecular dynamics to better characterize the dinitroaniline binding site. Although we have learned a great deal about this site using the aforementioned methods, a crystal structure of tubulin with a dinitroaniline bound would yield the most definitive information about the binding site. Traditionally, tubulin has been very difficult to crystallize, due in part to its lack of stability and it’s propensity to polymerize at higher conditions. Recently the crystal structure of vinblastine bound to mammalian tubulin has been elucidated. The authors utilized stathmin, a 17 kDa protein which acts to regulate dynamics, to sequester tubulin into tetramers and were able to crystallize a tubulin-stathmin complex. It was thought that this approach may be useful for crystallizing Leishmania tubulin.

Reports documenting the purification of mammalian stathmin indicate stathmin present in the soluble cell lysate of fetal bovine thymus will effectively inhibit tubulin polymerization by sequestering dimers. Although successful in inhibiting mammalian tubulin polymerization, preliminary attempts to sequester Leishmania tubulin with active stathmin containing fractions from fetal bovine thymus failed to show any activity. The lack of activity was not particularly surprising and it was hypothesized that variations in tubulin between Leishmania and were great enough to prohibit mammalian stathmin from interacting with Leishmania

iv tubulin. As such, the next step was an attempt to identify an active stathmin like protein present in Leishmania or the closely related parasite Crithidia fasciculata .

Efforts to isolate an active fraction by looking for activity in terms of inhibiting tubulin polymerization, per the protocol for isolating mammalian stathmin, were unsuccessful. It was discovered that upon warming, a dense precipitate formed in the Leishmania soluble cell lysates. Since tubulin requires heat to polymerize, and that polymerization is assessed by monitoring turbidity at 350 nm, the precipitate was preventing us from monitoring tubulin polymerization or the lack thereof. Attempts to remove the precipitate by repeated warming and centrifugation steps failed to decrease baseline levels to those at which tubulin polymerization could be observed.

Being as the protein of interest was likely a low molecular weight protein (LMW), size exclusion chromatography was used to fraction the soluble cell lysate by molecular weight. Although this method was useful in separating the present in the cell lysate, and removing the precipitate, the LMW fractions failed to demonstrate any activity against Leishmania tubulin. Concerns that column fractionation may have decreased the stathmin like protein concentration below its active threshold were addressed by concentrating LMW containing fractions. An iCON protein concentrator was used and effectively concentrated LMW containing fractions from

0.53 mg/mL to 2.7 mg/mL. Unfortunately, these concentrated LMW fractions still failed to show activity against tubulin. At this point the search for a Leishmania stathmin like protein was terminated. This work does not prove that there is no stathmin like protein present in Leishmania , however, it indicatess that if there is such a protein present, it likely does not as large of a role in regulating microtubule dynamics as its mammalian counterpart. Perhaps future studies utilizing

v coimmunoprecipitation coupled to mass spectrometry will be useful in identifying proteins that associate with and influence Leishmania microtubule dynamics.

GB-II-5 is a promising antileishmanial compound, however, it is desirable to have multiple leads due to later stage attrition often seen in the drug development process. As such, our lab has recently purchased the ChemBridge CNS set of

10,000 druglike molecules. A multifaceted approach is being employed with the library whereby compounds were screened both against axenic amastigotes and also against purified Leishmania tubulin. Compounds in this library conform to Lipinski’s rule of 5, thus, it is likely that any potential lead compound identified in this screen will possess oral availability.

The first stage of the screen against L. donovani axenic amastigotes identified 75 compounds which inhibited parasite growth by 50 % or more at a concentration of 10 µM. The specific activity, in terms of IC 50 , of each hit compound was then determined. Of the 75 identified active compounds, 47 were found to have

IC 50 values ≤ 5 µM. Surprisingly, 11 of the originally identified active compounds failed to show activity when they were reevaluated and had IC 50 values > 25 µM.

The 47 compounds with IC 50 values ≤ 5 µM were assayed against mammalian Vero cells to determine if they were selectively cytotoxic to Leishmaia cells. Of the 47 compounds, 17 were found to be at least 25-fold selective for inhibiting Leishmania proliferation. These compounds are currently being screened in our infected assay. The infected macrophage assay allows us to assess the compounds activity in clearing infection from mouse peritoneal ; this

vi system more closely mimics in vivo conditions. Furthermore, efforts are currently underway to examine the mechanism of action of some of the active compounds.

Hopefully this endeavor will bring us closer to our ultimate goal of producing an orally available, inexpensive antileishmanial chemotherapeutic agent.

vii

Dedicated to my Parents: Jeffrey and Kathleen Yakovich,

For everything I am and all that I have.

viii

ACKNOWLEDGMENTS

First and foremost, I would like to thank my advisor Dr. Karl Werbovetz for all of his support. Throughout the past few years I have learned a great deal from Karl, his guidance and advice were an invaluable part of my development process. I would also like to thank him for all of the assistance with the preparation of my dissertation and the work performed therein.

I would also like to thank Dr. Dan Sackett at the National Institutes of Health.

Dan served as my mentor for a month, however, we have collaborated closely for the majority of my time at OSU. I have learned a great deal from Dan and he continues to be there for me when I am trouble shooting or looking for new ideas.

I am appreciative of my Graduate Committee members: Drs. Pui-Kai Li,

Ching-Shih Chen, Werner Tjarks and my external committee member Dr. Jonathan

Godbout. Aside from serving as my committee members, they have provided me with support in various respects throughout the past few years.

I will truly miss and wish to thank my colleagues in “the Werbovetz Lab.” I have learned a great deal from my colleagues both in and outside of the lab. I would specifically like to thank Rachel Morgan for her support and assistance with the preparation of my dissertation, Dawn Delfin for her assistance with biological evaluation of selected compounds, Trupti Pandharkar for her assistance with the

ix infected macrophage assays performed herein and Manar Salem for serving as our

“lab mom” for a majority of my time in the lab.

I would also like to acknowledge and thank a group of individuals in the

College of Pharmacy whom although not directly connected with my education have gone out of their way to assist me in finding employment. Drs. Rajesh Balkrishnan,

Ken Hale, Milap Nahata and Robbert Brueggemeier have all been of great assistance in helping me to find employment either by serving as a reference or by forwarding my C.V. to the appropriate people. Additionally, I would like to thank

Kathy Wolken for her assistance with electron microscopy and confocal microscopy.

Finally, I would like to thank my family (Jeff, Kathy, Katie and Kristie

Yakovich) for their support throughout my entire life and my fiancé, Melissa Hayes, for her support and tolerance of me the past few months while I have been writing my dissertation. I would also like to thank God for blessing me with all of the wonderful people in my life and the abilities which have made my degree possible; may he continue to bless my family, loved ones and country.

x

VITA

March 8, 1980………………………………Born—Grand Rapids, Michigan

2002………………………………………….B.S. Biology, Baldwin-Wallace College Berea, Ohio

2002-Present……………………………….Graduate Research and Teaching Associate, Division of Medicinal Chemistry and Pharmacognosy The Ohio State University

PUBLICATIONS

1. George TG, Endeshaw MM, Morgan RE, Mahasenan KV, Delfin DA, Mukherjee MS, Yakovich AJ , Li C, Werbovetz KA. Synthesis and Biological Evaluation, and Molecular Modeling of 3,5-Substituted-N1-phenyl-N4, N4-di-n-butylsulfanilamides as Antikinetoplastid Antimicrotubule Agents. Bioorganic and Medicinal Chemistry (2007), Article in press.

2. Yakovich AJ , Ragone F, Alfonzo J, Sackett DL, Werbovetz KA. Leishmania tarentolae : Purification and characterization of tubulin and its sutability for antileishmanial drug screening. Experimental (2006), 114(4), 289-296.

3. Delfin DA, Bhattacharjee A, Yakovich AJ , Werbovetz KA. Activity of and Initial Mechanistic Studies on a Novel Antileishmanial Agent Identified through in Silico Pharmacophore Development and Database Searching. Journal of Medicinal Chemistry (2006), 49(14), 4196-4207.

4. George TG, Johnsamuel J, Delfin DA, Yakovich AJ, Mukherjee M, Phelps MA, Dalton JT, Sackett DL, Kaiser M, Brun R, Werbovetz KA. Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides. Bioorganic and Medicinal Chemistry (2006), 14(16), 5699-5710.

5. Das U, Gul HI, Alcorn J, Shrivastav A, George T, Sharma RK, Nienaber KH, De Clercq E, Balzarini J, Kawase M, Kan N, Tanaka T, Tani S, Werbovetz KA, Yakovich AJ , Manavathu EK, Stables JP, Dimmock JR. Cytotoxic 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4- pentadienes mounted on alicyclic scaffolds. European Journal of Medicinal Chemistry (2006), 41(5), 577-585.

xi

FIELDS OF STUDY

Major Field: Pharmacy

Area of Emphasis: Medicinal Chemistry and Pharmacognosy

xii

TABLE OF CONTENTS

Page

Abstract………..………………………………………………………………………...... ii

Dedication………..…………………………………………………………………..….....viii

Acknowledgments...... ix

Vita……………………..…………………………………………………………….…....…xi

List of Figures……….……………………………………………………………….……..xix

List of Tables…………….……………………………………………………….……...... xxii

List of Abbreviations……………..……………………………………….……………....xxiii

CHAPTER 1

1 Leishmaniasis...... 1

1.1 -borne Protozoan Parasitic Diseases Affecting

Humans in Developing Nations….…………………………………..……….1

1.2 Leishmaniasis, a Neglected Tropical Disease with Lethal

Consequences...... 4

1.2.1 Transmission and Life Cycle………………….…………...... …….4

1.2.2 Infectious Species and Clinical Manifestations…...... 9

xiii 1.2.3 Epidemiology……………...... 12

1.1.4 Leishmania /HIV Co-…………….……………...... ……14

1.2.5 Disease Diagnosis, Control and Chemotherapy…...... ………16

CHAPTER 2

2 Tubulin as a Target for Antileishmanial Chemotherapeutic Agents…...... …25

2.1 Tubulin and Antimitotic Agents…………………………………...... ……..25

2.1.1 Tubulin, A Critical Eukaryotic Protein…………………...... ……..25

2.1.2 Tubulin as a Chemotherapeutic Target………………...... ……..34

2.1.3 Antileishmanial Compounds Targeting Tubulin………...... …….40

2.2 Purification and Characterization of Tubulin from Leishmania

tarentolae and its Suitability for Antileishmanial Drug Screening...... 45

2.2.1 Drawbacks to Using L. amazonensis Tubulin for

Drug Screening……………………………….……….....………..45

2.2.2 Leishmania tarentolae Tubulin as a Potential Alternative

to L. amazonensis Tubulin…………………………….………….46

2.3 Materials and Methods...... 47

2.3.1 Chemicals……………………………………………….………….47

2.3.2 Parasites……………………………………………………………47

2.3.3 Purification of Tubulin……………………………………………..48

2.3.4 Tubulin/drug Assembly Assays…………………………………..50

xiv

2.3.5 Determination of Dissociation Constants by

Fluorescence Quenching Assays………………………….……..50

2.3.6 Genomic Cloning and Sequencing…………………………….…51

2.4 Results and Discussion...... 53

2.4.1 Purification of L. tarentolae Tubulin…………………….………..53

2.4.2 Sequences of L. tarentolae α- and β-tubulin

Genes and Comparison with Tubulins from Other

Leishmania Species and Other Organisms…………….…….....54

2.4.3 Tubulin-ligand Interactions……………...... ………...... …60

2.4.4 Fluorescence Quenching Results………………………..…....…61

2.5 Conclusions………………………….……………………………..…….……62

CHAPTER 3

3 The Search for a Stathmin Like Protein in Kinetoplastids Capable

of Affecting Microtubule Dynamics…………………………………………....……….66

3.1 Introduction to Stathmin…...………………………………………....………66

3.2 Materials and Methods...... 71

3.2.1 Chemicals...... 71

3.2.2 Blast Searching and Sequence Alignments…………....……….71

3.2.3 Preparation of Cell Lysate from Fetal Bovine

Thymus…………………………………………………....………..71

xv

3.2.4 Preparation of Cell Lysates from Leishmania

tarentolae and Crithidia fasciculata ………………………...... 72

3.2.5 Preparation and Calibration of Sephadex Column………....…..73

3.2.6 Fractionation, Concentration and Concentration

Determination of Cell Lysates…………………………....……….74

3.2.7 Leishmania and Mammalian Tubulin Polymerization

Inhibition Assays………………...………………………....………75

3.3 Results and Discussion...... 76

3.3.1 Blast Searching…………………………………………....……….76

3.3.2 Effects of Mammalian Cell Lysate on Tubulin

Assembly…………………………………………………....………80

3.3.3 Effects of C. fasciculata and L. tarentolae Lysates

on Tubulin Assembly……………………………………....………83

3.3.4 Column Calibration and Efficiency of Separation……....………89

3.3.5 Effects of parasite Lysate Fractions on Tubulin

Assembly………………………………………………....………...93

3.4 Conclusions……………………………...…………………………....………98

CHAPTER 4

4.1 Introduction to High Throughput Screening…………...... ….101

4.1.1 Current Drug Development Efforts………………..……...... …101

xvi 4.1.2 High Throughput Screening to Identify Novel

Therapeutic Agents and Molecular Targets…………….…..…103

4.1.2.1 High Throughput Screening…………………...... …..103

4.1.2.2 Target Based Approaches………...... …………..104

4.1.2.3 Phenotype Based Approaches……...... …107

4.1.3 Library Selection, Hit Identification and Lead

Optimization…………………………..………………………..…110

4.1.4 The Search for Novel Antileishmanial Compounds...... 113

4.2 Materials and Methods...... 117

4.2.1 Primary Screen, IC 50 Determinations and Vero Cell

Selectivity Screen……………………………………………..….117

4.2.1.1 Chemicals and Biochemicals…………………..…….117

4.2.1.2 Primary Screen Against L. donovani

Axenic Amastigotes………………………………..….118

4.2.1.3 Determination of IC 50 Values for

Hit Compounds…………………………………..…….120

4.2.1.4 Vero Cell Assay…………………………………..……122

4.2.1.5 Infected Macrophage Assay……………………...…..123

4.2.2 Biological Evaluation of Compound 4.45 Against

L. donovani Axenic Amastigotes…………………………..……125

4.2.2.1 Transmission Electron Microscopy……………..……125

4.2.2.2 Cell Cycle Analysis by Flow Cytometry…………..…126

xvii 4.3 Results and Discussion……………………………………………....…….127

4.3.1 Primary Screen, IC 50 Determinations and Vero

Cell Selectivity Screen………………….………...... ……..….127

4.3.1.1 Assay Validation and Optimization…………....…….127

4.3.1.2 Primary Screen…………………………………....…..129

4.3.1.3 Determination of the Activity of Primary

Hits……………………………………………….....…..133

4.3.1.4 Vero Cell Selectivity Screen………………...... ……135

4.3.1.5 Confirmation of Activity and Selectivity…….....…….142

4.3.1.6 Infected Macrophage Assay………………….....…...145

4.3.2 Biological Evaluation of Compound 4.45…………….....……..146

4.3.2.1 Transmission Electron Microscopy………….....……146

4.3.2.2 Flow Cytometry……………………………….....…….150

4.4 Conclusions and Future Directions…………...... ……151

References ……………………………………………………………………..………...154

xviii

LIST OF FIGURES

Page

1.1 The Life Cycle of Leishmania …………………………………………….…....…..7

1.2 Geographical Distribution of CL...... 13

1.3 Geographical Distribution of VL………...... 13

1.4 Current approved parenterally administered antileishmanial compounds...... 19

1.5 The chemical structure of the orally available antileishmanial drug ……………………………………………………………...... 23

2.1 The Tubulin dimer and the microtubule………...... 26

2.2 External factors which affect the equilibrium of purified tubulin…………...….27

2.3 Bead movement induced by microtubule disassembly…………………...…...33

2.4 The antimitotic drug colchicine…………………………………………...... 34

2.5 Putative binding regions of drugs which disrupt MT dynamics………...……..36

2.6 Structures of benomyl, oryzalin, and trifluralin…………………...………...... 40

2.7 Selected structures from [144] which were found to possess Antileishmanial activity and inhibit leishmanial tubulin Polymerization...... …..….43

2.8 Assessment of tubulin purity by SDS-PAGE……………………………....……53

2.9 Amino acid sequence alignment of L. tarentolae α-tubulin, etc…...... ………57

2.10 Amino acid sequence alignment of L. tarentolae β-tubulin, etc……....………58

xix

2.11 Fluorescence quenching profile of oryzalin and L. tarentolae tubulin……...... 62

2.12 Fluorescence quenching profile of GB-II-5 and L. tarentolae tubulin……...... 62

2.13 GB-II-5 SAR as predicted by molecular modeling and molecular docking studies………………………………………………………....………….64

3.1 Vinblastine and colchicine are shown bound to tubulin-RB3 complex…...... …69

3.2 Phylogenic tree illustrating divergences in stathmin like proteins between different species……………………………………………....……...... 77

3.3 The protein sequence of L. major glycoprotein 96-92...... 78

3.4 Alignment of mammalian and Xenopus stathmin protein sequences With their respective similar sequence regions on L. major gp 96-94…...... …79

3.5 Tubulin assembly in the presence of mammalian cell lysate top fraction...... 82

3.6 Tubulin assembly in the presence of Leishmania cell lysate top fraction...... 84

3.7 Tubulin assembly in the presence of Leishmania cell lysate bottom fraction………………………………………………………………………...... 85

3.8 Assembly profile for the soluble L. tarentolae cell lysate……………….....…..86

3.9 Leishmania tarentolae soluble cell lysate……………………………….....……87

3.10 Absorbance of samples after sequential warming/centrifugation cycles...... 88

3.11 Elution profiles of blue dextran and molecular weight standards…...... ……90

3.12 Calibration curve for the gel filtration column determined from standards………………………………………………………...... …..91

3.13 Fractions eluted from the gel filtration column analyzed by SDS-PAGE...... 92

3.14 10% SDS-PAGE of L. tarentolae fractions eluted from the column…….....…93

xx

3.15 Assembly of purified L. tarentolae tubulin in the presence of low molecular weight fractions from the soluble L. tarentolae cell lysate...... 94

3.16 4-20% SDS-PAGE of L. tarentolae cell lysate through preparative steps……………………………………………...... ……...96

3.17 Assembly profiles of L. tarentolae tubulin with and without concentrated lysate fractions……………………………………………...... 97

3.18 The chemical structure of GB-II-150 and its equipotent dicyano substituted analogue……………………………………………………...... 100

4.1 The structure of GB-II-5, an analogue of the antimitotic oryzalin……...... 101

4.2 Some representative structural classes of compounds which were/are being evaluated and/or optimized in our lab………...... …102

4.3 Therapeutic target classes………………………………………………...….…105

4.4 Stage by stage quality assessment to reduce costly late stage attrition…………………………………………………………………….……….112

4.5 Schematic of primary screen setup……………………………………….…....119

4.6 Determination of IC 50 values for hit compounds……………………….……...120

4.7 Assay plate following 6 hr incubation with colorimetric dye…………….……131

4.8 Electron micrograph images of Leishmania donovani axenic amastigotes treated with compound 4.45 ……………………………….……..149

4.9 Histogram of cell cycle analysis by flow cytometry…………...... ….……..150

4.10 Cyclical progression of compound development……………………….……..152

xxi

LIST OF TABLES

Page

1.1 Species of Leishmania which infect humans and their resulting clinical manifestations…...... 10

2.1 Activities of oryzalin and analogues against L. donovani promastigotes and amastigotes in vitro and purified tubulin from L. amazonensis ...... 43

2.2 Purification of tubulin from Leishmania tarentolae promastigotes...... 54

3.1 The percent amino acid identity of stathmin like proteins from different Species...... 76

3.2 Protein concentrations of L. tarentolae cell lysate fractions...... 94

4.1 An incomplete list of companies with compound collections for purchase...... 114

4.2 Comparison of 30 and 60 µL assay volumes...... 129

4.3 The average Z’-Factor and internal standard IC 50 values...... 132

4.4 The percent inhibition values from the primary screen and the IC 50 values of the 75 primary hits identified from the screen...... 134

4.5 Results from the selectivity screen against Vero cells...... 136-137

4.6 Structures, nomenclature and calculated partition coefficients of compounds displaying selectivity for L. donovani axenic amastigotes over mammalian Vero cells…………………………………………..……138-141

4.7 IC 50 values of selective compounds against L. donovani ……………………143

4.8 % inhibition of selective compounds against Vero cells……………………..144

xxii

LIST OF ABBREVIATIONS

Å Angstrom ADMET Absorption Distribution Metabolism Excretion Toxicity AIDS Acquired Immunodeficiency Syndrome AmB AMP Antimicrobial Peptides ATP Adenosine Triphosphate BLAST Basic Local Alignment Search Tool C Celsius CAC Critical Assembly Concentration cDNA cloned Deoxyribonucleicacid CL Cutaneous Leishmaniasis clogP Calculated log octanol/water partition coefficient CNS Central Nervous System CO 2 Carbon Dioxide DAT Direct Agglutination Test DCL Diffuse Cutaneous Leishmaniasis DEAE Diethylaminoethyl DEET diethyl toluamide diH 2O Deionized Water DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DTP Developmental Therapeutics Program DTT dichlorodiphenyltrichloroethane EDTA Ethylene Diamine Tetraacetic Acid EGTA Ethylene Glycol Tetraacetic Acid ELISA Enzyme Linked Immunosorbent Assay ER g Gram GDP Guanosine Diphosphate GDP ·Pi Guanosine Diphosphate + Inorganic Phosphate GMPCPP Guanylyl ( α,β)methylenediphosphate gp Glycoprotein GTP Guanosine Triphosphate h Hours HBSS Hank's Balanced Salt Solution HDX Hydrogen/deuterium Exchange

xxiii HIV Human Immunodeficiency Virus HTS High Throughput Screen i.m. intramuscular i.v. intravenous IC 50 Inhibitory Concentration of 50 % IFAT Indirect Immunofluorescence KATEX Latex Agglutination Test Kav Apparent Partition Coefficient Kd Dissociation Constant kDa Kilo Daltons L Liter L. Leishmania (genus) LC-ESI Liquid Chromatography-Electrospray Ionization LCL Localized Cutaneous Leishmaniasis LDL Low Density Lipoprotein LPG Lipophosphoglycan LTR Long Terminal Repeat M Moles MAP Microtubule Associated Protein MCL Mucocutaneous Leishmaniasis MD Molecular Dynamics MES 2-(N-morpholino)ethanesulfonic acid mg Milligram MgCl 2 Magnesium Chloride min Minutes mL Milliliter mM Millimolar (millimoles/liter) MM Molecular Modeling mRNA Messenger Ribonucleicacid MS Mass Spectrometry MTS Tetrazolium Dye NADPH Nicotinamide Adenine Dinucleotide Phosphate NCI National Cancer Institute nm Nanometer Op Oncoprotein ORF Open Reading Frames PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction pH log of the hydrogen cation concentration PI Phosphatidylinositol PIPES piperazine-N,N ′-bis(2-ethanesulfonic acid) PKADL Post-Kala Azar Diffuse Leishmaniasis PME 50 mM PIPES, 5 mM MgSO 4, 5 mM EGTA PMN Polymorphonuclear Neutrophil Granulocytes PMS N-methyl dibenzopyrazine methyl sulfate PMSF Phenylmethylsulfonylfluoride PNA Lectin Peanut Agglutination r2 Correlation Coefficient

xxiv RCL Recidivans Cutaneous Leishmaniasis rev Review RNA Ribonucleicacid ROS Reactive Oxygen Species rpm Revolutions Per Minute s.c. subcutaneous SAR Structure Activity Relationship Sb III Trivalent Sb v Pentavalent Antimony SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis sp Species spp species (plural) sspp Subspecies (plural) TEM Transmission Electron Microscopy TUNEL Terminal dUTP Nick End Lableing µM micromolar µm micron Ve Elution Volume VL Visceral Leishmaniasis Vo Void Volume VSG Variable Surface Glycoprotein Vt Total Volume W Watts WB Western Blotting WHO World Health Organization WT Wild Type

xxv

CHAPTER 1

INTRODUCTION TO LEISHMANIASIS

1.1. VECTOR-BORNE PROTOZOAN PARASITIC DISEASES AFFECTING

HUMANS IN DEVELOPING NATIONS

Although many people in Western nations are familiar with the spectra of diseases caused by bacteria and viruses, few are aware of the toll that parasitic infections take on developing nations. Parasites are defined as organisms which rely on a host for survival in a relationship which is not symbiotic, meaning that the parasite feeds at the host’s expense. Parasitic infections are endemic in, but not confined to, tropical, sub-tropical, and temperate climate regions. These infections are responsible for over 1 million deaths per year [1]. There are three main classes of parasites which can cause disease in humans [1]: 1. - single celled, microscopic organisms which can be free living or parasitic in nature. They may be found in the digestive tract (fecal-oral transmission) or in blood and tissue of humans (arthropod vector). Some examples of diseases caused by these protozoans include malaria ( Plasmodium ), African Sleeping Sickness ( Trypanosoma )

1

and leishmaniasis ( Leishmania ); 2. Helminths- large multicellular organisms which may be free living or parasitic in nature. The mature adult form cannot multiply within its human host. Some examples of these include flatworms (platyhelminths), thorny- headed worms (acanthocephalins), and roundworms (nematodes); and 3.

Ectoparasites- A broad category including blood sucking arthropods such as mosquitos, ticks, fleas, lice, and mites. It is important to note that these parasites may also serve as vectors for the aforementioned parasitic classes.

Protozoan parasites are of chief interest in our laboratory. The protozoa which are infectious to humans are classified based on their mode of motility [1].

The four groups are sarcodina (the amoeba), mastigophora (the ), ciliophora (the ciliates), and sporozoa (organisms whose adult stage is non-motile).

Although our laboratory has close ties and often collaborates with groups interested in the sporozoa Plasmodium falciparum (the etiological agent of malaria), our primary interests lie with the mastigophora, more specifically parasites belonging to the genera Leishmania and Trypanosoma . The Trypanosoma genus can be broken down into two categories, African trypanosomes and American trypanosomes, based on tissue tropism.

African (African sleeping sickness) is caused by trypanosomes belonging to the species (sp.) T. brucei . These parasites are transmitted by the blood sucking tsetse flies, distributed in 36 countries in sub-

Saharan and are estimated to affect over 70,000 people [2] There are two subspecies (sspp.) which infect humans that vary by both geographical distribution

2

and disease progression. T. b. rhodesiense , found mainly in Eastern and Southern

Africa, causes the acute form of the disease which progresses in weeks. T. b. gambiense , found mainly in West and Central Africa, causes the chronic form of the disease which often takes years to progress [3]. Early symptoms of these diseases include fever, headaches, and dizziness. If untreated, both diseases ultimately culminate in CNS invasion (meningoencephalitic phase), resulting in seizures, somnolence, coma, and eventually death. One notable feature of the trypanosomes is the presence of a variable surface glycoprotein (VSG), which allows it to evade host immune responses by “antigenic variation [3].”

American trypanosomiasis (Chagas’ disease) is caused by T. cruzi . Although these parasites are transmitted by the blood sucking reduviid bug, transmission occurs via fecal matter from the bug entering the host through the mouth, nose, eyes, or breaks. Chagas’ disease is estimated to affect 16-18 million people living mostly in Latin America [4]. The acute disease causes fever and inflammation of the lymph nodes, heart, and sometimes brain. Long term cardiac damage can result in heart failure and sudden death. Although our lab works intimately with trypanosomes, the main focus of my research efforts thus far have been directed towards Leishmania.

3

1.2. LEISHMANIASIS, A NEGLECTED TROPICAL DISEASE WITH LETHAL

CONSEQUENCES

1.2.1. Transmission and Lifecycle

As described previously, leishmaniasis is a spectrum of disease caused by protozoan parasites; these parasites belonging to the Trypanosomatidae family, which falls within the order. The Kinetoplastida refers to organisms which possess a , a mass of mitochondrial DNA located adjacent to the and the basal body.

The vector of Leishmania is the phlebotomine . Although any of the over 600 species could potentially be vectors, 30 spp. have been proven to be vectors [5]. Typically vectors fall within the subgeneras , Lutzomyia, and Psychodopygus . The parasites reside in the midguts and salivary glands of the female ; they enter their animal host as the sandfly withdraws its bloodmeal.

Occurrences of Leishmania transmission are either anthroponotic or zoonotic.

Anthroponotic transmission occurs when the parasites are transferred solely between the sandflies and humans and vice-versa. Zoonotic transmission involves intermediate animal reservoir hosts. Leishmania has many zoonotic reservoirs, including sloths, opossums, small , and [6,7,8].

4

Leishmania parasites have a dimorphic life cycle consisting of promastigote and amastigote stages. When residing within the midgut and salivary glands of the sandfly, they adopt a slender, flagellated morphology, 15-20 µm in length, designated the promastigote stage. Within the midgut, the promastigotes undergo sequential development from a non-infective, logarithmic growth cycle termed the procyclic stage to the stationary, infective metacyclic stage [9,10,11,12,13]. Although pro- and metacyclic parasites are morphologically identical, variations in their membrane composition have been observed. A 116 kDa surface protein, originally identified as metacyclic specific, was discovered by differential agglutination by lectin peanut agglutinin (PNA) [14]. PNA, which is very effective at agglutinating the non- infective procyclic forms, failed to agglutinate the infective metacyclic parasites. It has since been discovered that the surface protein, later identified as a lipophosphoglycan (LPG), is present on both parasite stages. As such, LPG structural variations between stages rather than its presence/absence account for the differential agglutination with PNA [15]. The cycle within the sandfly typically lasts between 8-20 days, at the conclusion of which the promastigotes are regurgitated into the salivary glands of the sandfly and injected into the animal host when the sandfly is withdrawing bloodmeal.

In mammalian hosts, Leishmania are obligate intracellular parasites which prefer phagocytes as host cells [15]. Polymorphonuclear neutrophil granulocytes

(PMN) are the first to arrive at the sub-cutaneous (s.c.) infection site, followed by macrophages [16]. The PMNs serve as intermediate hosts for the promastigotes

5

[17,18]. Apoptotic PMNs are phagocytized by macrophages allowing a silent “Trojan horse” entry mechanism for the promastigotes [18]. Within the macrophage, the parasites reside in the acidic phagolysosome. Multiple factors within this new environment, including an acidic pH (~5) and an increase in ambient temperature from ~25 °C (within the poikilothermic sandfly) to 37 °C facilitate a morphological change from promastigote to amastigote. During this metamorphosis, the parasites retract their flagella, lose motility and adopt a smaller (2-5 µm in length) rounded phenotype. In susceptible hosts, the amastigotes are able to suppress the production of cytotoxic factors such as nitric oxide, which are typically produced to clear infections [19]. Within the host macrophages, the amastigotes replicate and eventually lyse the macrophage thereby allowing them to reenter the blood stream.

It is thought that the amastigotes mediate macrophage lysis by the production of cytolysin, which aggregates and forms a pore in the plasma membrane [20]. While in the bloodstream these amastigotes are either phagocytized by other macrophages, in which they continue to propagate, or enter another sandfly vector if it feeds on an infected host. Upon reentry to the vector, the amastigotes convert to promastigotes and the cycle is perpetuated. The lifecycle of Leishmania is summarized in Figure 1.1.

6

Figure 1.1 The life cycle of Leishmania .

(Reproduced by permission of the Centers for Disease Control and Prevention.)

7

It is important to note that upon entry to the bloodstream the promastigotes encounter and must evade the host’s innate immune response. as such, Leishmania have evolved multiple mechanisms to accomplish this task. As previously discussed, the promastigotes are coated with a thick LPG layer which is essential for survival within both the vector and host [21]. Perhaps the most important roles for LPG occur within the sandfly midgut, where LPG has been suggested to promote survival by inhibiting the release of proteases [22], protecting the parasite surface from proteolytic attack [23], and by helping the parasites attach to the gut wall such that they are not excreted with the passage of a bloodmeal [24]. Within the host, LPG has been recognized as favoring the intracellular survival and establishment of the parasite [25]. LPG has been shown to inhibit several important macrophage functions including the generation of oxygen radicals [26]. During conversion to the amastigote form, the parasite loses most of its LPG and retains only the intramembrane component phosphatidylinositol (PI) [27].

The parasites also possess a 63 kDa membrane bound protease called gp63 or leishmanolysin. Gp63, a zinc-metalloprotease, is the major surface protein on

Leishmania promastigotes [28]. Aside from cleaving host antimicrobial peptides

(AMP), gp63 has displayed affinity for cellular fibronectin receptors [29]. These interactions with fibronectin receptors have been shown to cooperate with -dependent adhesion; this most likely provides the parasite with an efficient attachment and entry mechanism into macrophages [30]. Identification

8

and characterization of these Leishmania specific proteins is essential not only to better understand host/ interactions, but also may prove useful in developing a Leishmania [31,32].

1.2.2 Infectious Species and Clinical Manifestations

At least 20 species of Leishmania are known to infect humans [33].

Leishmania infections vary between species, both in terms of tissue tropism and severity. Leishmaniasis is a broad spectrum disease which can range from asymptomatic to disfiguring forms of tegumentary and potentially fatal visceral disease [34,35]. There are three main clinical manifestations of leishmaniasis, the causative parasite spp. are further classified as “old world” or “new world” parasites based upon geographical distribution and vector genus. Old World parasites are found primarily in Europe, Asia and Africa and are transmitted by sandflies belonging to the Phlebotomus genus. New World parasites are found primarily in the Western regions of Central and South America; they are transmitted by sandflies belonging to the genus Lutzomyia . The three primary manifestations are cutaneous leishmaniasis

(CL), mucocutaneous leishmaniasis (ML) and visceral leishmaniasis (VL). CL can be further broken down into four categories: localized (LCL), diffuse (DCL), recidivans

(RCL), and post-kala azar dermal leishmaniasis (PKADL) [38]. Leishmania spp. responsible for the aforementioned clinical manifestations are shown in Table 1.1

[37].

9

Clinical Manifestation Old World Parasites New World Parasites Cutaneous leishmaniasis L. major (LCL) L. mexicana (LCL) (CL) L. tropica (LCL/RCL) L. venezuelensis (LCL/DCL) L. aethiopica (LCL/DCL) L. amazonensis (LCL/DCL) L. infantum (LCL/PKADL) L. braziliensis (LCL/RCL) L. donovani (PKADL) L. panamensis (LCL) L. guyanensis (LCL) L. peruviana (LCL) L. chagasi (LCL/PKADL)

Mucocutaneous L. aethiopica (rare) L. mexicana leishmaniasis (ML) L. major L. amazonensis L. braziliensis L. guyanensis L. panamensis

Visceral leishmaniasis (VL) L. infantum L. chagasi L. donovani L. tropica (rare)

Table 1.1 Species of Leishmania which infect humans and thei r resulting clinical manifestations

Cutaneous leishmaniasis, the most common form, causes 1-200 simple skin lesions which self heal within a few months but often leave unsightly scars [5]. LCL typically appears at the site of infection. It typically begins as an inflammatory papule, which later progresses to an . DCL often occurs in patients who cannot mount a sufficient immune response to contain LCL. These patients develop hundreds of papules, nodules, and plaques covering the body. DCL is often resistant to therapy and thus may assume a chronic course. Although rare, RCL is a recurrence of lesions at a previously healed site. These recurrences typically occur on the facial region, tend to be resistant to therapy and often result in severe morbidity. PKADL, which is endemic to and Sudan, develops months to years after the patient’s recovery from VL. PKADL often manifests as widespread

10

cutaneous papules reminiscent of DCL and often persist for decades. Treating

PKADL is extremely difficult due to its chronic nature and requires protracted treatment regiments which are extremely expensive, often prohibitively so [5].

Mucocutaneous leishmaniasis is predominantly a New World disease which often results in great morbidity [5,36]. MCL often takes years to manifest clinically and is thought to result from LCL which has migrated to the upper respiratory tract.

The resulting destruction of the oro- and nasopharynx membranes causes extensive facial morbidity and may be fatal.

Visceral leishmaniasis (kala azar, or black fever), a systemic parasitic infection, is the most severe manifestation of Leishmania infection. In this form, the parasites localize to the reticuloendothelial system rather than to the skin. The hallmarks of this disease form include hepatosplenomegaly, abdominal ascites, fever, malaise, anorexia, pancytopenia, and hypergammaglobulinemia [5,36]. In the absence of successful treatment, the mortality rate of VL is near 100%.

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1.2.3 Epidemiology

According to the World Health Organization (WHO) the overall prevalence of people affected by leishmaniasis is 12 million. Annual is estimated to be 2 million (75% CL infection, 25% VL infection), with an additional estimated at risk population of 350 million [38]. It is important to note that there is likely a discrepancy between the aforementioned numbers and actual numbers [5]. Several factors may account for this: 1.) The distribution of transmission sites within endemic areas is often discontinuous, 2.) numerous cases are undiagnosed, misdiagnosed or unreported and 3.) leishmaniasis is not always a notifiable disease; official recognition of disease often creates expectations for intervention by health authorities, these authorities are often unable or unwilling to act to meet the demands of the population [39]. As such, there is little doubt that the number of actual cases are much higher than reported and the number of infections even greater.

The geographical distribution of leishmaniasis is world-wide, affecting 88 countries on five continents – Africa, Asia, Europe, North America and South

America. 90% of CL cases occur in seven countries: Afghanistan, Algeria, Brazil,

Iran, Peru, and Syria, whereas 90% of VL cases occur in five countries:

Bangladesh, India, Nepal, Sudan and Brazil. The geographical distributions of CL and VL are illustrated in Figures 1.2 and 1.3 respectively.

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Cutaneous Leishmaniasis Highly Endemic Countries (90% of cases)

Endemic Regions

Figure 1.2 Geographical distribution of CL

(Reproduced by permission of the World Health Organization (WHO))

Visceral Leishmaniasis Highly Endemic Countries (90% of cases)

Endemic Regions

Figure 1.3 Geographical distribution of VL

(Reproduced by permission of the World Health

Organization (WHO))

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Several of the aforementioned areas have recently seen a disturbing increase in infection trends. In Brazil, CL cases have nearly doubled (21,800 cases in 1998 to

40,000 cases in 2002) and VL cases in the northeastern region of the country have more than tripled (1,840 cases in 1998 to 6,000 cases in 2002)[5]. Furthermore, in

Kabul, Afghanistan CL has increased from 14,200 cases in 1994 to 65,000 cases in

2002 [5]. Factors responsible for these increases include both an increase in human exposure to the vector and changes in individual risk factors, some examples of these include: increased migration from rural to urban areas, movement of seasonal workers and refugees due to civil unrest, large-scale migrations of populations for occupational reasons, increases of non-immune manpower for the development of new projects (road building, oil prospecting, mining, tourism, military activity, etc.) and deterioration in social and economic conditions [40]. Poor social and economic conditions not only serve to increase the occurrence, prevalence and severity of

Leishmania infections, but also increase the hosts’ susceptibility to other infections.

1.2.4 Leishmania/HIV Co-infections

As the vast majority of Leishmania infections occur in developing countries, socioeconomic factors play a large role in its prevalence. Not only do indigent people often lack access to medical care and medicine but they are often immunocompromised as a result of malnourishment; the end result is a population with an increased risk of contracting diseases and a decreased probability that adequate medical care will be available. In environments such as these, Leishmania acts as an opportunistic pathogen [41]. The WHO estimates that there are over 40

14

million people infected with the human immunodeficiency virus (HIV) and that approximately one-third of these infections occur in Leishmania endemic regions

[42].

HIV-1 propagates mainly in T- because these cells express CD4, the primary cellular receptor for viral entry [43,44,45]. However, within the lymph nodes, lungs and central nervous system (CNS), the macrophage has been recognized as the predominant cell line productively infected with HIV [46]. The progression of the HIV infection to full-blown acquired immunodeficiency syndrome

(AIDS) varies in individuals based largely on their immunocompetence [47]. As such, individuals infected with HIV are not only more susceptible to contracting symptomatic leishmaniasis, but once infected, the Leishmania infection progresses more rapidly with increased morbidity and mortality rates. Aside from increasing the severity of the parasitic infection, it appears that the co-infection is a symbiotic relationship in which the Leishmania infection can expedite the progression from HIV to AIDS. Many infections, Leishmania included, induce host inflammatory immune responses; these along with other signaling events may act to promote viral replication [26]. For example, it is known that certain stimuli can activate regulatory elements located within the HIV long-terminal-repeat (LTR) region which can induce a latent virus to begin replication [48,49]. More specifically, both Leishmania LPG and its core PI have been shown to activate complex biological pathways such as

NF-κB, which in turn can activate HIV-1 LTR which acts as a trigger for the activation of a latent virus.

15

Thirty four countries around the world have already reported cases of

Leishmania/HIV co-infections [50]. To combat the problem, the WHO has set up a world-wide surveillance network which includes 28 institutions. The discussion of

Leishmania /HIV symbiosis presented herein serves solely as a brief summary to highlight the increased risks to populations within Leishmania /HIV endemic regions.

For a detailed discussion of the pathogenesis of Leishmania /HIV co-infections see

Oliver et al. [27].

1.2.5 Disease Diagnosis, Control and Chemotherapy

Diagnosis of Leishmania can be broken down into four categories: serological, etiological, PCR and xenodiagnosis [37]. Serological techniques are most useful in diagnosing VL and MCL, both which cause a notable immune response. The most common serological techniques include indirect immunofluorescence (IFAT), dot enzyme-linked immunosorbent assay (ELISA),

Western blotting (WB) and direct agglutination test (DAT). Etiological diagnosis involves examination of prints, smears or histological sections of biological material.

These samples are stained and examined microscopically for the presence of the parasite. The polymerase chain reaction (PCR) is a fairly non-invasive procedure which allows for the identification of all forms of leishmaniasis. The use of specific probes has made it possible to discriminate between the causative species of CL

[51,52]. Xenodiagnosis involves utilizing the sandfly to diagnose VL. In this method, the sandfly is examined for the presence of promastigotes 48 hours after ingesting a bloodmeal from the patient.

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Unfortunately, all of the aforementioned diagnostic methods have drawbacks, most of which originate from the requirement for diagnostic centers with trained personnel and expensive equipment. Despite the limitation, several advances in detection show promise for future diagnosis. A DAT kit using lyophilized antigen is now commercially available [53]. This will allow for testing in remote areas which often lack access to centralized testing facilities. Furthermore, a dipstick called K39 which detects a surface antigen specific for VL is available for field use [54] and a new latex agglutination test (KATEX) which is capable of detecting antigens in urine is now available [55]. Increasing diagnostic capabilities is an important facet in the fight against leishmaniasis. Accurate data regarding the distribution, prevalence and occurrence of Leishmania infections will serve to increase awareness and assist governments in endemic regions in lobbying for external financial support [56].

As with most vector borne diseases, the best method for control is reducing human exposure to the vector. The sandfly is most active from dusk to dawn, is small enough to fit through standard mosquito netting and is a relatively poor flyer

[37]. As such, avoiding nighttime activities, using insecticides such as diethyl toluamide (DEET) on exposed skin, wearing clothes treated with wash resistant insecticides such as permethrin, using a fine-mesh bed net impregnated with permethrin and sleeping with a fan on all work well to reduce human-vector exposure. Indoor and outdoor spraying with insecticides such as dichlorodiphenyltrichloroethane (DDT) continues to be the method of choice for reducing sandfly numbers [57]. Another method for reducing numbers is to reduce

17

breeding. Sandflies prefer to breed in dark, moist environments [57]. Eliminating these environments by using moisture absorbing building materials such as limestone works well in reducing breeding. Although all of the aforementioned methods have drawbacks including expense and developing resistance to insecticides, when used in conjunction they greatly reduce the risk of exposure to the sandfly.

Although many promising results have been obtained towards developing a

Leishmania vaccine (See 32 for review), chemotherapy remains the frontline defense in disease management. Presently there are about 25 compounds which demonstrate efficacy in treating leishmaniasis, however, all of these compounds have one or more limitations and/or drawbacks [58]. As previously discussed, leishmaniasis is endemic primarily in developing countries, so ideal chemotherapeutic agents should be orally available, inexpensive, stable and demonstrate efficacy with short treatment regimens. There are currently four classes of parenterally administered drugs approved for the treatment of leishmaniasis

(Figure 1.4): The pentavalent antimonials (Sb v) such as Pentostam () ( 1.1 ) remain the drug of choice for treating all forms of leishmaniasis; the polyene macrolide antibiotic amphotericin B ( 1.2 ) has been used to successfully treat both CL and VL; diamidines such as ( 1.3 ) have been used to treat

CL and VL; and ( 1.4 ), a aminocyclitol-aminoglycoside antibiotic is currently used to treat CL and is in phase III clinical trials for treatment of VL

[59,60,61,62,63].

18

OH OH OH OH H3C OH O O O O O HO Sb Sb OH HO O OH OH OH OH O CH3 CO2H O O H HO OH H3C CH3 OH HO OH O O OH OH NH2 1.1 1.2 OH

H2N HO NH2 HO OH NH NH O HO O O O H2N NH2 O O OH H N OH O(CH ) O 2 HO NH 2 5 OH 2 NH2

1.3 1.4

Figure 1.4 Current approved parenterally administered antileishmanial drugs. Sodium stibogluconate ( 1.1 ), amphotericin B ( 1.2), pentamidine ( 1.3) and paromomycin ( 1.4).

Despite drawbacks, the pentavalent antimonials (Sb V) have long been and continue to be the drugs of choice for treating both CL and VL. The antimonials are administered intravenously (i.v.) or intramuscularly (i.m.) at doses between 15-20 mg

Sb v/kg of body weight per day for 21-28 days [64]. Although the antimonial drugs

19

have long been used to treat leishmaniasis, their exact mechanism of action has yet to be fully elucidated. It is known that the pentavalent antimonials are prodrugs; in vivo they are reduced to the active trivalent form [65], however, the site and mechanism of reduction remains unclear [66]. Recent work by Wyllie et al [66] indicatess that Sb V exerts its leishmaniacidal activity by interfering with trypanothione metabolism. Trypanothione, an enzyme found exclusively in the Kinetoplastida order, plays an analogous role to glutathione which is found in mammalian systems.

Much like its mammalian counterpart glutathione, trypanothione is an important electron donor in the antioxidant metabolic pathway for hydrogen peroxide removal

[67]. This metabolic pathway is a crucial for parasite survival as it protects them from a cytotoxic buildup of reactive oxygen species (ROS). To donate electrons, trypanothione must first be reduced, this is carried out by the NADPH-dependent flavoenzyme trypanothione reductase [67]. Recently, pentavalent trivalent antimony

(Sb III ) has been shown to interfere with trypanothione metabolic pathway by two mechanisms [66]. First, Sb III decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione and second, Sb III inhibits trypanothione reductase which results in accumulation of the di-sulfide forms of trypanothione [66]. Additional suggested targets of Sb III include [68], purine transport [69], and DNA topoisomerases [70]. Although there have been reports of the development of resistance [71], the Sb v response rate still ranges from

70-90% [60]. Drawbacks associated with treatment failure include the poor quality of the product, inadequate dosing and non-availability [59]. Furthermore, the higher doses required to treat non-responsive disease increase the expense and duration of

20

treatment required and have also been associated with an increased incidence of side effects [72].

The diamidines such as pentamidine have been used since the early 1940s to treat leishmaniasis [73]. Pentamidine is administered three times weekly i.v. or i.m. at 4 mg/kg body weight for 3-4 weeks. Pentamidine inhibits parasite growth by inhibiting replication and at the mitochondrial level [58]. Crystal structures have shown that pentamidine makes hydrogen bonds with O 2 of thymidine or N 3 of adenine within the minor groove of DNA [74]. It has also been suggested that pentamidine disrupts mitochondrial membrane potential in the parasite [75].

Aside from the obvious drawbacks such as availability, price and route of administration, pentamidine toxicity is a major factor limiting its use. Due to toxicity and reported decreases in efficacy, pentamidine is rarely used in India [36].

Amphotericin B (AmB) remains the gold standard for treating fungal infections and is widely used as a second line treatment for leishmaniasis [58]. Unfortunately,

AmB requires i.v. administration with a dosing regimen of 1 mg/kg body weight per day for twenty days. AmB exerts its antileishmanial effects by binding to ergosterol, the major component of parasitic cell membranes; this results in changes in membrane permeability which facilitates cell lysis. Although AmB is useful in treating leishmaniasis cases which are refractory to pentamidine, it still suffers from drawbacks including toxicity. Toxicity results from internalization of AmB-lipoprotein complexes mediated by low density lipoprotein receptors (LDL) [58]. AmB- lipoprotein complexes have been shown to be toxic to renal cells [76]. In an effort to

21

increase the therapeutic index and reduce toxicity of AmB, lipid formations of AmB have been developed. Liposomal preparations (AmBisome, Amphocil and Abelcet) are more active and better tolerated, however, the increased price associated with their preparation reduces their availability in endemic regions [72]. It should also be noted that although no clinical AmB resistance has been reported, drug resistant L. donovani mutants have been cultured in vitro [77]; this indicatess the possibility of developing clinical resistance as AmB use becomes more widespread.

Paromomycin, an antibacterial agent, was first shown to have antileishmanial activity thirty years ago [78]. Since the discovery of its antileishmanial activity, paromomycin has shown efficacy in treating both CL [79] and VL [80]. For treatment of VL, paromomycin is administered i.m. at 15 mg/kg body weight for twenty one days; for treating CL topical formulations are available. It has been shown that paromomycin acts by inducing the destabilization of parasite membranes [81]. As with the other antileishmanial drugs, parenteral administration of paromomycin has drawbacks including ototoxicity and myocarditis [80].

As all of the aforementioned drugs suffer from various disadvantages, the search for orally available, inexpensive and non-toxic antileishmanial drugs continues. The discovery of the alkylphospholipid, miltefosine ( 1.5 , Figure 1.5), has offered hope for an efficacious oral therapy. Miltefosine has been found to be 94-

97% effective for treating VL when administered orally at 2.5 mg/kg body weight for twenty eight days [58]. Miltefosine has been approved in India and by the WHO for treatment of VL and is in phase III clinical trials in Columbia for treatment of CL. The

22

mechanism of action of miltefosine has yet to be elucidated, however, there are several proposed targets including: damage to the flagellar membrane [82], perturbation of alkyl-phospholipid metabolism and glycosylphosphatidylinositol anchor biosynthesis [83] and interference with ether-lipid remodeling through the inhibition of alkyl-lyso-phosphatidylcholine specific acyltransferase [84]. Side effects include nausea, vomiting and teratogenicity. Although no clinical resistance has been observed thus far with miltefosine, resistant parasites which have been cultured in laboratories [85] indicate that resistance may loom in the future as miltefosine use increases.

O N+ O P O O-

Figure 1.5 The chemical structure of the orally available antileishmanial drug miltefosine (1.5).

It should be noted that there are a variety of other drug classes currently being investigated as antileishmanial agents. Orally available azoles (ketoconazole, fluconazole and itraconazole), purine analogues and quinolines continue to be investigated for antileishmanial activity. Additionally, numerous natural products including quinones (lapachol), alkaloids (berberine), phenolic derivatives (chalcones, flavonoids) and terpenes are being investigated [see 58 and 86 for revs.] Despite recent advances, the necessity for novel antileishmanial drugs persists. Strategies

23

towards drug development will continue to focus not only on optimizing current drugs and characterizing identified targets but also on identification of novel drug candidates and targets.

24

CHAPTER 2

TUBULIN AS A TARGET FOR ANTILEISHMANIAL CHEMOTHERAPEUTIC

AGENTS

2.1 TUBULIN AND ANTIMITOTIC AGENTS

2.1.1 Tubulin, A Critical Eukaryotic Protein

Tubulin is a heterodimeric protein composed primarily of α- and β-subunits; each monomer is approximately 55 kDa. Under the appropriate conditions, the dimers polymerize to form (Fig 2.1). Microtubules are long, cylindrical tubes approximately 24 nm in diameter composed α/β-tubulin dimers which associate longitudinally in a head-to-tail arrangement to form protofilaments, thirteen of which associate laterally in a parallel fashion to form the microtubule (MT)[87]. As such, both longitudinal and lateral interactions are required for successful microtubule formation. The intact microtubule has two surfaces, the external surface and the luminal (internal) surface. Microtubules exhibit a structural polarity and are dynamic in nature, constantly switching between growing and shrinking phases [88].

Elongation rates at each end of the microtubule differ due to its intrinsic polarity; the

25

faster growing end is referred to as the plus (+) end and the slower growing end is referred to as the minus (-) end [89]. During the shrinking phase, protofilament fragments are peeled from microtubule ends [90]. These growing and shrinking phases of MTs are essential to their function and are thus highly regulated (See

Chapter 3 for a detailed discussion of proteins which affect microtubule dynamics).

Microtubules play diverse, vital roles in eukaryotic cellular processes including cellular motility, chromosomal segregation, and organelle transport.

(-) A B α

β α β- t u b u l i n Pl u s e n d

β

α- t u b ul i n M i n us e nd

2 5 n m

( +)

Figure 2.1. The tubulin dimer and the microtubule. A. The ribbon diagram of α-tubulin with GTP bound and β-tubulin with GDP and taxotere bound. B. The structure of a microtubule.

26

It is important to note that tubulin can be purified and assembled in vitro . This phenomenon was first observed by Weisenberg who observed the reversible self- assembly of tubulin in the presence of zwitterionic sulfonate buffers containing GTP

[91]. Shelanski et al. later exploited tubulin’s dynamic nature to purify the protein by cycles of assembly/disassembly [92]. Methods of purification now often employ anion-exchange chromatography to increase the purity of tubulin, which is rich in acidic amino acids at the C-terminus of both α- and β-subunits. A great deal of work has since been carried out in characterizing purified tubulin and factors which affect its polymerization state (see [93] for review). Some of the major factors which affect the polymerization state of tubulin are summarized in Figure 2.2. Polymerization can either be observed microscopically or spectrophotometrically by observing increases in turbidity at 350 nm as tubulin polymerizes. Studies that have made use of tubulin’s ability to assemble in vitro have been crucial in increasing our understanding of MT dynamics.

Heat/DMSO/GTP/Mg2+ Dimer Polymer 2+ Cold/Ca

Figure 2.2 External factors which affect the equilibrium of purified tubulin.

27

Tubulin α- and β-monomers are homologous and are ~50% identical at the amino acid level [94]. Each monomer is formed by a core of two β-sheets surrounded by α-helices and has three functional domains: the amino-terminal domain, the intermediate domain and the carboxy-terminal domain. The latter two domains contribute to polymerization interfaces. The C-terminal domain comprises the exterior surface of the protofilament and serves as the binding site for motor proteins such as kinesins and dyneins [95]. Each monomer has a guanine nucleotide-binding site; the N-site (non exchangeable) located on α-tubulin has guanosine triphosphate (GTP) permanently bound while the E-site (exchangeable) on β-tubulin can bind either GTP or guanosine diphosphate (GDP) [96]. While the N- site on α-tubulin is buried in the intradimer interface, the E-site is partially exposed on the surface of the unbound dimer until it is covered by the α-tubulin subunit of a subsequent dimer during polymerization [97]. These nucleotide containing subunits play vital roles in the regulation of microtubule dynamics.

During polymerization, the exposed β-tubulin GTP subunit on the elongating microtubule is hydrolyzed shortly after the addition of the next α/β dimer. Although

GTP hydrolysis is closely coupled with polymerization, it is not required for it.

Interestingly, GTP hydrolysis confers dynamic instability to the microtubule [98]. In the absence of GTP, tubulin has a propensity to polymerize into ring structures [99].

These results, coupled to evidence which shows that microtubules capped with GDP rapidly depolymerize by peeling [88], indicate that tubulin dimers can exist in two different curvature states based on the phosphorylation state of the β-tubulin bound guanosine nucleotide. Melki et al. [100] proposed that the tubulin dimers conform to

28

a curved (low energy state) when bound to GDP and a straight (high energy) microtubule forming conformation when bound to GTP. This hypothesis was later refined when M ϋller-Reichert et al. [101] found that depolymerizing microtubules containing bound guanylyl (alpha,beta)methylenediphosphonate (GMPCPP), a non- hydrolyzable GTP analogue, yielded curled oligomers. Although slightly curved, the oligomers had significantly less curvature than those formed using traditional hydrolysable GTP. Thus, the current evidence indicatess that both GTP and GDP bound dimers are curved with the latter being curved to a greater extent. These results raise two important questions: how are straight microtubules constructed from slightly curved and curved dimers and what are the structural consequences of and rationale for GTP hydrolysis?

To adequately address the former question, it is necessary to give some consideration to the interactions involved in microtubule stability. Microtubules are maintained by both lateral and longitudinal dimeric interactions. The longitudinal interactions, which are stronger based on the observation that microtubules depolymerize by the peeling of protofilaments [90], are formed between the exposed

β-subunit on the elongating protofilament and the α-subunit of the dimer being added. The major areas of longitudinal contacts, which are formed primarily by hydrophobic and polar interactions, are broken down into three zones: A, B, and C

[102]. Zone A involves interactions of the H10-S9 loop in one monomer with the H11 and loop T5 in the next monomer down. H10 also interacts with H6 and the H6-H7 loop of the previous monomer. In zone B, H8 interacts with T5, T3, and the H11-H12 loop. Zone C, near the of the MT, involves direct interaction of loop T7 with

29

the nucleotide and adjacent regions of the previous monomer, T1, H2, and H7. It should also be noted that similar interactions occur between α- and β-subunits within each dimer, however, the specifics will not be discussed. Lateral interactions, which are mostly electrostatic in character, occur between adjacent protofilaments. The

S7-H9 loop (M loop), a central element for interaction, interacts with H3, the C- terminal part of the H2-S3 loop, and part of the H1-S2 loop (N loop) on the adjacent protofilament [102]. The protofilaments form a B-type lattice with a seam [103]. In this lattice α- and β-monomers interact with the corresponding α- and β-monomers of the adjacent protofilament respectively. The lateral dimers deviate horizontally by

10° which results in a left handed 3-start helix which travels up the microtubule. In the B-type lattice, there is discontinuity where in each 3-start helix an α-subunit interacts laterally with a β-subunit or vice-versa; this is referred to as the seam.

Although this will be discussed in greater depth, it should be noted that although MTs are in a helical arrangement, they do not polymerize in that manner. The aforementioned interactions are extremely important and serve to unravel the seemingly paradoxical phenomena of straight MTs formed from curved dimers and the rationale for GTP hydrolysis.

The GTP cap model, proposed by Mitchinson & Kirschner [88] (reviewed by

Desai et al. [103]), provided a large step forward in understanding microtubule dynamics. The model indicatess the presence of an unhydrolyzed GTP cap on the end of polymerizing dimers. The straighter ends at the termini of elongated microtubules reduce outward longitudinal curvature while also favoring lateral bonds between other subunits being added [104]. Although still slightly curved, the GTP

30

capped protofilament ends still allow for lateral interaction while increasing longitudinal interactions. The result from these increased interactions is the formation of an open sheet at the end of elongating microtubules which bends slightly outwards [105]. These sheets then close along the seam, forming the MT and completing the straightening process. This cap is necessary to maintain elongation; loss of the cap results in rapid depolymerization. GTP is hydrolyzed upon longitudinal addition of the next dimer, however, lattice energy keeps the dimer from adopting the preferred bent confirmation. It has since been determined that the cap is actually composed of GDP·P i rather than GTP [106]; thus it is the release of the P i rather than the hydrolysis which drives the conformational change. This phenomenon is not uncommon and has been observed with ATP hydrolyzing motor proteins and GTP hydrolyzing G proteins [107]. The event of nucleotide hydrolysis during polymerization may seem counterintuitive; energy is wasted each time a dimer adds, and microtubule stability is decreased when compared to its straighter, more stable counterpart polymerized with GMPCPP [101]. However, further examination of this event reveals an ingenious regulatory mechanism.

For successful cellular proliferation, tubulin polymerization and depolymerization are equally important. For example Taxol, an anti-cancer drug isolated from the bark of the Pacific Yew tree, induces mitotic arrest via stabilization of microtubules (for review see [108]). Furthermore, it is believed that energy released during hydrolysis is stored kinetically in the microtubule lattice [109].

Grishchuk et al. [110] recently showed that a depolymerizing microtubule can generate ~10 times the force generated by the motor proteins. Briefly, biotinylated

31

microtubules were capped with rhodamine conjugated tubulin by extending the microtubules with GMPCPP. Glass microbeads coated with streptavidin were allowed to bind the biotinylated tubulin. Laser tweezers were used to apply a steady force to the beads away from a trap and towards the (+) end of the microtubule. The beads were then released via depolymerization which was induced by photoexcitation of the rhodamine conjugated caps. Bead motion was detected by an additional laser focused on the center of the bead. It was found that the bead moved differently depending on the nature of microtubule depolymerization. If the protofilaments attached to the bead depolymerized along with or before the rest of the protofilaments, the bead moved further from the trap. If the protofilaments not attached to the bead depolymerized first, the bead was shifted towards the trap

(Figure 2.3).

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Figure 2.3 Bead Movement Induced by Microtubule Disassembly. a, the bead attached to the microtubule, α- and β-tubulin are shown in dark and light green respectively, red indicates the rhodamine GMPCPP cap. The center of the trap is indicated by the red cross. b, the protofilaments attached to the bead peel away 1 st , pushing the bead further from the trap. c, the protofilaments which are not connected to the bead depolymerize 1 st causing the bead to move towards the trap. Reprinted by permission from Macmillan Publishers Ltd: Nature Publishing Group, Vol 438:384-388, 2005.

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In vivo evidence of the energy released by depolymerizing MTs was provided by Westermann et al [111] during real time microscopy observations of the Dam1 complex. The Dam1 complex, which binds to the kinetochore and oligomerizes into a ring that binds microtubules, moves processively during microtubule depolymerization. For a recent review of microtubule intermediates and their implications in biological systems see Nogales et al. [112]. In summation, it has been shown that dephosphorylation of β-tubulin subunits during polymerization is an essential process, both in terms of dynamicity and locomotion.

2.1.2 Tubulin as a Chemotherapeutic Target

As alluded to in the previous section with the mention of Taxol, the critical cellular roles played by tubulin, its tight regulation, and

H CO O its dynamic character are all characteristics which 3 H AB N C CH3 make it a highly exploitable chemotherapeutic target. H3CO H3CO C Interestingly, tubulin was first identified as the subunits O OCH comprising microtubules based upon its interaction with 3 2.1 the antimitotic drug colchicine ( 2.1, Fig 2.4). Figure 2.4 The antimit otic drug colchicine

Microtubules were first definitively identified in 1963 by electron microscopy.

It was later suggested that they were structural elements due to their localization at sites where cells were changing their shape [113]. In 1965, Taylor showed that colchicine, a drug known to destroy the mitotic spindle, bound with high affinity and

34

simple kinetics to cells [114]. Later, colchicine binding cellular extracts were described [115]. It was discovered that all of the cellular extracts to which colchicine bound contained an abundant source of microtubules [116]. It was further noted that colchicine-binding activity in extracts resulted in the disappearance of microtubules.

After in vitro binding kinetics with colchicine were shown to match those seen in intact cells, the authors concluded that colchicine binding site is the subunit of microtubules. The name “tubulin” was later assigned by Mohri [117].

Since its identification, there has been great interest in identifying compounds which interact with tubulin. Drugs which bind to tubulin and affect microtubule dynamics have broad applications ranging from the treatment of human cancer

[118,119,120] to use as antiparasitic agents [121]. Tubulin binding drugs are classified by the region of the protein to which they bind. To date, there are three well established and characterized binding regions, however, recent work discussed herein indicates that additional sites likely exist. The three established binding regions are the taxol site, colchicine site, and the vinca alkaloid domain (Fig 2.5).

Compounds which bind to these domains vary in size, have diverse structures, and include both synthetic compounds and natural products. Treatment of susceptible cells with these compounds induces mitotic arrest. Cells treated with tubulin binding compounds often have condensed chromosomes, lack a nuclear membrane, and either have no mitotic spindle or a highly aberrant spindle morphology [93]. When examined by flow cytometry, a large accumulation of such cells in the G 2+M phases

(tetraploid) is often found.

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Figure 2.5 Putative binding regions of drugs V which disrupt MT dynamics (adapted from [22]). E Binding to the vinca alkaloid domain (V) results in an interdimer wedge which destabilizes lateral and T β longitudinal interactions. Binding to the taxol site (T) results in increased MT stability by resisting outward C N peeling of protofilaments. Compounds which bind the colchicine site (C) result in unfolding in the β- α tubulin monomer which interferes with intradimer contacts. The exchangeable (E) and non- exchangeable (N) nucleotide binding sites are also shown.

Although drugs targeting the different tubulin binding regions elicit the same end result of mitotic arrest, the mechanisms by which they achieve their effects vary greatly. Presently, there appear to be four distinct mechanisms by which drugs bind to and disrupt MT dynamics [93].

Drugs which bind to the taxol site bind preferentially to polymerized tubulin as opposed to dimeric tubulin [93]. As opposed to other classes, taxol site ligands induce mitotic arrest by increasing MT assembly and stability. Often times treated cells show an increase in MT density; these MTs also appear shorter and more bundled than MTs in untreated cells [93]. Drugs which bind to the taxol binding site include paclitaxel, epothilone B, discodermolide, eleutherobin and laulimalide [93].

Paclitaxel is one of the most popular taxol site ligands due to its applications in cancer treatment [119,120]. The structure of taxol bound to the mammalian tubulin

36

dimer in zinc polymerized tubulin sheets has been determined by electron crystallography at 3.5 Å resolution [122]. This crystal structure revealed that taxol binds to the intermediate domain on β-tubulin. In intact microtubules, this binding site is found on the luminal side of the MT wall and is lined by several hydrophobic residues [122]. A recent study by Xia et al. [123] investigated the mechanism by which taxol increases MT stability by monitoring hydrogen/deuterium exchange

(HDX) by liquid chromatography-electrospray ionization (LC-ESI) coupled to mass spectrometry (MS). Briefly, the authors noted a reduction in HDX when tubulin was treated with taxol. They were able to map the sites where there was a reduction in

HDX. In summation, it was found that taxol applies pressure on the βH10 (on the M loop) at the intradimer interface. This results in a loss of flexibility thereby prohibiting a “roll out” of lateral contacts which would lead to opening of the MT wall. It is interesting to note that the MT stabilizing drugs laulimalide and peloruside A have been shown to bind to a different site on tubulin than paclitaxel [124]. More research is required to determine the tubulin binding site(s) of these compounds and how they exert their MT stabilizing effects.

The remaining mechanisms by which drugs disrupt MT dynamics all involve

MT inhibition and/or destabilization. As previously discussed, tubulin was first identified as the subunit of MTs based on its interaction with and inhibition by colchicine. Molecules which bind to the colchicine site tend to be structurally diverse, simple molecules [93]. Some examples of molecules which bind to the colchicine site include podophyllotoxin, steganacin, the combrestatins, 2-methoxyestradiol, the curacins, carbamates such as nocodazole, heterocyclic ketones including some

37

flavones, and benzoylphenylureas [93]. Colchicine inhibits microtubule formation and at high concentrations disrupts MTs, indicatinging that the colchicine site is not completely buried within the polymerized MT [93]. Proteolysis studies on tubulin incubated with colchicine reveal that colchicine binds β-tubulin and induces the unfolding of a small region in the c-terminal domain near Arg390 [125]. This unfolding likely interferes with contacts necessary for assembly.

The third well established binding region on tubulin is the vinca alkaloid domain. Drugs which bind this domain tend to be larger and many are natural products. The two classical drugs which are used to define the vinca domain are vinblastine and vincristine [93]. Vinblastine and other vinca alkaloids have had a good deal of success in clinics when used as antitumor chemotherapeutic agents

[126]. Other drugs which bind to this domain inhibit the binding of radiolabeled vinblastine or vincristine in either a competitive or noncompetitive manner. As such, drugs which bind competitively are said to bind to the vinca site whereas drugs which bind in a noncompetitive manner are said to bind to the vinca domain. Some examples of competitive inhibitors include maytansine and rhizoxin. Noncompetitive inhibitors include the halichondrins, spongistatins, depsipeptides, dolastatins, cryptophycins and the hemiasterlins [93]. Compounds which bind the vinca domain

(vinca site included) induce self-association of tubulin into spiral aggregates as opposed to MT growth [127]. The mechanism of action of vinblastine and other vinca agents had remained largely elusive until recently. Gigant et al . used electron crystallography to study the structure of vinblastine bound to tubulin complexed with the RB3 protein stathmin [128]. The authors found that vinblastine binds to the end

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of elongating MTs and induces a wedge in the interdimer interface. This longitudinal displacement interferes with lateral interactions by displacing the M-loop.

The fourth established mechanism by which MT dynamics are disrupted is by agents which form a covalent crosslink with tubulin cysteine residues. Some examples of drugs in this class include 2,4-dichlorobenzyl thiocyanate, 2-fluoro-1- methoxy-4-pentafluorophenylsulfonamidobenzene, and ottelione A [93]. The former two compounds interact specifically with the Cys-239 on β-tubulin; the sulfhydryl group that ottelione A interacts with has yet to be identified. These covalent interactions inhibit tubulin’s ability to polymerize.

It should be noted that several classes of compounds bind to tubulin at sites which have not yet been fully characterized. Examples of these compounds include the benzamidazoles such as benomyl (2.2, Fig. 2.6) (used as an antifungal) [129] and the dinitroaniline herbicides such as oryzalin (2.3, Fig. 2.6) [130] and trifluralin

(2.4, Fig. 2.6) [129]. It is also noteworthy that many of these compounds possess phylogenic selectivity in MT disruption. Although there are non-selective benzamidazoles (e.g. nocodazole), others have been shown to be selective for inhibiting yeast and nematode tubulin assembly [131,132]. Oryzalin has been shown to selectively inhibit taxol-induced polymerization of rose tubulin while showing little if any effect on taxol-induced polymerization of bovine brain tubulin at equivalent concentrations [133].

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O CH 3 N N N O NH O2N NO2 O2N NO2 N CH3 N O H SO2NH2 CF3

2.2 2.3 2.4

Figure 2.6 Structures of benomyl (2.2), oryzalin (2.3), and trifluralin (2.4)

As our understanding of the tightly regulated and essential process of tubulin polymerization continues to increase, it becomes more evident that tubulin is indeed an extremely valuable chemotherapeutic target. Drugs interfering with MT dynamics continue to be invaluable tools against diseases such as cancer. As discussed earlier tubulin is not unique to mammalian cells, but rather it is a critical eukaryotic protein. Thus, subtle interspecies tubulin heterogeneity could be selectively exploited as chemotherapeutic targets.

2.1.3 Antileishmanial Compounds Targeting Tubulin

As outlined in Chapter 1, kinetoplastid parasites continue to contribute to significant morbidity and mortality in developing countries, and current therapies all have drawbacks. Our lab is interested in identifying and developing antikinetoplastid drugs. Reports that dinitroaniline herbicides, mentioned briefly earlier, possess activity against protozoan parasites [134,135,136,137] indicates that protozoan tubulin may be an exploitable chemotherapeutic target.

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Tubulin is a major protein of Leishmania sp. [138], and also the closely related kinetoplastid parasites Crithidia fasciculata [139] and

[140]. Aside from chromosomal segregation and organelle transport, MTs play other crucial roles in kinetoplastid parasites; they are the main components of both the flagella (in certain life stages) and the subpellicular corset which serves as a (for an in depth review of MT roles within the kinetoplastid parasites see

[141]). Furthermore, although tubulin is fairly conserved between species, kinetoplastid tubulin shows a divergence from mammalian tubulin. α-tubulin amino acid sequences from L. major and T. brucei are 94% identical whereas they share only 82% and 84% identity with α-tubulin from S. scrofa (pig) respectively. L. major and T. brucei β-tubulin are 94% identical while each is only 84% identical to S. scrofa

β-tubulin [142]. These subtle differences coupled with the observations that 1) radiolabeled trifluralin was shown to bind Leishmania tubulin with greater affinity than rat brain tubulin [87,143] and 2) in the presence of 5 µM of oryzalin, MT assembly from partially purified L. mexicana tubulin was inhibited by 73% whereas oryzalin had no effect on the assembly of partially purified rat brain tubulin at 50 µM [130], serve to validate kinetoplastid tubulin as a potential chemotherapeutic target.

In our laboratory, oryzalin was shown to have moderate activity against purified Leishmania tubulin (54% inhibition of assembly at 20 µM) and L. donovani parasites in vitro (IC 50 values of 44 and 73 µM against promastigotes and amastigotes, respectively) [144]. In an attempt to obtain a structure activity relationship (SAR) for oryzalin against kinetoplastid parasites and to draw a

41

correlation between oryzalin’s leishmanicidal activity and its effects on purified

Leishmania tubulin, 13 analogues were synthesized with variations on oryzalin’s N1

(sulfonamide nitrogen) and N4 (aniline nitrogen) positions [144]. Four of the 13 analogues showed a marked increase in potency against the parasites in vitro , three of which demonstrated increased activity against purified leishmanial tubulin. The structures of the four compounds are shown in Figure 2.7 and their activities against

L. donovani and purified tubulin from L. amazonensis are summarized in Table 2.1.

The promising antileishmanial and antimicrotubule results obtained with the oryzalin analogues warranted further investigation. As such, two of the more potent compounds, 2.5 (GB-II-46) and 2.8 (GB-II-5) were further evaluated in terms of both efficacy and selectivity; furthermore, the underlying mechanisms of cytotoxicity were more closely examined [145]. The compounds were reevaluated against L. donovani axenic amastigotes and were also tested against two strains of Trypanosoma brucei brucei (variant 221 and Lab 110 EATRO), J774 macrophages (murine macrophages), and PC3 prostate cells. GB-II-5 demonstrated good activity against both strains of trypanosomes, with IC 50 values of 0.41 µM and 0.73 µM against variant 221 and Lab 110 EATRO, respectively.

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R1 2.5 R 1 = N((CH 2)3CH 3)2 O2N NO2 R2 = H 2.6 R 1 = N(C 5H11 )2 R 2 = H 2.7 R = N((CH ) CH ) 1 2 5 3 2 R 2 = H SO 2.8 R 1 = N((CH 2)2CH 3)2 2 R2 = C 6H5 NHR2

Figure 2.7 Selected structures from [144] which were found to possess antileishmanial activity and inhibit leishmania tubulin polymerization

% Inhibition of IC 50 vs. L. donovani IC 50 vs. L. donovani leishmanial tubulin Com pound promastigotes a amastigotes a assem bly at 20 µM b 2.3 44 73 54 2.5 18 20 89 2.6 8 9 95 2.7 12 12 48 2.8 15 5 108

Table 2.1 Activities of oryzalin and analogues against L. donovani promastigotes and amastigotes in vitro and purified tubulin from L. amazonensis , adapted from [144]. a Values are given in µM units, and are the mean of at least two independent experiments b Values given are the mean of at least two independent experiments

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GB-II-46 was less active against the trypanosomes with IC 50 values of 2.6 µM and 1.9 µM against variant 221 and Lab 110 EATRO respectively. Both GB-II-5 and

GB-II-46 demonstrated selectivity for kinetoplastid parasites over mammalian cells; the IC 50 values against J774 macrophages were 29 µM and 9.4 µM respectively, while their IC 50 values against PC3 prostate cells were 35 µM and 23 µM respectively. The binding affinities of GB-II-5 and GB-II-46 for leishmania tubulin and mammalian tubulin were determined by fluorescence quenching. The dissociation constants (K d) of GB-II-5 for leishmania and mammalian tubulin was determined to be 1.7 and 13 µM respectively, whereas GB-II-46 had lower selectivity with K ds of 8.3 and 13 µM respectively. Cell cycle analysis of L. donovani promastigotes treated with GB-II-5 shows an accumulation of cells in the G 2+M phase which is indicative of mitotic arrest. These results indicate that GB-II-5 possesses selective antileishmanial and antitrypanosomal activity and exerts its effects via mitotic arrest mediated by inhibition of tubulin polymerization. Furthermore, the decrease in the antimitotic effects, selectivity and K ds observed with GB-II-46 indicatess that N1 aryl substitution is an important aspect of the dinitroaniline SAR. GB-II-5 appeared to be a promising lead which warranted further synthetic endeavors to improve selectivity and activity and to optimize pharmacokinetic properties.

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2.2 PURIFICATION AND CHARACTERIZATION OF TUBULIN FROM

LEISHMANIA TARENTOLAE AND ITS SUITABILITY FOR ANTILEISHMANIAL

DRUG SCREENING

2.2.1 Drawbacks to Using L. amazonensis Tubulin for Drug Evaluation

Ongoing drug discovery efforts against Leishmania tubulin will require the continued in vitro evaluation of new candidate compounds against purified parasite tubulin. Reports detailing the expression of tubulin from parasites have appeared

[146,147], some of which have demonstrated the assembly of recombinant α/β- tubulin [148,149]. No such expression system currently exists for the leishmanial protein, however, and antileishmanial drug discovery work will continue to rely on the purification of tubulin directly from the parasite in the near future. Previous evaluations of antimitotic compounds in our laboratory were performed against tubulin purified from the pathogenic species L. amazonensis .

There are several drawbacks to purifying tubulin from L. amazonensis . This parasite is an etiological agent of cutaneous leishmaniasis and is classified as a

BSL-2 pathogen. To achieve significant yields of tubulin for compound screening, these parasites must be grown to high cell densities and large quantities of cells must be collected (~ 5 × 10 11 parasites), increasing the risk to laboratory personnel of accidental exposure. To meet the continual need for purified Leishmania tubulin, it would be ideal to find an alternative source of this protein which does not infect

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humans, yet where the tubulin possesses sequence similarity to and drug suceptibility properties in common with tubulin from a pathogenic species.

2.2.2 Leishmania tarentolae Tubulin as a Potential Alternative to L. amazonensis

Tubulin

Leishmania tarentolae ’s natural host is the Gecko lizard ( Tarentola mauritanica ) [150], and this parasite species does not infect humans. Furthermore,

L. tarentolae can be cultured to higher cell densities than L. amazonensis ; typical achievable cell densities are 1.8 × 10 8 parasites/ml [151] and 5 × 10 7 parasites/ml

[152], respectively. In addition, L. amazonensis requires Schneider’s Drosophila medium supplemented with bovine serum for high density growth [152], whereas L. tarentolae can be grown in Brain Heart Infusion supplemented with hemin [151], with the latter medium being considerably less expensive. To help assess the suitability of L. tarentolae tubulin as viable alternative to the corresponding protein from L. amazonensis for compound screening, both the α- and β-tubulin genes were sequenced for comparison with the genes from other species of Leishmania . The effects of selected antimicrotubule agents on tubulin isolated from L. tarentolae and

L. amazonensis were also examined. Our studies demonstrate that purification of L. tarentolae tubulin is a valid alternative to obtaining the protein from pathogenic

Leishmania species.

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2.3 MATERIALS AND METHODS

2.3.1 Chemicals

Where not otherwise specified, materials were obtained from Sigma.

Oryzalin analogues were synthesized as described in Bhattacharya et al. [145]. All of the compounds assayed against tubulin were prepared as stock solutions in 100%

DMSO and were stored in the dark at -4 °C.

2.3.2 Parasites

Leishmania tarentolae (UC strain) were a generous gift from Dr. Larry

Simpson (Department of Microbiology, Immunology and Molecular Genetics,

University of California, Los Angeles, CA). The parasites were maintained in Brain

Heart Infusion medium (Becton Dickinson) at 37 g/L which was sterilized by autoclave and supplemented with 10 mg/mL filter sterilized hemin (added from a 2 mg/ml stock dissolved in 0.05N NaOH). Parasites were maintained by serial passage in T-25 flasks (Corning) at 25 °C. Large scale cultures were grown in a 1 liter flask in an orbiting shaker incubator set at 125 rpm and 25 °C. Cells were harvested in late log phase (~ 1-2 × 10 8 parasites/ml) by centrifugation at 1200 × g at

4 °C. Cell pellets were then washed twice with phosphate buffered saline (PBS) solution and stored at -80 °C until use.

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2.3.3 Purification of tubulin

A modification of the earlier protocol for the purification of tubulin from L. amazonensis was employed for the isolation of L. tarentolae tubulin [152]. PBS washed parasites (7.3 × 10 11 cells) were thawed and resuspended in three 40 mL portions in PME + P buffer (PME plus protease inhibitors, consisting of 0.1M PIPES

(pH 6.9), 1 mM MgCl 2, 1 mM EGTA, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin) on ice. These cell suspensions were extensively sonicated on ice using a Misonix (Microson XL) probe sonicator

(Farmingdale, NY) using ten 30 s bursts at full power (~ 25 W) with two minute cooling intervals between bursts. The resulting sonicated suspensions were allowed to cool on ice for 30 min, then were centrifuged in an Optima L-90K Ultracentrifuge

(Beckman, Fullerton, CA) using a Ti-70 rotor at 40,000 × g for 40 min at 4 °C. The resulting supernatant was filtered through glass wool, then loaded via peristaltic pump at a rate of 2.5 ml/min on a column containing 13 ml DEAE-Sepharose Fast

Flow matrix (Amersham Biosciences) that was previously equilibrated with 2 column volumes PME + P. The column was then washed with two column volumes PME +

P, followed by 4 column volumes PME + P containing 0.1 M KCl and 0.25 M glutamate (pH 6.9). Tubulin was eluted with two column volumes PME + P containing 0.3 M KCl and 0.75 M glutamate (pH 6.9). Approximately 3 ml fractions were collected after beginning the 0.3 M KCl / 0.75 M glutamate elution. Fractions were assayed for the presence of tubulin by performing assembly reactions. Briefly,

20 µl of each fraction were added to the 96-well half-area microplates (Costar) on ice, along with distilled H2O and 5× PME (to restore the final PME concentration to

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1×). Assembly was initiated by the addition of 10 µl of 5 mM GTP in 50% DMSO; the final assay volume was 50 µl. Assembly was assessed by measuring the turbidity at

351 nm over a twenty minute period in a SpectraMax Plus microplate reader

(Molecular Devices, Sunnyvale, CA) with the thermostat control set at 30 °C.

Assembly competent fractions were pooled and diluted in PME buffer supplemented with GTP, dimethylsulfoxide and additional MgCl 2 (final concentrations 1× PME, 10 mM MgCl 2, 8% DMSO (v/v), 2 mM GTP). The solution was then incubated at 37 °C for 30 min to promote assembly, then spun at 50,000 × g at 30 °C for 30 min.

Following centrifugation, the supernatant was removed, then the remaining pellet consisting of microtubules was rinsed once with warm PME (~37 °C) and resuspended in ~1.5 ml cold PME. The remaining pellet was further solubilized via probe sonication (three ~5 s bursts at 10 W) using the Misonix sonicator. The tubulin-rich solution was incubated on ice for an additional 30 min, then spun at

50,000 × g at 4 °C for 30 min to remove denatured tubulin. The supernatant containing heterodimeric tubulin was stored at –80 °C in 50 µl aliquots. Protein purity was assessed by SDS-PAGE using 12% polyacrylamide Ready Gels

(BioRad). Gels were stained with Coomassie Brilliant Blue R250 (0.25 g dissolved in

20% MeOH, 8% acetic acid in distilled H2O) for 30 min, then destained with 20% methanol and 8% acetic acid in distilled H2O.

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2.3.4 Tubulin/drug Assembly Assays

Tubulin/drug assembly assays were performed as outlined previously [145].

Assembly reactions were carried out in 96-well half-area microplates (Costar) in a final volume of 50 µL and contained final concentrations of 1.5 mg/ml (15 µM) tubulin, 0.1 mM PIPES (pH 6.9), 1 mM EGTA, 1 mM MgCl 2, 10% (v/v) DMSO, and 1 mM GTP with and without compounds of interest. Solutions containing PIPES,

EGTA, MgCl 2, and compounds were added to the microplate on ice. The compounds were added to reaction mixtures in 5 µl volumes containing 50% DMSO, assembly was initiated by adding 10 µL of 5 mM GTP in 25% DMSO. Absorbance values were recorded in a SpectraMax Plus microplate reader at 351 nm with the thermostat control set at 30 °C. IC 50 values are reported as the mean ± range of two duplicate experiments.

2.3.5 Determination of Dissociation Constants by Fluorescence Quenching Assays

The strong 300-350 nm absorbance of the dinitroaniline compounds makes them useful acceptors of resonance energy transfer from photoexcited tryptophan residues within tubulin. Quenching of intrinsic tubulin fluorescence by dinitroaniline compounds was assessed based on a previous method [145]. Samples were studied using sub-micro 50 µL quartz spectrophotometer cells (Starna Cells).

Tubulin samples (1 µM) containing varying concentrations of oryzalin analogues were excited at 290 nm and tryptophan fluorescence was measured between 310 and 340 nm. Spectra were recorded using a Perkin Elmer LS 50B luminescence

50

spectrometer with excitation and emission slits of 2.5 nm and 15 nm, respectively.

Emission intensity at 325 nm was taken for calculation of quenching. In contrast to the previous study [145], multiple tubulin samples with varying concentrations of compound in a fixed volume of solvent (DMSO, 9.7% v/v) were prepared. Since the

DMSO concentrations were fixed and the fluorescence was only measured once per sample, it was unnecessary to correct for solvent effects and photobleaching. Inner filter effects were corrected by measuring the quenching of N-acetyl-L- tryptophanamide (8 µM) fluorescence in the presence of the compounds. K d values were calculated by plotting decrements in fluorescence as a function of oryzalin analogue concentration. These values were fit to a single-site binding model using the nonlinear fitting routines of Prism software (GraphPad Software, San Diego, CA) and are reported as the fitted K d ± standard error of the fit.

2.3.6 Genomic Cloning and Sequencing

To determine the sequence of Leishmania tarentolae α-tubulin, genomic DNA from L. tarentolae was amplified by the polymerase chain reaction (PCR) using 40 pmol forward (5’-ACCCCTCTTTCTTCTTTTCAGCCATG-3’) and 40 pmol reverse (5’-

TTAGTACTCCTCGACGTCCTCCTCACC-3’) oligonucleotide primers (Integrated

DNA Technologies, Coralville, IA) designed to match the 5’-untranslated region and

3’-terminal region of the L. donovani α-tubulin gene [153], respectively. β-tubulin was PCR amplified using 40 pmol forward (5’-

ATGCGTGAGATCGTTTCCTGCCAGGC-3’) and 40 pmol reverse (5’-

CTAGTAGGCTTCCTCCTCCTCGTCG-3’) oligonucleotide primers matching the 5’-

51

terminal and 3’-terminal regions of the β-tubulin gene (cβT2.2 isotype) from L. major

[154], respectively. PCR reactions were performed using PCR SuperMix (Invitrogen) and incubated in a thermal cycler using a program consisting of a 94 °C denaturation step, 40 °C and 50 °C annealing steps for 40 s, and an elongation step of 72 °C repeated for a total of 25 cycles, following the manufacturer’s instructions (Perkin

Elmer).  Insert containing clones were isolated after transformation into Escherichia coli DΗ5α and sequenced at the Plant-Microbe Genomics Facility at The Ohio State

University using a 3730 DNA analyzer (Applied Biosystems, Inc.).

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2.4 RESULTS AND DISCUSSION

2.4.1 Purification of L. tarentolae tubulin

L. tarentolae tubulin was kDa purified based on a previous protocol 103- 77- for the isolation of L. amazonensis tubulin [152]. The initial step in the 50- earlier protocol involved lysis of cells 34.3- by sonication, followed by 28.8 - centrifugation to generate a Figure 2. 8 Assessment of tubulin purity by SDS-PAGE: Lane 1, crude cytoplasmic fraction and a pellet rich in whole cell lysate obtained by sonication (30 µg); lane 2, cell particulate free tubulin (most likely from subpellicular supernatant obtained by ultracentrifugation (30 µg); lane 3, pooled microtubules and flagella). Since fractions from DEAE chromatography (30 µg); lane 4, cycled leishmanial tubulin there was little tubulin in the (10 µg); lane 5, purified porcine brain tubulin (10 µg); lane 6, cycled cytoplasmic fraction, it was discarded leishmanial tubulin (30 µg). and the pellet was resuspended and sonicated to solubilize tubulin. In our current protocol, the initial fractionation was eliminated in favor of extensive sonication, thus shortening the purification protocol. Figure 2.8 indicates that the

DEAE-Sepharose column was nonetheless capable of providing nearly homogeneous tubulin despite the presence of cytoplasmic proteins. Further purification and concentration of the protein using one cycle of assembly- disassembly provided tubulin with approximately 1% overall recovery based on total

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cellular protein. Fong and Chang determined that tubulin comprised 11.4% of the total cellular protein in L. mexicana promastigotes [138]. Assuming this to be a reasonable approximation for L. tarentolae tubulin, the overall yield of the present purification is roughly 9 – 10%. Protein recoveries obtained at different stages of the protocol are summarized in Table 2.2; a typical procedure can be completed in ~11 hours.

Fraction Volume Protein conc. Total protein Recovery (%) (ml) (mg/ml) (mg) Cell lysate 135 11.7 1580 100 Centrifuged supernatant 85 14.1 1200 75.6 Pooled DEAE fractions 9 2.75 24.8 1.56 Cycled tubulin 1.55 10.7 16.5 1.04 Table 2.2 Purification of tubulin from Leishmania tarentolae promastigotes as described in Section 2.3

2.4.2 Sequences of L. tarentolae α- and β-tubulin genes and comparison with tubulins from other Leishmania species and other organisms

To investigate the similarity between L. tarentolae tubulin and tubulin from other Leishmania species and other organisms, both α- and β-tubulin genes of L. tarentolae were sequenced. The L. tarentolae α-tubulin gene (accession number

DQ309032 ) and β-tubulin gene (accession number DQ309033 ) share 97.3% and

97.5% nucleotide sequence identity with the corresponding genes from the L. major

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Friedlin strain, respectively [155]. As expected, the tubulin amino acid sequences from L. tarentolae show high identity to the tubulin sequences from other Leishmania species and with other organisms. Leishmania species possess a single mRNA for

α-tubulin [156,157], while multiple β-tubulin mRNAs have been observed in this parasite. Three distinct β-tubulin mRNAs have been detected in L. amazonensis

[157] and L. major [154], while multiple β-tubulin mRNAs were also found in L. mexicana [156]. The identity between amino acid sequences for L. tarentolae α- tubulin and α-tubulins from L. major and L. infantum present in available databases is 98%, while the identity between the L. tarentolae β-tubulin sequence reported here and the various β-tubulin sequences (from L. infantum , L. brazilensis , L. major , L. mexicana , and L. amazonensis ) is at least 96%. For L. tarentolae β-tubulin, this includes 98% identity with L. major β-tubulin cβT2.8 and over 99% identity with L. mexicana β-tubulin A850, the predominant β-tubulin isotypes from amastigotes of these species [154,158]. Based on the high amino acid identity observed between L. tarentolae tubulin and tubulins from other Leishmania species, the former protein would be expected to be suitable for antimitotic drug screening against this parasite.

Alignments of the L. tarentolae α- and β -tubulin amino acid sequences with the corresponding sequences from L. major [155], Trypanosoma brucei [159],

Trypanosoma cruzi [160], Plasmodium falciparum [161], and Sus scrofa (pig)

[162,163]are shown in Figures 2.9 and 2.10, respectively. A comparison of these sequences provides some insight into similarities and differences in antimitotic drug susceptibility among these organisms. Compounds that are effective antimitotic

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agents against Leishmania would also be expected to be effective against African trypanosomes, as has been observed for GB-II-5 and GB-II-150 [145,164] based on the 94% amino acid identity between the α- and β-tubulin sequences from T. brucei and L. tarentolae . The lower amino acid sequence identity between L. tarentolae and mammalian tubulin (81% and 84% for α- and β-tubulin, respectively) is reflected by differences in susceptibility to antimitotic ligands. Although vinca domain agents and the microtubule stabilizer taxol affect microtubules in mammalian cells and in

Leishmania [152,165], mammalian colchicine-site agents have little effect on

Leishmania [152] while kinetoplastid tubulin is much more sensitive to dinitroaniline sulfanilamides than porcine tubulin [145,164].

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L. tarentolae MREAICIHIGQAGCQVGNACWELFCLEHGIQPDG SMPSDKCIGVEDDAFNTFFSETGAGK 60 L. major MREAICIHIGQAGCQVGNACWELFCLEHGIQPDGSMPSDKCIGVEDDAFNTFFSETGAGK 60 T. brucei MREAICIHIGQAGCQVGNACWELFCLEHGIQPDG AMPSDK TIGVEDDAFNTFFSETGAGK 60 T. cruzi MREAICIHIGQAGCQVGNACWELFCLEHGIQPDG AMPSDK TIGVEDDAFNTFFSETGAGK 60 P. falciparum MRE VISIH VGQAG IQVGNACWELFCLEHGIQPDG QMPSDK ASRANDDAFNTFFSETGAGK 60 S. scrofia MRE CISIH VGQAG VQIGNACWEL YCLEHGIQPDG QMPSDK TIG GG DD SFNTFFSETGAGK 60

L. tarentolae HVPRCLFLDLEPTVVDEVRTGTYRQLFNPEQLVSGKEDAANNYARGHYTIGKEIVDLALD 120 L. major HVPRC IFLDLEPTVVDEVRTGTYRQLFNPEQLVSGKEDAANNYARGHYTIGKEIVDLALD 120 T. brucei HVPR AVFLDLEPTVVDEVRTGTYRQLF HPEQL ISGKEDAANNYARGHYTIGKEIVDL CLD 120 T. cruzi HVPR AVFLDLEPTVVDE IRTGTYRQLF HPEQL ISGKEDAANNYARGHYTIGKEIVDL CLD 120 P. falciparum HVPRC VFVDLEPTVVDEVRTGTYRQLF HPEQL ISGKEDAANN FARGHYTIGKE VIDVCLD 120 S. scrofia HVPR AVFVDLEPTV IDEVRTGTYRQLF HPEQL IT GKEDAANNYARGHYTIGKEI IDL VLD 120

L. tarentolae RIRKLADNCTGLQGFMVFHAVGGGTGSGLGALLLERLSVDYGKKSKLGYTVYPSPQVSTA 180 L. major RIRKLADNCTGLQGFMVFHAVGGGTGSGLGALLLERLSVDYGKKSKLGYTVYPSPQVSTA 180 T. brucei RIRKLADNCTGLQGF LVYHAVGGGTGSGLGALLLERLSVDYGKKSKLGYTVYPSPQVSTA 180 T. cruzi RIRKLADNCTGLQGF LVYHAVGGGTGSGLGALLLERLSVDYGKKSKLGYTVYPSPQVSTA 180 P. falciparum RIRKLADNCTGLQGF LMFSAVGGGTGSG FGCLMLERLSVDYGKKSKL NFCCW PSPQVSTA 180 S. scrofia RIRKLAD QCTGLQGF SVFH SFGGGTGSG FTSLL MERLSVDYGKKSKL EFSI YP APQVSTA 180

L. tarentolae VVEPYNCVLSTHSLLEHTDVATMLDNEAIYDLTRRSLDIERPSYTNVNRLIGQVVSSLTA 240 L. major VVEPYNCVLSTHSLLEHTDVATMLDNEAIYDLTRRSLDIERPSYTNVNRLIGQVVSSLTA 240 T. brucei VVEPYN SVLSTHSLLEHTDVA AMLDNEAIYDLTRR NLDIERP TYTN LNRLIGQVVSSLTA 240 T. cruzi VVEPYN SVLSTHSLLEHTDVA AMLDNEAIYDLTRR NLDIERP TYTN LNRLIGQVVS ALTA 240 P. falciparum VVEPYN SVLSTHSLLEHTDVA IMLDNEAIYD IC RR NLDIERP TYTN LNRLI AQV ISSLTA 240 S. scrofia VVEPYN SI LTTH TTLEH SDCAFMVDNEAIYD IC RR NLDIERP TYTN LNRLIGQ IVSS ITA 240

L. tarentolae SLRFDGALNVDLTEFQTNLVPYPRIHFVLTSYAPVVSAEKAYHEQLSVSDITNSVFEPAG 300 L. major SLRFDGALNVDLTEFQTNLVPYPRIHFVLTSYAPVVSAEKAYHEQLSV ADITNSVFEPAG 300 T. brucei SLRFDGALNVDLTEFQTNLVPYPRIHFVLTSYAPV ISAEKAYHEQLSVS EISNAVFEPA S 300 T. cruzi SLRFDGALNVDLTEFQTNLVPYPRIHFVLTSYAPV ISAEKAYHEQLSVS EISNAVFEPA S 300 P. falciparum SLRFDGALNVD VTEFQTNLVPYPRIHF MLSSYAPVVSAEKAYHEQLSVS EITNS AFEPA N 300 S. scrofia SLRFDGALNVDLTEFQTNLVPYPR AHF PLATYAPV ISAEKAYHEQLSV AE ITN ACFEPA N 300

L. tarentolae MLTKCDPRHGKYMSCCLMYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKCGINYQPP 360 L. major MLTKCDPRHGKYMSCCLMYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKCGINYQPP 360 T. brucei MMTKCDPRHGKYM ACCLMYRGDVVPKDVNAA VATIKTKRTIQFVDW SPTGFKCGINYQPP 360 T. cruzi MMTKCDPRHGKYM ACCLMYRGDVVPKDVNAA VATIKTKRTIQFVDW SPTGFKCGINYQPP 360 P. falciparum MMAKCDPRHGKYM ACCLMYRGDVVPKDVNAA VATIKTKRTIQFVDWCPTGFKCGINYQPP 360 S. scrofia QMVKCDPRHGKYM ACCLLYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFK VGINY EPP 360

L. tarentolae TVVPGGDLAKVQRAVCMIANSTAIAEVFARIDHKFDLMYSKRAFVHWYVGEGMEEGEFSE 420 L. major TVVPGGDLAKVQRAVCMIANSTAIAEVFARIDHKFDLMYSKRAFVHWYVGEGMEEGEFSE 420 T. brucei TVVPGGDLAKVQRAVCMIANSTAIAEVFARIDHKFDLMYSKRAFVHWYVGEGMEEGEFSE 420 T. cruzi TVVPGGDLAKVQRAVCMIANSTAIAEVFARIDHKFDLMYSKRAFVHWYVGEGMEEGEFSE 420 P. falciparum TVVPGGDLAKV MRAVCMI SNSTAIAEVF SRMDQKFDLMY AKRAFVHWYVGEGMEEGEFSE 420 S. scrofia TVVPGGDLAKVQRAVCM LS NTTAIAE AW AR LDHKFDLMY AKRAFVHWYVGEGMEEGEFSE 420

L. tarentolae AREDLAALEKDYEEVGAESADDMGEED--VEEY 451 L. major AREDLAALEKDYEEVGAESADDMGEED--VEEY 451 T. brucei AREDLAALEKDYEEVGAESAD MD GEED--VEEY 451 T. cruzi AREDLAALEKDYEEVGAESAD ME GEED--VEEY 451 P. falciparum AREDLAALEKDYEEVG IES NEAE GE DE GY EADY 453 S. scrofia ARED MAALEKDYEEVG VD SVEGE GEE E-- GEEY 451

Figure 2.9 Amino acid sequence alignment of L. tarentolae α-tubulin ( DQ309032 ) with the corresponding sequences from L. major ( CAJ02503 ), T. brucei (XP_846749 ), T. cruzi (XP_802499 ), P. falciparum ( CAD51722 , tubulin α-I isotype, expressed in both asexual and sexual blood stages), and S. scrofa (pig, P02550 , 99% identical to the human major neural isotype, b α1). Identical and conserved amino acids are shaded in black and grey, respectively. Alignments were performed with the aid of the program ClustalW [166].

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L. tarentolae MREIVSCQAGQCGNQIGSKFWEVISDEHGVDPTGTYQGDSDLQLERINVYFDESSGGRYV 60 L. major MREIVSCQAGQCGNQIGSKFWEVI ADEHGVDPTG SYQGDSDLQLERINVYFDES AGGRYV 60 T. brucei MREIV CVQAGQCGNQIGSKFWEVISDEHGVDPTGTYQGDSDLQLERINVYFDE AT GGRYV 60 T. cruzi MREIV CVQAGQCGNQIGSKFWEVISDEHGVDPTGTYQGDSDLQLERINVYFDE AT GGRYV 60 P. falciparum MREIV HI QAGQCGNQIG AKFWEVISDEHG IDP SGTY CGDSDLQLER VDVFYNEAT GGRYV 60 S. scrofia MREIV HI QAGQCGNQIG AKFWEVISDEHG IDPTG SYHGDSDLQLERINVY YNEAA GNK YV 60

L. tarentolae PRAVLMDLEPGTMDSVRAGPYGQLFRPDNFIFGQSGAGNNWAKGHYTEGAELIDSVLDVC 120 L. major PRAVLMDLEPGTMDSVRAGPYGQLFRPDNFIFGQSGAGNNWAKGHYTEGAELIDSVLDVC 120 T. brucei PR SVL IDLEPGTMDSVRAGPYGQ IFRPDNFIFGQSGAGNNWAKGHYTEGAELIDSVLDVC 120 T. cruzi PRAVL IDLEPGTMDSVRAGPYGQ IFRPDNFIFGQSGAGNNWA QGHYTEGAELIDSVLDVC 120 P. falciparum PRA ILMDLEPGTMDSVRAGP FGQLFRPDNF VFGQ TGAGNNWAKGHYTEGAELID AVLDV V 120 S. scrofia PRA ILVDLEPGTMDSVR SGP FGQ IFRPDNF VFGQSGAGNNWAKGHYTEGAEL VDSVLDV V 120

L. tarentolae RKEAESCDCLQGFQLSHSLGGGTGSGMGTLLISKLREEYPDRIMMTFSVIPSPRVSDTVV 180 L. major RKEAESCDCLQGFQLSHSLGGGTGSGMGTLLISKLREEYPDRIMMTFSVIPSPRVSDTVV 180 T. brucei C KEAESCDCLQGFQ IC HSLGGGTGSGMGTLLISKLRE QYPDRIMMTFS IIPSP KVSDTVV 180 T. cruzi RKEAESCDCLQGFQ IC HSLGGGTGSGMGTLLISKLREEYPDRIMMTFS IIPSP KVSDTVV 180 P. falciparum RKEAE GCDCLQGFQ IT HSLGGGTGSGMGTLLISK IREEYPDRIM ETFSV FPSP KVSDTVV 180 S. scrofia RKE SESCDCLQGFQL THSLGGGTGSGMGTLLISK IREEYPDRIM NTFSV VPSP KVSDTVV 180

L. tarentolae EPYNTTLSVHQLVENSDESMCIDNEALYDICFRTLKLTTPTFGDLNHLVAAVMSGVTCCL 240 L. major EPYNTTLSVHQLVENSDESMCIDNEALYDICFRTLKLTTPTFGDLNHLVAAVMSGVTCCL 240 T. brucei EPYNTTLSVHQLVENSDESMCIDNEALYDICFRTLKLTTPTFGDLNHLV SAV VSGVTCCL 240 T. cruzi EPYNTTLSVHQLVENSDESMCIDNEALYDICFRTLKLTTPTFGDLNHLV SAV VSGVTCCL 240 P. falciparum EPYN ATLSVHQLVEN ADE VQV IDNEALYDICFRTLKLTTPT YGDLNHLV SAAMSGVTC SL 240 S. scrofia EPYN ATLSVHQLVEN TDE TYCIDNEALYDICFRTLKLTTPT YGDLNHLV SATMSGVT TCL 240

L. tarentolae RFPGQLNSDLRKLAVNLVPFPRLHFFMMGFAPLTSRGSQQYRGLSVAELTQQMFDAKNMM 300 L. major RFPGQLNSDLRKLAVNLVPFPRLHFFMMGFAPLTSRGSQQYRGLSVAELTQQMFDAKNMM 300 T. brucei RFPGQLNSDLRKLAVNLVPFPRLHFFMMGFAPLTSRGSQQYRGLSV PELTQQMFDAKNMM 300 T. cruzi RFPGQLNSDLRKLAVNLVPFPRLHFFMMGFAPLTSRGSQQYRGLSV PELTQQMFDAKNMM 300 P. falciparum RFPGQLNSDLRKLAVNL IPFPRLHFFM IGFAPLTSRGSQQYR ALTVPELTQQMFDAKNMM 300 S. scrofia RFPGQLN ADLRKLAVN MVPFPRLHFFM PGFAPLTSRGSQQYR ALTVPELTQQMFDAKNMM 300

L. tarentolae QAADPRHGRYLTASALFRGRMSTKEVDEQMLNVQNKNSSYFIEWIPNNIKSSICDIPPKG 360 L. major QAADPRHGRYLTASALFRGRMSTKEVDEQMLNVQNKNSSYFIEWIPNNIKSSICDIPPKG 360 T. brucei QAADPRHGRYLTASALFRGRMSTKEVDEQMLNVQNKNSSYFIEWIPNNIKSS VCDIPPKG 360 T. cruzi QAADPRHGRYLTASALFRGRMSTKEVDEQMLNVQNKNSSYFIEWIPNNIKSSICDIPPKG 360 P. falciparum CASDPRHGRYLTA CAMFRGRMSTKEVDEQMLNVQNKNSSYF VEWIP HNTKSS VCDIPPKG 360 S. scrofia A ACDPRHGRYLT VA AVFRGRMS MKEVDEQMLNVQNKNSSYF VEWIPNN VKTAV CDIPP RG 360

L. tarentolae LKMSVTFIGNNTCIQEMFRRVGEQFTGMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420 L. major LKMSVTFIGNNTCIQEMFRRVGEQFTGMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420 T. brucei LKM AVTFIGNNTCIQEMFRRVGEQFT LMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420 T. cruzi LKM AVTF VGNNTCIQEMFRRVGEQFT AMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420 P. falciparum LKM AVTFVGN STAIQEMF KRV SD QFT AMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420 S. scrofia LKMS ATFIGN STAIQE LFKRIS EQFT AMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS 420

L. tarentolae EYQQYQDATVEEEGEYDEEEEAY -- 443 L. major EYQQYQDATVEEEGEYDEEEEAY -- 443 T. brucei EYQQYQDAT IEEEGE FDEEE QY--- 442 T. cruzi EYQQYQDAT IEEEGE FDEEE QY--- 442 P. falciparum EYQQYQDAT AEEEGE FEEEE GDVEA 445 S. scrofia EYQQYQDAT AD EQGE FEEE GEEDEA 445

Figure 2.10 Amino acid sequence alignment of L. tarentolae β-tubulin ( DQ309033 ) with the corresponding sequences from L. major ( CAJ06134, tubulin c βT2.2 isotype, the predominant β-tubulin isotype in promastigotes), T. brucei ( XP_846748 ), T. cruzi (XP_816690 ), P. falciparum ( NP_700558 ), and S. scrofia ( P02554 , a class II β-tubulin, the major β-tubulin isotype expressed in neural tissues). Identical and conserved amino acids are denoted as in Figure 3, and alignments were again performed with the aid of ClustalW.

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Interestingly, kinetoplastid tubulin appears to be nearly as distant from

Plasmodium tubulin as it is from porcine tubulin. This may help to explain differences in susceptibility to dinitroaniline sulfanilamides between the kinetoplastids and malaria parasites. Oryzalin has been shown to possess greater potency against

Plasmodium than against kinetoplastids [145,146], while GB-II-5 is approximately 5- fold more active against L. donovani axenic amastigotes and about 60-fold more active against T. brucei bloodstream forms than against intraerythrocytic P. falciparum (K. Werbovetz, unpublished data). Experimental evidence indicates that the binding site for oryzalin is on α-tubulin [167,168,169]. In the apicomplexan parasite Toxoplasma gondii , molecular simulations indicate that the oryzalin binding site consists of α-tubulin residues Arg2, Glu3, Val4, Trp21, Phe24, His28, Ile42,

Asp47, Arg64, Cys65, Thr239, Arg243, and Phe244, which are located just below the

N-loop [167]. Only one of these residues, where Val4 is replaced with Ala, differs between Toxoplasma and Leishmania α-tubulin. This does not necessarily account for the susceptibility differences between kinetoplastid and apicomplexan tubulin to dinitroanilines, as the amino acid at position 42 differs between Plasmodium α- tubulin and all the other tubulins listed in Figure 3 and the amino acid at position 65 differs between the Leishmania and Trypanosoma α-tubulin. Morrissette et al. have suggested that amino acid differences at positions other than the binding site causing allosteric effects are responsible for the selectivity of dinitroanilines for plant and protozoal tubulin [167]. The aforementioned argument is further supported by observations with oryzalin resistant T. gondii mutants [167]. Briefly, serial pressure with oryzalin was used to create resistant T. gondii mutants. All resistant mutants

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had point mutations in the single α-tubulin gene, however, not all mutations which conferred resistance were on the M- or N-loops. In fact, a vast majority of the mutations were within the core of α-tubulin; this supports the argument that distal allosteric modulation can affect the conformation of the dinitroaniline binding site.

More recently, docking studies were done with several dinitroanilines and α-tubulin from T. gondii , P. falciparum and L. major [169]. Molecular docking results of oryzalin, trifluralin and GB-II-5 to all three organisms coincided with the previous results obtained with T. gondii that indeed, the dinitroanilines bind in a pocket located below the N-loop on α-tubulin. It should be noted that although the compounds all shared a common binding site on the three tubulins, they adopted slightly different orientations on each; this offers a possible explanation of the differences in dinitroaniline susceptibility between the organisms. Further structure-activity studies and molecular modeling data will be useful in developing a refined binding site model for the dinitroaniline sulfanilamides on kinetoplastid tubulin which will facilitate the design of improved ligands for this important drug target.

2.4.3 Tubulin-ligand interactions

The amino acid sequences of L. tarentolae α- and β-tubulin indicate that this heterodimeric protein would be a suitable alternative to leishmanial tubulin isolated from a pathogenic species for compound screening. This was verified by comparing data generated in assembly assays and fluorescence quenching assays performed with tubulin from L. amazonensis and L. tarentolae. The IC 50 values for GB-II-5, GB-

II-150, and oryzalin in the assembly assay with tubulin purified from L. tarentolae

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were 6.8 ± 1.4 µM, 5.8 ± 0.6 µM, and >40 µM, respectively. Previously, the IC 50 values for these same compounds were shown to be 6.7 ± 0.7 µM, 6.9 ± 0.0 µM, and

>40 µM against L. amazonensis tubulin assembly when assayed under identical assay conditions [145,164]. Thus, the activities of these compounds against both tubulins are comparable in this assay.

2.4.4 Fluorescence quenching results

To further investigate the similarity of ligand binding properties between tubulin from L. tarentolae and from L. amazonensis , we examined the binding affinities of GB-II-5 and oryzalin for the two proteins by employing fluorescence quenching to determine dissociation constants (K d). The K ds determined for oryzalin and GB-II-5 against L. tarentolae tubulin (Figures 2.11 and 2.12 respectively) were

17 ± 8 µM and 2.4 ± 0.9 µM, respectively, which were similar to the K d values of 19 ±

3 µM and 1.7 ± 0.4 µM reported for these compounds against tubulin purified from the pathogenic L. amazonensis [145].

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Oryzalin Quenching GB-II-5 Quenching Data 20 20 R2 = 0.94 R2 = 0.94 15 15

10 10 % Quenching % Quenching 5 5

0 0 0 25 50 75 100 125 0 25 50 75 100 125 Concentration ( µµµM) Concentration ( µµµM)

Figu re 2.11 Fluorescence Figu re 2.12 Fluorescence quenching profile of oryzalin and L. quenching profile of GB-II-5 and L. tarentolae tubulin (K d = 17 µM) tarentolae tubulin (K d = 2.4 µM)

2.5 CONCLUSIONS

The purification of L. tarentolae tubulin described here proved advantageous in that the isolation procedure can be shortened by ~ 1.5 hours compared to the previous protocol without sacrificing yield or purity. Furthermore, by utilizing L. tarentolae the overall process is shortened in that the parasites can be grown to high cell densities, reducing the time required to harvest adequate numbers of parasites for a large-scale purification of tubulin. The L. tarentolae α- and β -tubulin genes were 97.3% and 97.5% identical to L. major respectively. A majority of the nucleotide sequence substitutions are silent in that the α- and β-tubulin protein sequences are 99.6% and 99.4% identical to the corresponding sequences from L. major , respectively. Fluorescence quenching and assembly inhibition assays further illustrated the interchangeability of L. tarentolae tubulin with the corresponding

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protein from L. amazonensis , as differences in the results between L. tarentolae and

L. amazonensis tubulin for the dinitroaniline sulfanilamides were negligible. Tubulin from L. tarentolae is thus suitable for tubulin-ligand assays performed as part of an antileishmanial drug discovery program. Given that the L. tarentolae protein can be obtained more cheaply and quickly than tubulin from pathogenic species of

Leishmania , these findings should be a great benefit to medicinal chemistry efforts targeting this essential parasite protein.

Although we have developed a means to safely and cheaply acquire large quantities of biologically relevant Leishmania tubulin, the exact mechanism by which our compounds bind tubulin and inhibit polymerization remains somewhat elusive.

Mutagenesis studies by Morrissette et al. [167] on the Apicomplexan parasite T. gondii revealed that resistance to dinitroanilines was conferred by point mutations in

α-tubulin. These mutations, found primarily on the M or N loops, were sufficient to confer oryzalin resistance when transfected into wild type (WT) parasites.

Furthermore, some of the mutated residues corresponded to the dinitroaniline binding site as predicted by molecular modeling. These results led the authors to speculate that the dinitroanilines act by disrupting M-N loop contacts which are necessary for lateral interactions. To further examine and characterize the binding site of dinitroanilines, Mitra and Sept [169] have recently performed molecular dynamics (MD) simulations and molecular docking studies with several dinitroaniline analogues bound to T. gondii , P. falciparum , and L. major α-tubulins. The MD simulations were useful in that they allow predictions how the macromolecule responds to the ligand. The results support the earlier findings that the binding site is

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located beneath the H1-S2 loop and includes residues on helix H7 and on the T7 loop on all of the organisms. Binding results in decreased flexibility of the H1-S2 loop, this loss of flexibility results in the loop being pulled toward the interior of α- tubulin which displaces it from the M-loop of the α-tubulin subunit on the adjacent protofilament. The models also served to predict protein-ligand molecular interactions involved in binding which are useful in gathering SAR data which can be used to optimize analogues. Figure 2.13 shows a summary of the proposed interactions between dinitroanilines and their tubulin binding site as determined by

Mitra and Sept [169].

O2 atoms have electrostatic N Linked via solvent network to interactions with O2N NO2 the salt-bridge b/t Asp47 and Glu3, Thr239 and Arg64* Arg243 and H- bond with Ala4*

O2 atoms involved in H-bonding SO2 with Cys41 H-bonding with Ala240 HN

Fits snugly at entrance of binding pocket and is involved in pi-stacking interactions with His28 ring

Figure 2.13 GB -II -5 (2.8) SAR as predicted by molecular modeling and molecular docking studies [169]

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The SAR as predicted by molecular modeling and MD corresponds well with experimental data generated in our lab by in vitro analysis of 30 dinitroaniline analogues [164]. Further SAR data generated by our lab shows that activity is decreased when: the N1 hydrogen is replaced with a methyl or phenyl group, the nitro group on the sulfanilamide aromatic ring is replaced with a hydrogen or another functional group and when the conformation of the N4-alkylamino chain is restricted

[164].

These findings shed some light on a previously uncharacterized binding site on α-tubulin and the possible mechanisms by which ligands of this site inhibit microtubule polymerization. Although these findings are extremely valuable tools for optimizing future generations of analogues, a tubulin-dinitroaniline crystal structure, which would provide the most definitive and telling information about the binding site, has yet to be determined.

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CHAPTER 3

THE SEARCH FOR A STATHMIN LIKE PROTEIN IN KINETOPLASTIDS

CAPABLE OF AFFECTING MICROTUBULE DYNAMICS

3.1 INTRODUCTION TO STATHMIN

As discussed in Chapter 2, microtubules, comprised of α/β heterodimers, exist in a perpetual dynamic state. This property, known as dynamic instability, allows both polymerizing and depolymerizing microtubules to exist in the same population and to infrequently interconvert between these two states [170].

Transition from a state of growth to a state of degradation is termed “catastrophe,” whereas transition from a state of catastrophe to a state of elongation is termed

“rescue [171].” Proper control of microtubule dynamics is essential for many microtubule-dependent processes[172]. As microtubules play vital roles in growth, development, and replication of eukaryotic cells their polymerization dynamics are highly regulated [173].

Microtubule associated proteins (MAPs) are major regulators of microtubule dynamics. MAPs stabilize microtubules mainly by suppressing catastrophes and

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increasing rescues [138,174]. However, microtubule stabilizing proteins alone don’t account for the regulation of microtubule dynamics during the cell cycle. The catastrophe rate observed in cells and cell extracts is much higher than the rate observed with purified tubulin [175,176]. These results indicate the presence of additional regulatory components which act inversely to MAPs by increasing the catastrophe rates of microtubules.

Oncoprotein 18 (Op18), also known as stathmin, was first identified as a 17 kDa, heat stable, cytosolic protein that is rapidly phosphorylated when HL60 leukemia cells are induced to undergo terminal differentiation and cease to proliferate [177,178]. Further studies in several other leukemia cell lines also showed that stathmin expression is drastically decreased when the cells ceased to proliferate upon exposure to a variety of differentiation agents [179]. A correlation between the expression of stathmin and the regulation of cell cycle progression was clearly established when it was shown that antisense RNA inhibition of stathmin expression in K562 leukemia cells resulted in decreased proliferation and an accumulation of cells in the G2/M stage [180]. Furthermore, overexpression of stathmin in K562 cells also resulted in decreased proliferation and an accumulation of cells in the G 2/M stage [181]. It is evident from the aforementioned studies that stathmin is an essential protein in the cell cycle and expression levels must be tightly regulated for cellular proliferation to properly occur. The method by which stathmin plays a role in regulation of the cell cycle was established by a study designed to directly identify microtubule catastrophe factors [172]. A low molecular weight

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protein, identified as stathmin, was shown to cause microtubule destabilization both in Xenopus egg extracts and in tissue culture cells [139,172,182].

Currently, there are two models which explain how stathmin acts to destabilize microtubules. The first model to explain the mechanism by which stathmin exerts its effects on tubulin indicatess that it does so by increasing catastrophe rates of microtubules [172]. It has been shown that during this process, stathmin binds tubulin heterodimers at the ends of elongating microtubules and increases the rate of catastrophe by a GTP hydrolysis-dependent mechanism

[183,184] Subsequently, a different model was proposed in which stathmin interacts with tubulin by sequestering two dimers, resulting in a ternary stathmin-tubulin complex (T2S) [185]. The sequestration of tubulin diminishes microtubule elongation by depleting the intracellular pool of tubulin available for polymerization. Electron microscopy [186] and crystal structure analysis [187] have shown that stathmin binds two tubulin heterodimers in a longitudinal arrangement similar to that observed during microtubule formation. Since the proposals regarding the two mechanisms of stathmin were published, it has been shown that both occur and the ratio of occurrence is dictated by pH [183].

The formation of a ternary stathmin-tubulin complex has been utilized as a method for stabilizing tubulin in solution for crystallization [187]. Traditionally, three- dimensional crystal structures of tubulin have been difficult to obtain; these difficulties have been attributed to (1) the heterogeneity of tubulin preparations caused by posttranslational modifications of tubulin isoforms, (2) the instability of tubulin in

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solution, and (3) the heterodisperse nature of tubulin preparations [187].

Furthermore, at high concentrations tubulin polymerizes, this further complicates crystallization. Although the near-atomic 3.7 Å resolution structure of zinc stabilized antiparallel arranged crystallized tubulin sheets has been determined [95]; scanning transmission electron microscopy combined with digital image processing have shown that the T2S complex more closely mimics the biologically relevant protofilament [186]. Gigant et al. [187] subsequently employed an expressed stathmin-like domain of RB3 (RB3-SLD), a stathmin family protein, to stabilize tubulin and elucidate the 4 Å, three-dimensional crystal structure. Recently, the crystal structure of vinblastine bound to a tubulin-RB3 complex has been reported (Figure

3.1); these studies showed that vinblastine, a tubulin-targeting Vinca alkaloid with a previously undetermined binding site and mechanism of antimitotic action, binds at the interface of two tubulin dimers and introduces a wedge. Once formed, the wedge interferes with lateral dimer interactions thus interfering with the stability of the microtubule.

Figure 3.1 Vinblastine and c olchicines are shown bound to a tubulin - RB3 complex. The complex consists of the RB3-SLD bound to two tubulin α/β heterodimers. Reprinted by permission from Macmillan Publishers Ltd: Nature Publishing Group, Nature 2005 , 435, 519-522 69

Our lab is currently interested in oryzalin analogues which bind leishmanial tubulin and inhibit polymerization (see Chapter 2). Although molecular modeling attempts have given us some insight into where our compounds bind, this novel site remains largely uncharacterized. Furthermore, the mechanism by which our compounds inhibit polymerization has yet to be elucidated. Thus, for future compound development and optimization, it would be of great value to have a crystal structure of our compounds bound to leishmanial tubulin. Herein, the efforts to identify a stathmin like protein with the ability to sequester Leishmania tubulin are reported. The first stages in this search included: (1) examining active, stathmin containing fractions isolated from fetal bovine thymus against Leishmania tubulin and

(2) Blast searching of the recently completed genome database for stathmin encoding regions based on stathmin sequences from human [188], mouse [189], chicken [190], rat [191], Xenopus [192], and Schistosoma [193] species. The second stage of this project consisted of efforts to isolate an active, stathmin like protein directly from Leishmania tarentolae .

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3.2 MATERIALS AND METHODS

3.2.1 Chemicals

Unless otherwise specified, materials were purchased from Sigma.

3.2.2 Blast Searching and Sequence Alignments

A BLAST [194] search was performed to determine if any of the predicted protein sequences from the recently elucidated Leishmania major genome [155] shared similarity with known stathmin like protein sequences from mouse, human, chicken, Xenopus , and Schistosoma using GeneDB (http://www.genedb.org/).

Sequence alignments and phylogenic trees were produced using ClustalW [166].

3.2.3 Preparation of Cell Lysate from Fetal Bovine Thymus

Crude stathmin containing fractions were prepared based on the protocol described by Belmont et al. [172]. Briefly, fetal bovine thymus (HyClone Biologicals,

Logan, UT) which had been quick frozen in liquid nitrogen was broken up with a hammer. 30 grams of frozen thymus were suspended in 30 mL assembly buffer (80 mM PIPES [pH 7.5], 5 mM MgCl 2, 1 mM EGTA) along with the protease inhibitor

PMSF (1 mM) and reducing agent 2-mercaptoethanol (0.1%). After thawing, the suspension was homogenized in a chilled Waring blender with 5 × 5-second pulses.

The homogenate was spun at 4000 rpm in a Marathon 3200-R centrifuge (Fisher

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Scientific) for 20 minutes; then the resulting supernatant was filtered through glass wool to remove particulates. The remaining filtrate was centrifuged in a Optima L-

90K ultracentrifuge (Beckman Coulter) in a 50.2 Ti rotor at 50,000 rpm for 4 hours at

4 °C. Following centrifugation a soluble bilayer was observed. The bottom layer

(reddish color) and top layer (clear color) were carefully removed and stored separately at -80 °C.

3.2.4 Preparation of Cell Lysates from Leishmania tarentolae and Crithidia fasciculata

Leishmania tarentolae (UC strain) from Dr. Larry Simpson (Department of

Microbiology, Immunology and Molecular Genetics, University of California, Los

Angeles, CA) were maintained as described in Chapter 2. Crithidia fasciculata

(American Type Culture Collection) were maintained by serial passage in 355

Crithidia medium (trypticase peptone [Fisher] 6 g, yeast extract [Fisher] 1 g, liver concentrate [Fisher] 0.1 g, sucrose [Fisher] 15 g, distilled water 1 L, and 5 mL hemin solution (triethanolamine 2.5 mL, hemin 25 mg, distilled water 2.5 mL) pH 7.8 and autoclaved at 121 °C for 20 minutes) in a T-25 culture flask (Corning) at 25 °C.

Large scale cultures of L. tarentolae and C. fasciculata were grown in 1 L flasks in an orbiting shaker incubator set at 125 rpm and 25 °C. Cells were harvested in late log phase (1-2 × 10 8 cells/mL) by centrifugation at 1200 × g at 4 °C. Cell pellets were then washed twice with phosphate buffered saline (PBS). ~6 g of L. tarentolae and

C. fasciculata pellets (6 mL volume, ~1.5 × 10 11 cells) were resuspended in 6 mL

PME with protease inhibitors PMSF (0.5 mM) and leupeptin (25 ug/ml) to give a final

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volume of ~11 mL. The cells were sonicated on ice using a Misonix (Microson XL) probe sonicator (Farmingdale, NY) with 5 × 5 second bursts at power level 11 (~18

W). The cell lysates were then spun for 3 hr at 50,000 rpm in the previously mentioned Optima L-90K ultracentrifuge using a 50.2 Ti rotor. The soluble bilayers

(clear upper layers, brown lower layers) were carefully removed with a syringe and passed through a 0.22 µm filter to provide a final volume of 8 mL. Fractions were stored at -80 °C until use. Where indicated, samples were subjected to batch chromatography and multiple warming and centrifugation steps. For batch chromatography, 400 µL of soluble cell lysate were combined with 100 µL of DEAE

Sephadex which had previously been equilibrated in PME. The resulting sample was mixed for 5 min by pipetting, the sample was then centrifuged at 1500 × g in an

5415 D centrifuge (Eppendorf) to sediment DEAE. The resulting supernatant (pH

6.9) was carefully removed by pipette and stored at 4 °C until use. Sequential warming/centrifugation steps were performed by incubating samples for 15 minutes in a water bath at 37 °C followed by centrifugation at 50,000 rpm in the aforementioned ultracentrifuge and rotor at 30 °C for 30 min. The resulting supernatant was carefully removed by pipetting and stored at 4 °C prior to use.

3.2.5 Preparation and Calibration of Sephadex Column

Sephadex G-75 superfine beads (10-40 µm dry bead diameter, globular protein fractionation range M r 3,000-70,000 Da) were swelled overnight under vacuum in PME, pH 6.9. The beads were poured into a 1 × 50 cm column (BioRad) as a slurry resulting in a settled bed height of 45 cm. The column was connected to

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a conical 500 mL reservoir positioned directly above and was connected to a fraction collector (BioRad model 2110) placed below; the column, reservoir, and fraction collector were placed in a cold room at 4 °C prior to use. To determine the void volume of the column (V 0), 1mL of blue dextran (1 mg/mL in PME pH 6.9) was added to ~1cm of buffer above the gel bed and was allowed to run into the column. To prevent bed disruption, ~5 cm of buffer was then added to the column. The column was then connected to the reservoir and 20 drop (~0.5 mL) fractions were collected.

A low molecular weight gel filtration calibration kit (Amersham Biosciences) containing ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and albumin (35.5 kDa) was used to calibrate the column. 5 mg of each standard (total volume 1 mL in PME) were added to the column and fractions were collected as previously described for blue dextran. Eluted fractions were analyzed with the aid of a SPECTRAmax PLUS spectrophotometer (Molecular Devices); blue dextran was measured at 620 nm while ribonuclease A, chymotrypsinogen A, ovalbumin, and albumin were all measured at 280 nm. To further evaluate column efficiency, protein rich fractions were analyzed by SDS-PAGE electrophoresis on a

12% ReadyGel (BioRad).

3.2.6 Fractionation, Concentration, and Concentration Determination of Cell Lysates

Following calibration, the L. tarentolae cell lysate (3 mL, prepared as previously described) was loaded onto the column and 0.5 mL (20 drop) fractions were collected and read at 280 nm beginning with fraction 15 and terminating with fraction 65. The protein rich, low molecular weight fractions (fractions 35-52) were

74

pooled to give a combined volume of 7 mL. The eluent was then concentrated using an iCON concentrator (Pierce) with a 7 mL capacity and a molecular weight cut off of

9 kD. The pooled fractions were added to the concentrator and spun at 3000 × g for

30 minutes at 4 °C. The resulting concentrated supernatant (total volume 1.2 mL) and aliquots saved from all stages of the preparation were analyzed by SDS-PAGE using a gradient ReadyGel 5-20% (BioRad); the concentrations were determined using the BioRad Bradford protein assay and stored at -80 °C.

3.2.7 Leishmania and Mammalian Tubulin Polymerization Inhibition Assays

Assembly assays were performed in 96-well half-area microplates (Costar) in a final volume of 50 µL with either Leishmania tarentolae tubulin prepared as described in Chapter 2 or with purified porcine brain tubulin (prepared as described by Werbovetz et al. [145]. Reactions were performed with 26 µL of crude cell lysates, fractions of interest or PME (control ), 10 % (w/v) DMSO, 1 mM GTP, and either mammalian tubulin (1.5 mg/mL) or Leishmania tubulin (1 mg/mL) on ice.

Reaction mixtures excluding DMSO and GTP were added on ice and the volume of each well was adjusted to 40 µL with PME. Assembly was initiated by the simultaneous addition of a 10 µL GTP/DMSO solution to each well with a multi- channel pipette. The change in turbidity at 350 nm was measured in a

SPECTRAmax PLUS microplate reader (Molecular Devices) at 37 °C for 20 min.

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3.3 RESULTS AND DISCUSSION

3.3.1 Blast Searching

To investigate the homology between known stathmin like proteins, the sequences were aligned to determine percent amino acid identity (Table 3.1) and a phylogenic tree (Figure 3.2) was produced to illustrate the divergence of the proteins.

SeqA Length (aa) SeqB Length (aa) % Identity Mouse 149 Human 149 98 Mouse 149 Chicken 148 91 Mouse 149 Xenopus 145 79 Mouse 149 Rat 179 66 Mouse 149 Schistosoma 117 11 Human 149 Chicken 148 92 Human 149 Xenopus 145 77 Human 149 Rat 179 66 Human 149 Schistosoma 117 11 Chicken 148 Xenopus 145 79 Chicken 148 Rat 179 66 Chicken 148 Schistosoma 117 11 Xenopus 145 Rat 179 64 Xenopus 145 Schistosoma 117 11

Rat 179 Schistosoma 117 15

Table 3.1 The percent amino acid identity of stathmin like proteins from different species. Shown are the sequences of (GenBank™ accession numbers in parentheses) mouse stathmin ( P54227 ), human Op18 (M31303 ), chicken stathmin ( P31395 ), Xenopus stathmin (Q09006 ), rat SCG10 ( P21818 ), and Schistosoma SmSLP ( AF091509 ).

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Figure 3.2 Phylogenic tree illustrating divergences in stathmin like proteins between different species.

With the exception of mouse, human, and chicken, there is a fair amount of amino acid sequence heterogeneity between species, especially with the

Schistosoma stathmin like protein. BLAST searching of the L. major database revealed a number of proteins which share some identity with the previously identified stathmin like proteins. The protein with the highest score, L. major glycoprotein (LmjF28.2210), has partial similarities to mouse, human, chicken, and

Xenopus sequences. The smallest sum probability values (P values) for each were

2.0 e -8, 1.2 e -8, 1.5 e -8, and 8.7 e -8 respectively, however, glycoprotein (gp) 96-92 is a much larger protein (87.1 kDa) than the aforementioned low molecular weight proteins. As such, the alignment identities were only based on partial amino acid sequences within gp 96-92 (Fig 3.3). Interestingly, the regions of homology between the stathmin like proteins and gp 96-92 vary; for example, Xenopus stathmin appears near the C-terminus whereas the human stathmin alignment occurs near the N- terminal region. The regions of gp 96-92 which are similar to mammalian and

Xenopus stathmins is highlighted in Fig. 3.3, the sequence alignments can be seen in Fig. 3.4. Rat stathmin showed the highest probability (8.9 e -6) of being related to the conserved hypothetical protein LmjF34.0690, a 358 kDa protein. Schistosoma

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stathmin showed the highest probability (0.96) to being related to an ATP-dependent

Clp protease subunit, heat shock protein 78 (LmjF27.2630), a 90.8 kDa protein. As

BLAST failed to identify any LMW stathmin like proteins within Leishmania , the next step was to search for stathmin like proteins by looking for activity. It should be noted, however, that stathmins from different species do appear to show a fairly high identity with segments of Leishmania gp 96-92. Future studies examining interactions between expressed and/or purified gp 96-92 and Leishmania tubulin would be useful in investigating the significance, if any, of the sequence segments which resemble stathmin.

1 msaapsdvaeqhfdpapannvaplanaaqqeesatnkfhsgstgngkvlpgnsttkkrll 61 rknrsvatveaakkckeedkkladeiakareaearaakekakrireaeaesrkkrdqkdv 121 riqkdvaeerkqreelqrqreeeekqriemvrkqreeaqkkreeiqkqreeeikrrkaei 181 eaerqklkelqeehereqeearqrrvaeekeaqkkaekkaeeaedelaatrrqrkgelee 241 lqrqrekeekqriemvrkqreeaqkkreeiqkqreeeikrrkaeieaerqklkelqeehe 301 reqeearqrrvaeekeaqkkaekkaeeaedelaatrrqrkgeleelqrqreeeekqriem 361 vrkqreeaqkkreeiqkqreeeikrrkaeieaerqklkelqeehereqeearqrrvaeek 421 eaqkkaekkaeeaedelaatrrqrkgeleelqrqreeeekqriemvrkqreeaqkkreei 481 qkqreeeikrrkaeieaerqklkelqeehereqeearqrrvaeekeaqkkaekkaeeaed 541 elaatrrqrkgeleelqrqreeeekqriemvrkqreeaqrkreklkerdikeaeeikrqr 601 keelaelqkrrereqevqrkkveelrtkgkkdskkeqilkekrrtaaaererleeqrrkq 661 keeeekeleakhkrvmeqleknynivdeeeyererekarqmreeqekaaavalgla

Figure 3.3 The protein sequence of L. major gp 96 -92 . The sequence segments with which mammalian and Xenopus stathmins were aligned are highlighted in red and yellow respectively.

78

L. major IQKDVAEERKQREELQRQREEEEKQRI-EMVRKQREEAQKKREEIQKQREEEIKRRKAEI 59 Human IQVKELEKRASGQAFELILSPRSKESVPEFPLSPPKKKDLSLEEIQKKLEAAEERRKSH- 59 ** . *:* . : :: . ..*: : *: . :: : . *****: * :***:.

L. major EAERQKLKELQEEHEREQEEARQRRVAEEKEAQKKAEKKAEEAEDELAATRRQRKGELE- 118 Human EAE--VLKQLAEKREHEKE-VLQKAIEENNNFSKMAEEKLTH---KMEANKENREAQMAA 113 *** **:* *::*:*:* . *: : *::: .* **:* . :: *.:.:*:.::

L. major ELQRQREKEEKQRIEMVRKQRE 140 Human KLERLREKDK--HIEEVRKNKE 133 :*:* ***:: :** ***::*

L. major KKAEKKAEEAEDELAATRRQRKGELEELQRQREEEEKQRIEMVRKQREEAQRKREKLKER 60 Xenopus KKKECSLEEIQKKLEAAEERRKLHEAEILKQLAEKREHEKEVLQKAIEENNNFSKMAEEK 60 ** * . ** :.:* *:..:** . *: :* *:.::. *:::* ** :. : :*:

L. major DIKEAEEIKRQRKEELA-ELQKRREREQEVQRKKVEELRTKGKK 103 Xenopus LTTKMETIKENREAQIAAKLERLREKD-----KKVEEIR-KGKE 98 .: * **.:*: ::* :*:: **:: *****:* ***:

Figure 3.4 Alignment of mammalian and Xenopus stathmin protein sequences with their respective similar sequence regions on L. major gp 96-94. Identical residues (*), conserved residues (:) and semi-conserved residues (.) are shown.

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3.3.2 Effects of Mammalian Cell Lysate on Tubulin Assembly

As tubulin is a highly conserved protein among species (81 and 84 % amino acid sequence identity between L. tarentolae and S. scrofa α- and β-tubulin respectively), the first logical approach was to attempt to utilize well-characterized mammalian stathmin to sequester leishmanial tubulin. A mammalian stathmin- leishmanial tubulin interaction would allow us to bypass identifying and purifying leishmanial stathmin and pursue the ultimate goal of tubulin crystallization. Fetal bovine thymus was chosen as a source of mammalian stathmin because it contains many dividing cells and serves as a readily available source of starting material

[172]. To insure that the isolated soluble cell fractions possessed activity observed by Belmont et al. [172], they were incubated with exogenous mammalian tubulin to demonstrate assembly inhibition. The upper (clear) isolated fraction was incubated with purified mammalian and leishmania tubulins (Fig. 3.5). As expected, the lysate completely inhibited mammalian tubulin polymerization, however, no effect was observed on leishmania tubulin. Interestingly, the bottom (red) fraction had no effect on mammalian tubulin or leishmania tubulin; in some cases it increased mammalian tubulin polymerization levels to greater than those of controls (data not shown). One possible explanation for the increase in polymerization is that endogenous tubulin present in the cell lysate may be present at higher levels in the bottom fraction while stathmin remains mostly in the top layer. Although the fetal bovine thymus soluble cell lysate effectively inhibits mammalian tubulin polymerization, it has little to no effect on the assembly of purified leishmania tubulin (Fig. 3.5). It should be noted

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that although mammalian and leishmania tubulins are highly conserved, they exhibit markedly different assembly profiles. Aside from a faster kinetic profile (in terms of initiation of polymerization), leishmania tubulin polymerizes readily at cold temperatures and has a lower critical assembly concentration (CAC) than mammalian tubulin. It thus appears that the equilibrium between dimer and polymer is shifted towards the latter when compared with its mammalian counterpart.

Therefore, it should be noted that these results do not determine whether or not mammalian stathmin interacts with leishmania tubulin, but rather that the affinity of mammalian stathmin for Leishmania tubulin is not high enough to influence its assembly profile.

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0.25

0.2

0.15

350

A 0.1

0.05

0 0 200 400 600 800 1000 1200 Time (s)

Figure 3.5 Tubulin assembly in the presence of mammalian cell lysate top fraction. Leishmania and mammalian tubulins at concentrations of 1 mg/mL (10 µM) and 1.5 mg/mL (15 µM), respectively, were assembled with 26 µL of the upper mammalian soluble cell lysate (TF) or PBS (control). Initiation of assembly and assessment of polymerization were performed as described in Materials and Methods . The assembly curves are indicated as follows: Leishmania tubulin control ( ♦), Leishmania tubulin + TF ( ▲), mammalian tubulin control ( ■), mammalian tubulin + TF (x).

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3.3.3 Effects of C. fasciculata and L. tarentolae Lysates on Tubulin Assembly

As active mammalian stathmin-containing fractions failed to sequester leishmanial tubulin, the next step was to assay for the presence of stathmin activity in a Leishmania cell lysate. Purified leishmanial and mammalian tubulins were incubated with both the upper (Fig 3.6) and the lower (Fig. 3.7) lysate fractions obtained from L. tarentolae . Neither the upper nor lower fractions appeared to have any inhibitory effects on tubulin polymerization. In both instances, the fractions appear to enhance tubulin polymerization. C. fasciculata soluble whole cell fractions were examined against both mammalian and Leishmania tubulins as well, the results

(not shown) were comparable to those obtained with the L. tarentolae soluble cell lysates. Three possible explanations for this occurrence are 1) the endogenous tubulin present in the soluble lysate rapidly copolymerized with the exogenous tubulin prohibiting any sequestration 2) no stathmin homologue is present in

Leishmania which effects tubulin dynamics by sequestration 3) there is a stathmin homologue present which sequesters Leishmania tubulin, however, the low critical concentration for Leishmania tubulin assembly requires any stathmin present in the lysate to be enriched to observe activity.

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0.7 0.6 0.5

0.4 x 350 0.3 A 0.2 0.1 0 0 200 400 600 800 1000 1200 TimeTime (s)(s)

Figure 3. 6 Tubulin assembly in the presence of Leishmania lysate top fraction. Leishmania and mammalian tubulins at concentrations of 1 mg/mL (10 µM) and 1.5 mg/mL (15 µM), respectively, were assembled with 26 µL of the upper L. tarentolae soluble whole cell lysate layer (TF) or PBS (control). Initiation of assembly and assessment of polymerization were performed as described in Materials and Methods . The assembly curves are indicated as follows: Leishmania tubulin control ( ♦), Leishmania tubulin + TF ( ▲), mammalian tubulin control ( ■), mammalian tubulin + TF (x). Results are the average of two independent experiments.

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0.7

0.6

0.5

0.4

350 0.3 A

0.2

0.1

0 0 200 400 600 800 1000 1200 TimeTime (s)

Figure 3.7 Tubulin assembly in the presence of Leishmania lysate bottom fraction. Leishmania and mammalian tubulins at concentrations of 1 mg/mL (10 µM) and 1.5 mg/mL (15 µM), respectively, were assembled with 26 µL of the lower L. tarentolae soluble whole cell lysate layer (BF) or PBS (control). Initiation of assembly and assessment of polymerization were performed as described in Materials and Methods . The assembly curves are indicated as follows: Leishmania tubulin control ( ♦), Leishmania tubulin + BF ( ▲), mammalian tubulin control ( ■), mammalian tubulin + BF (x). Results are the average of two independent experiments.

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To test the first hypothesis, the soluble cell lysates were investigated to determine if the endogenous tubulin in the lysate alone was capable of assembly. 50

µL of soluble cell lysate was added to a 96-well plate and monitored at 350 nm for 30 minutes (Fig. 3.8). As seen in Fig. 3.8, there is a lag in assembly onset and atypically high absorbance in such samples. This aberrant assembly profile indicatess that perhaps the increasing turbidity was not solely a byproduct of tubulin polymerization.

Turbidity is simply measured light scattering, which is a non-specific event.

Therefore, this detection method does not specifically measure tubulin polymerization and will record any phenomenon which causes light to scatter.

0.7

0.6

0.5

0.4

350

A 0.3

0.2

0.1

0 0 200 400 600 800 1000 1200 1400 1600 1800 Time (s)

Figure 3.8 Assembly profile for the soluble L. tarentolae cell lysate

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In an effort to determine the cause of the turbidity, an attempt was made to remove any endogenous tubulin present in the sample by batch chromatography. The resulting supernatant was removed and

50 µL were placed in a 96-well plate which Figure 3.9 Leishmania was monitored at 350 nm for 30 minutes. tarentolae soluble cell lysate. (a) The lysate before warming. The resulting turbidity profile closely (b) The lysate following incubation at 37 °C for 15 resembled that which was observed in minutes

Fig. 3.8 (data not shown). These results indicate that tubulin polymerization is not responsible for the turbidity observed.

Much like tubulin, however, the component in the sample responsible for the turbidity increase appeared to be thermally driven. Before proceeding with the search for a stathmin like protein which sequesters tubulin, it was necessary to remove this interference. 200 µL of soluble Leishmania lysate were warmed to 37 °C in a water bath for 15 minutes. Following incubation, a cloudy precipitate was observed (Fig.

3.9). An attempt was made to remove the precipitate by centrifugation, however, upon spectrophotometric analysis the precipitate returned. The warming and centrifugation steps were repeated three additional times (see materials and methods) and the absorbance of these samples was recorded as a function of time

(Fig. 3.10). Although centrifugation proved useful in reducing the precipitate, it was incapable of reducing to interference to levels at which tubulin polymerization could be observed. Attempts were made to resolubulize the precipitate such that it could

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be characterized and removed. Several detergents including CHAPS, SDS, Triton X, and Nonident P-40 at concentrations ranging from 0.5-10 % all failed to solubilize the precipitate. As the precipitate could not be solubilized for identification, the next step was to attempt to circumvent the precipitate by fractionating the soluble Leishmania lysate.

1 0.8

0.6 350

A 0.4 0.2 0 0 500 1000 Time (s)

Figure 3.10 Absorbance of samples after sequential warming/centrifugation cycles. The turbidity of original sample ( ♦) and that sample after the 1 st ( ■), 2 nd ( ▲), 3 rd ( ) and 4 th ( □) centrifugation cycles was monitored for 20 minutes at 37 °C.

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3.3.4 Column Calibration and Efficiency of Separation

The void volume (V o) of the gel filtration column was determined to be 11.5 mL by blue dextran absorbance at 620 nm. Molecular weight standards were then added two at a time to calibrate the column. Albumin and chymotrypsinogen A were added concomitantly and eluted, then ovalbumin and ribonuclease A were added.

The elution volume (Ve) of the standards was determined by adding 50 µL of each collected fraction to a 96-well half-area plate (Costar); protein containing fractions were detected by measuring the absorbance at 280 nm in a 96-well plate reader

(Molecular Devices). The elution profiles for the standards can be seen in Figure

3.11.

89

0.6

0.5

0.4

0.3

Absorbance 0.2

0.1

0 0 5 10 15 20 25 mLs Eluted

Figure 3.11 Elution profiles of blue dextran and molecular weight standards . The series 1 peak ( ♦) at 11.5 mL (fraction 23) corresponds to blue dextran. The series 2 peaks ( ■) at 12.5 (fraction 25) and 16 mLs (fraction 32) correspond to albumin (35.5 kDa) and chymotrypsinogen A (25 kDa) respectively. The series 3 peaks ( ▲) at 14.5 (fraction 29) and 18.5 mLs (fraction 37) correspond to ovalbumin (43 kDa) and ribonuclease A (13.7 kDa) respectively.

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2 3 The total volume (V t) of the system ( πr h) was determined to be 35.3 cm . A calibration curve (Fig. 3.12) was created by plotting the apparent partition coefficient

(Ve - Vo) (K av ), defined as , vs the log (molecular weight) of the standards. To ()Vt - Vo assess the column’s efficiency of separation, the protein rich containing fractions were analyzed by SDS-PAGE (12%). As can be seen in Figure 3.13, the column effectively separated the molecular weight standard proteins.

0.35

0.3 y = -0.367x + 1.8188 R2 = 0.993 0.25

0.2 av av

K 0.15

0.1

0.05

0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Log MW

Figure 3.12 Calibration curve for the gel fi ltration column determined from standards.

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1 2 3 4 5

Figure 3.13 Fractions eluted from the gel filtration column were analyzed by SDS-PAGE. Lane 1 contains 7 µL of each protein rich containing fraction (fractions 25, 29, 32, and 37). Lane 2 contains 15 µL of fraction 37 (ribonuclease A). Lane 3 contains 15 µL of fraction 32 (chymotrypsinogen A). Lane 4 contains 15 µL of fraction 29 (ovalbumin). Lane 5 contains 15 µL of fraction 25 (albumin).

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3.3.5 Effects of Parasite Lysate Fractions on Tubulin Assembly

As removal of the precipitate 1 2 3 4 5 6 7 8 9 10 proved challenging, it was kDa

103- determined that the most 77- appropriate step was to attempt to 50- work around the precipitate by 34- fractionating the lysate. A soluble 29-

L. tarentolae cell lysate was 21- prepared as described previously. Fig ure 3.14 10% SDS -PAGE of L. tarentolae fractions eluted from the As can be seen in Figure 3.14 the column. 5 µL of fraction was loaded per well. The molecular weight standards are in column efficiently separated the lane one, fractions 26, 28, 30, 32, 34, 36, 38, 40 and 42 are in lanes 2-10 respectively. soluble lysate by molecular weight.

The low molecular weight fractions (36-42) were assayed for their effect on purified

L. tarentolae tubulin. As can be seen in Figure 3.15, none of the low molecular weight fractions elicited a pronounced effect on tubulin polymerization. Although there does appear to be a decrease in polymerization, that decrease falls well within the variability observed in Leishmania tubulin polymerization assays performed in our lab. One major concern was that while fractionation was effective in removing the precipitate, it may have decreased the concentration of any stathmin like protein present. Low molecular weight containing fractions were therefore concentrated using the iCON protein concentrator. The concentrations of all of the samples

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throughout the entire procedure were determined by the Bradford protein concentration assay (Table 3.2).

0.05

0.04

0.03

350

A 0.02

0.01

0 0 100 200 300 400 500 600 Time

Figure 3.15 Assembly of purified L. tarentolae tubulin in the presence of low molecular weight fractions from the soluble L. tarentolae cell lysate. Shown are the control ( ♦), fraction 36 ( ■), fraction 38 ( ▲), fraction 40 (×) and fraction 42 ( ). Results are the average of two independent experiments.

Volume Conc. Fraction (mL) mg/mL Crude Lysate 11 22 Soluble Lysate 8 13 LMW Fractions 7 0.53 LMW Fractions After Concentration 1.2 2.7

Table 3.2 Protein concentrations of L. tarentolae cell lysate fractions . Shown are the concentrations of the crude lysate, soluble lysate, low molecular weight (LMW) fractions and LMW fractions after concentration.

*Only three mLs of soluble lysate (39 mg of protein) were loaded onto the column.

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To assess the efficiency of fractionation and degree of concentration, the pooled and concentrated LMW fractions were analyzed by SDS-PAGE (Fig. 3.16).

As can be seen in Fig. 3.16, the column provided adequate separation of the proteins by molecular weight and these fractions were successfully concentrated.

Due to volume constraints in gel loading volumes, it was not possible to load equivalent amounts (30 µg) of the pooled, unconcentrated low molecular weight fractions. Lanes 5 and 6 in Fig. 3.16 show a side-by-side comparison of equivalent volumes (20 µL) of low molecular weight proteins before and after concentration.

The SDS-PAGE confirms the results from the Bradford protein concentration determination that indeed, efforts at concentration were successful; the next step was to examine the concentrated fractions for their ability to block tubulin assembly.

Although the protein concentration of the low molecular weight fractions was 2.7 mg/mL, the concentration of low molecular weight proteins employed in the assay was 1.4 mg/mL due to the addition of the other reagents required for tubulin assembly. As shown in Figure 3.17, the fractions did not exhibit a pronounced effect on tubulin polymerization even though the concentration of low molecular weight proteins had been increased to 1.4 mg/mL. Although the low molecular weight fractions were a mixture of proteins, the suggested binding ratio for stathmin and tubulin of 1:2 M respectively [185,195] should have been met to a degree in which some biological activity would be observed.

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1 2 3 4 5 6 kDa 209- 124-

80-

49.1-

34.8- 28.9-

20.6-

7.1- Figure 3.16 4 -20% SDS -PAGE of Leishmania tarentolae cell lysate throughout preparative steps. Unless otherwise indicated 30 µg of protein were loaded per well in 20 µL volumes. Crude cell lysate (lane 1), soluble cell lysate (lane 2), ~11 µg pooled low molecular weight fractions (lane 3), concentrated fractions (lane 4), ~11 µg pooled low molecular weight fractions (lane 5), undiluted concentrated fractions (lane 6)

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0.09 0.08 0.07

0.06

0.05

0.04 350

A 0.03

0.02

0.01 0 0 200 400 600 800 1000 1200 Time (s)

Figure 3.17 Assembly profiles of L. tarentolae tubulin with and without concentrated lysate fractions. Shown are the control ( ♦), L. tarentolae tubulin with concentrated fraction ( ■) and without concentrated fraction ( ▲). Results are the average of two independent experiments

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3.4 CONCLUSIONS

After comprehensive efforts to identify a stathmin like protein capable of sequestering and thereby inhibiting L. tarentolae tubulin polymerization failed, the project was terminated. It should be noted that aside from the investigation of mammalian and Leishmania cell lysates, cell lysates from the trypanosome Crithidia fasciculata were also investigated (see materials and methods, data not shown). The rationale for this investigation was based on an observation made by Russell et al.

[139] who during the purification of tubulin from C. fasciculata noted that “despite the abundance of tubulin in the cytoplasm of C. fasciculata , attempts to purify it by assembly from cytoplasmic extracts were unsuccessful. This may represent the presence of an assembly inhibitor, similar to that reported for Polytomella sp. [196].”

Not only did soluble cell lysates fail to inhibit L. tarentolae tubulin polymerization, but a precipitate similar to the one described previously in Leishmania soluble cell lysates was observed. Efforts to remove the precipitate by size exclusion chromatography were successful, however, no effect on L. tarentolae tubulin polymerization was observed (data not shown). Being that stathmin is a critical protein present in diverse organisms, it would be surprising if it did not exist in the

Leishmania sp. Regardless if it is present or not, the results obtained herein indicate it does not play a significant role in regulating tubulin dynamics as seen in the mammalian system [172]. It has not escaped attention, however, that if the

Leishmania stathmin like protein is a high molecular weight protein, the molecular weight fractionation employed herein would not have been capable of separating it from the precipitate. Furthermore, self aggregation or association with other

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proteins, potentially tubulin, may have caused stathmin to co-elute with the precipitate. Perhaps future studies utilizing co-immunoprecipitation coupled to mass spectrometry [197] will serve to identify tubulin associated proteins and potentially proteins which play a role in regulating microtubule dynamics within the Leishmania species.

In the mean time, our lab is proceeding with alternative attempts to better characterize the dinitroaniline binding site. As discussed in Chapter 2, molecular modeling/docking studies combined with in vitro biological activity studies with dinitroaniline analogues have provided us with valuable information about the site

[164,169]. A recent success story utilizing the aforementioned approaches was the elimination of the nitro groups from GB-II-150 ( 3.1 Fig. 3.18) [198]. Previous experimental data indicateded that the nitro groups on the aniline ring were essential for activity [164], however, several nitroaromatic compounds have been shown to be mutagenic [199,200,201]. Therefore, substitution of the nitro groups with a more desirable functional group without sacrificing the activity of the compound would be a marked advancement in the drug development process. The molecular modeling and molecular dynamics studies performed by Mitra and Sept indicateded that the nitro groups were serving as hydrogen bond acceptors [169]. The authors predicted substitution of the nitro groups with moieties which could act as hydrogen bond acceptors would still fit well within the dinitroaniline binding site. As such, our laboratory recently synthesized analogues of ( 3.1 ) substituted with amino, chloro, cyano, carboxylate, methyl ester, amide and methyl ketone moieties in the nitro positions [198]. It was found that dicyano substitution yielded a compound ( 3.2 , Fig.

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3.18) which possessed activity tantamount to ( 3.1 ) in terms of antileishmanial activity, antimicrotubule activity and selectivity.

N((CH ) CH ) N((CH2)3CH3)2 2 3 3 2 O2N NO2 NC CN

SO2NHPh SO2NHPh 3.1 3.2

Figure 3.18 The chemical structures of GB -II -150 (3.1) and its equipotent dicyano substituted analog (3.2).

While the departure from the dinitroaniline scaffold is a notable advancement in the development of these antimitotic compounds, there are still hurdles which must be overcome. Although the dinitroaniline compounds and analogues show good in vitro activity, we have yet to identify an analogue which demonstrates activity in vivo .

Pharmacokinetic studies indicate extensive metabolism and rapid excretion as a possible explanation for the lack of correlation between in vitro and in vivo activities

[164,202]. Work is currently underway to produce future generation analogues with more desirable pharmacokinetic parameters. While the dinitroaniline story continues to unfold, it is imperative that our lab continue to exploit alternative avenues in hopes of identifying novel lead compounds possessing antileishmanial activity.

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CHAPTER 4

HIGH THROUGHPUT SCREENING AS A TOOL TO IDENTIFY NOVEL

ANTILEISHMANIAL COMPOUNDS

4.1 INTRODUCTION TO HIGH THROUGHPUT SCREENING

4.1.1 Current Drug Development Efforts

Current treatment strategies for leishmaniasis, their drawbacks, and the unmet need for novel antileishmanial agents are detailed in chapter 1. Although optimizing activity and improving the N pharmacokinetic parameters of our lead antimitotic O2N NO2 leishmaniacidal and trypanocidal compounds, more specifically derivatives of the antimitotic herbicide oryzalin SO2NHPh such as N1-phenyl-3,5-dinitro-N4, N4-di-n- Figure 4.1 The propylsulfanilamide ( 4.1 , Fig. 4.1) [145], have been the structure of GB-II-5 (4.1), an analogue of main focus of our lab, we are also interested in identifying the antimitotic herbicide oryzalin and developing other novel antileishmanial and

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antitrypanosomal compounds. Some examples of recent endeavors of our laboratory include the investigation of the antikinetoplastid activity of 3-aryl-5- thiocyanatomethyl-1,2,4-oxadiazoles [203], the isolation and evaluation of antiprotozoal activities of isoflavonoids and other compounds found in extracts from

Psorothamnus polydenius [204] and Psorothamnus aborescens [205], and the antiparasitic activities of bis-2,5-[4-guanidinophenyl]thiophenes [206]; some representative structures from the aforementioned compounds can be seen in Figure

4.2.

N O MeO O N SCN

Cl O Cl 4.2 4.3

HO O NH S NH OH N N N N H H H H OH O OH 4.4 4.5

Figure 4.2 Some representative structural classes of antiparasitic compounds which were/are being evaluated and/or optimized in our lab. Compound 4.2 (3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole inhibits L. donovani and T. b. brucei growth by 50% at concentrations of 2.3 and 5.2 µM respectively. Compound 4.3 (dalrubone) inhibits L. donovani and T. b. brucei growth by 50% at concentrations of 7.5 and 22 µM respectively. Compound 4.4 (5,7,3’,4’-tetrahydroxy-2’-(3,3-dimethylallyl)isoflavone) inhibits L. donovani and T. b. brucei growth by 50% at concentrations of 13 and 12 µM respectively. Compound 4.5 (2,5-Bis[4-(N-cyclopentylguanidino)phenyl]- thiophene dihydrochloride) inhibits T. b. brucei, P. falciparum and L. donovani growth by 50% at concentrations of 0.31, 0.13, and 1.5 µM respectively.

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Although optimization efforts are still underway and promising results have been obtained with the aforementioned compounds, work continues to achieve the ultimate goal of identifying preclinical candidates for consideration as inexpensive, orally available antikinetoplastid drugs. As such, we are continuing with a multifaceted approach to both optimize current leads as well as identify novel ones.

4.1.2 HIGH THROUGHPUT SCREENING TO IDENTIFY NOVEL THERAPEUTIC

AGENTS AND MOLECULAR TARGETS

4.1.2.1 High Throughput Screening

The screening of chemical compounds for pharmacological activity has occurred in various forms for at least 40 years [207]. Considering a representative target portfolio, high throughput screening (HTS) is presently the most widely applicable technology delivering chemistry entry points for drug discovery programs

[208]. Today many pharmaceutical companies are screening libraries ranging from

100,000 up to 1,000,000 compounds. With increasing technology and access to libraries, throughputs have increased from ~10,000 assays/year to current levels of ultra-throughput which can carry out 100,000 assays/day. The paradigm for screening is such that when a compound elicits a desired effect above a set threshold, it has passed the first milestone in becoming a clinically useful therapeutic agent. Compounds which fail the primary screen are retained for potential future screening against different targets/cell types. The term “desired effect” is a fairly ambiguous in screening. Desired effects, or endpoints, can be investigated for by

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either a target based approach to identify activity against a specific enzyme or protein to a phenotype based approach.

4.1.2.2 Target Based Approaches

The target based approach to high throughput screening involves screening against a previously identified and validated target. It is also a requirement that when an active compound interacts with the target a discernable response can be measured in a manner conducive to high throughput. All drugs which are presently on the market are estimated to target less than 500 biomolecules, some examples of target classes and their market share can be seen in Fig. 4.3. The obvious advantage to this screen is that target identification is not necessary. A major disadvantage with target based screens are that molecular interactions with other potential targets are often not observed; this can result in either producing hits which have undesirable interactions with other cellular components or neglecting to identify potentially active compounds which produce the desired end result by interacting with an alternative target.

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Figure 4.3 Therapeutic target classes. Reprinted by permission from Macmillan Publishers Ltd. Nature Reviews Drug Discovery. 2:369-78 (2003)

One example of a target based approach is reverse chemical genetics.

Chemical genetics is a research approach that uses small molecules, or compounds, as probes to study biological functions in cells or whole organisms [209]. Chemical genetics is grouped into two categories, reverse chemical genetics and forward chemical genetics. Reverse chemical genetics relies on the selection of a protein target of interest and screening for compounds which affect the proteins activity

[145]. This is followed by the use of the compound to study the protein(s) function in a biological context. Some recent examples of success in this field include but are not limited to Chang et al. (2001, and references therein); Feng et al. (2003), Cheung et al. (2002); and Peterson et al. (2001) [210,211,212,213]. Chang et al. [210] screened a small molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. They identified a potent and

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selective compound, N-benzyl-p-toluene sulphonamide (BTS), which will be useful in investigating the validity of motor proteins as therapeutic targets. Feng et al. [211] screened for compounds capable of interfering with specific steps in membrane traffic. They identified a compound, 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), which induces a rapid collapse of the Golgi to the endoplasmic reticulum (ER). Cheung et al. [212] screened a synthetic library of myoseverin analogues. Analogues which inhibited MT assembly, like myoseverin, were evaluated against 60 cancer cell lines. Peterson et al. [213] screened a chemical library against Xenopus cell free extracts to identify compounds which inhibit signaling pathways involved in actin polymerization. They identified a cyclic peptide which interferes with N-WASP, a protein that has been investigated for its role in the actin signaling network. All of the aforementioned screens identified a compound which acts at a specific target. These compounds can now be used to study the respective targets’ role within the biological system. Forward chemical genetics, which is an example of a phenotype approach, involves identifying an aberrant phenotype or disruption of a biological process in an organism or cell caused by a compound, then identifying the target(s) of that compound.

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4.1.2.3 Phenotype Based Approaches

The phenotype based approach involves screening to look for a desired phenotype such as aberrant cell morphology, inhibition of motility, and live/dead assays which are based solely on cell survival. One example of this approach in parasitology is the tritiated hypoxanthine incorporation assay performed to determine the activity of a compound against Plasmodium falciparum (the etiological agent of malaria) [214].

One drawback, and often the rate limiting step, of the phenotype based approach is target identification. With the exception of compounds which give a distinct or unique phenotype such as the monastrol [215], often little target information is provided, as such, further experimentation is required before the crucial stage of lead candidate selection. There are three general approaches to identify a target: the guess and test approach, the biochemical and cDNA expression-based approaches, and genetic approaches [216].

The “guess and test” approach, although the most rudimentary of the three, can be a useful tool nonetheless. In this method a hypothetical target is proposed and tested based on all available information, including information procured by biological assays and analytical tools (e.g. TEM, flow cytometry, fluorescent staining, etc.) as well as information which can be gleaned via structure similarity searching of available literature and databases such as SciFinder Scholar (Chemical Abstracts

Service, American Chemical Society). This approach will become increasingly

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powerful as databases documenting the systematic gene disruption in model organisms are further expanded [217,218]. Perhaps the recent elucidation of the tritryp genome [science 2006 ref.] will bring about the construction of gene disruption models for these parasites. These would go a long way in helping us to learn more about these organisms both in the identification of novel therapeutic targets and also the identification of the targets of previously identified antiparasitic agents.

Biochemical and cDNA based approaches to target identification include a variety of diverse techniques; a few examples such as affinity chromatography, photoaffinity labeling, radiolabeled analogue synthesis, protein microarrays, and the use of genetic methods [216,219,220] will be discussed briefly herein. Affinity chromatography relies on immobilizing the compound of interest to a matrix.

Following immobilization, crude or fractionated cell lysates are run through the matrix in hopes of capturing proteins with affinity for the compound. Although this method can be successful [221,222], there are numerous drawbacks including loss of affinity during the linking process, high levels of non-specific binding, and low concentrations of protein(s) of interest; these obstacles are discussed in greater breadth by Burdine et al. (2004)[220]. Photoaffinity labeling (reviewed by Dorman & Prestwich (2000))

[223], which is closely related to affinity chromatography, relies on attaching a photoreactive group to the compound of interest. The conjugated compound is then incubated with the cell lysate of interest allowing the molecule to interact with its respective target. The sample is then irradiated, this activates the photoreactive group which allows for irreversible binding to the target(s). This method shares similar drawbacks with affinity chromatography such as nonspecific binding and

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reduction of compound affinity by conjugation. Radiolabeled analogue synthesis involves either synthesizing the biologically active molecule with a radiolabeled isotope or conjugating it with one following synthesis. This allows the protein(s) to which the molecule binds to be tracked through separation techniques including 1- and 2-D gel and chromatography. Once identified, the protein can be identified by techniques such as mass spectrometry. One example of success utilizing this method was performed by Merck Research Laboratories [224]. Some drawbacks associated with this method include the necessity to use radioactive materials, as well as the potential of reducing the affinity of the molecule during conjugation.

Protein microarrays are multiple proteins which are spotted onto glass slides at a high spatial density [225,226]. These arrays can be used to identify protein-protein interactions, enzyme-substrate interactions, and more importantly in this context, targets of biologically active molecules. An example of this was done by Zhu et al.

(2001) [226]. Briefly, 5800 yeast open reading frames (ORFs) were expressed, purified and spotted onto a slide at a high spatial density. This yeast proteome microarray was screened for its ability to interact with proteins and phospholipids.

Utilizing this method, the authors identified many new calmodulin- and phospholipid- interacting proteins; furthermore, they were able to identify a common binding motif for calmodulin. cDNA expression based approaches can be problematic if the protein of interest is not in the expression library, if the expressed protein is not folded properly, or the expressed protein requires interaction with other proteins or lipids for the small molecule to bind [216]. Genetic methods are particularly useful in identifying the region(s) on the target protein(s) which confer activity. Mutagenesis involves creating mutant cells/organisms which are resistant to the compound of

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interest. Resistant mutants can be created by inducing random point mutations with a mutagen, or by serial passage under drug pressure. An example of success employing this approach in parasitology was performed by Morrissette et. al. (2004)

[167] who showed that oryzalin resistant Toxoplasma gondii had point mutations in

α-tubulin. Although none of the aforementioned methods are ideal for every instance, and each has limitations; they are all validated tools offering investigators multiple options for target identification.

4.1.3 Library Selection, Hit Identification, and Lead Optimization

To begin any small molecule screen it is necessary to acquire a collection of chemically diverse, highly pure compounds. Library selection is a crucial step; it is important to obtain the number and quantity of compounds which will be amenable to the type of screen employed. Perhaps equally important is the consideration of the properties of the molecules within the library. The maxim of past high throughput screens was to identify compounds with high affinity/activity and selectivity.

Unfortunately, structural modifications to improve the pharmacokinetic properties of a drug often come at the expense of marginalized activity and/or selectivity. A recent analysis of launched drugs indicates that, generally, relatively minor changes in structural and physical molecular properties take place between the lead and the launched drug candidate [227]. As such, there has been an increasing awareness of the need for developing compounds with drug-like properties [228] which are pharmacologically relevant and synthetically tractable. As such, in an attempt to

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reduce attrition in latter stages of development, attention has shifted back to the selection of the library.

Following the development of suitable screening conditions, the first milestone in HTS, identification of hits, is somewhat arbitrary as compound concentrations and hit threshold limits can be set in such a manner that the ratio of

“active” to ”inactive” compounds can be controlled. As such, a delicate balance exists between excluding a potential lead compound and maintaining a succinct, rigorous screen which is delivers a practical number of hits. The method for detecting hits should be discrete and capable of analyzing large volumes of samples.

Statistical tools such as the Z- and Z’-factor [229] (see “Results and Discussion”) are useful for evaluating the suitability of an HTS assay for hit identification. Many target based screens and some phenotype based assays can be readily analyzed in plate reader format using absorbance, luminescence, radioactivity, scintillation proximity or various fluorescence-based assays as a readout [230,231,232,234]. Examples of alternative methods for detection range from the “cytoblot” assay if antibodies are available [215,235] to visual inspection of images collected by microscopy [223].

Once hits are identified and confirmed, they must be carefully examined both in terms of chemical properties and often times selectivity for the particular target or cell type to determine which hits will become leads.

Hit-to-lead selection is another crucial stage in the development process; it is desirable to have multiple leads should one fail due to unforeseen complications

(e.g. poor bioavailability, toxicity, etc.), however, the development of multiple leads

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increases the financial burden as well as the ramifications of failure. One important aspect of HTS or any lead development strategy is to reduce attrition, especially in later stages of development. The late stage attrition of chemical entities in development and beyond is very costly and therefore must be kept to a minimum by setting in place rigorous quality assessment at key points [208] (Fig. 4.4).

Figure 4. 4 Stage by stage quality assessment to reduce costly late stage attrition. Some important milestones in drug development are identified here including the validated hit series (VHS), the lead series identified (LSI), and the clinical candidate selection (CCS). Reprinted by permission from Macmillan Publishers Ltd. Nature Reviews Drug Discovery. 2:369-78 (2003)

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4.1.4 The Search for Novel Antileishmanial Lead Compounds

As access to compound collections and HTS technology have improved

[232], the screening of compound libraries, once only feasible for pharmaceutical companies, has become increasingly popular in academic research institutions [236].

Funding institutions such as the National Cancer Institute (NCI) have formally recognized the value and potential of HTS through a variety of initiatives and programs [216], including the Discovery Services of the Developmental Therapeutics

Program [237], Molecular Targets Drug Discovery grants, and the Molecular Target

Laboratories initiative (see Science 2002, 295). Aside from the NCI developmental therapeutics program (DTP) library which contains more than 140,000 synthetic and natural product agents which have been evaluated as potential anticancer and anti-

HIV agents [238], numerous companies in the industry sector have compound libraries available for purchase; some examples of these companies and the relative size of their libraries were compiled by Morgan et al. [219] and can be seen in table

4.1.

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Number of Company Compounds

Biofocus 25K Bionet/Key Organics Ltd. 41K

Cerep 23K Chembridge 220K

Interbioscreen 350K Maybridge 58K Peakdale 18K Specs & Biospecs 220K Tripos 100K

Table 4.1 An incomplete list of companies with compound collections for purchace. Adapted from [219]

As the majority of parasitology research is conducted in academic laboratories, HTS as a tool to identify novel antiparasitic compounds is a relatively recent approach [219]. Recently, Baldwin et al. and St. George et al. have performed HTS using unbiased compound collections against the protozoan parasites P. falciparum and L. major respectively [239,240]. The phenotype based live/dead screen performed by St. George et al. assayed 15,000 compounds from the aforementioned NCI Developmental Therapeutics Program repository of synthetic and natural products against both L. tarentolae and L. major . Compounds possessing antileishmanial activity which were not synthetically tractable or had

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previously reported cytotoxicities against mammalian cells were discarded. Of the

786 compounds identified which inhibited L. tarentolae growth by > 90% at either 25

µM or 12.5 µM, 692 were removed due to cytotoxic, antifungal, or antibacterial properties. The 43 most potent compounds were assayed against L. major promastigotes in vitro . Compounds which demonstrated activity against L. major promastigotes were next screened against L. major amastigotes in the infected macrophage assay [241]. This assay, which more closely mimics the in vivo infection, uses isolated murine peritoneal macrophages as a host for the amastigote

(see Results and Discussion). Three compounds were found to clear amastigotes better than the standard antileishmanial drug Pentostam; these results are promising and warrant in vivo evaluation in the mouse [176,242,243,244] or hamster models[245].

Recently, Baldwin et al. and Grant et al. have performed target based HTS against Plasmodium falciparum and L. mexicana amastigotes respectively [239,246].

The screen by Grant et al. was performed against L. mexicana CRK3 cyclin dependent kinase; previous studies have shown that inhibition of CRK3 histone H1 kinase activity by flavopiridol resulte in cell cycle arrest in the G 2/M phase of the cell cycle [247]. The screen assayed a mixture of 634 diverse synthetic and natural product isolates against purified transgenic his-tagged CRK3 (CRK3his). Inhibition of activity was assessed by incubating CRK3his with histone H1, the reaction was initiated by the addition of [ γ-32 P]ATP; phosphorylation was detected by SDS-PAGE and autoradiography. Twenty-seven potent CRK3 inhibitors were identified, sixteen of which were shown to inhibit L. donovani growth in vitro with IC 50 values < 10 µM.

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Future studies including X-ray crystallography and active-site molecular modeling are planned to further characterize the binding site.

Our laboratory has recently purchased the ChemBridge CNS-Set™ of 10,000 compounds in an effort to identify novel compounds possessing antileishmanial activity. The library is a collection of pharmacophore diverse, druglike small molecules. This library contains compounds which conform to Lipinski’s rule of 5

[248] and possess appropriate clogP and polar surface area values. Thus it is likely that any leads which are identified will possess good oral bioavailability. Our lab is conducting two distinct screens employing both the phenotype based approach

(live/dead assay) against L. donovani axenic amastigotes and target based approach against purified L. tarentolae tubulin [142] to identify compounds which affect tubulin polymerization [152]. This will allow us to screen against a previously validated molecular target [152] while not neglecting any potential leads which exert antileishmanial activity via mechanisms other than inhibition of tubulin polymerization. It is important to employ alternative hit identification strategies that are able to effectively address a variety of biological macromolecular targets, and to identify proprietary, synthetically tractable and pharmacologically relevant compounds [208]. Hits identified from these HTS which possess selectivity for

Leishmania will be advanced as lead compounds. Lead compounds will be investigated in vitro with the infected macrophage assay [249], and ultimately in vivo with the infected mouse model [176,242,243,244]. Analoges of promising leads will be synthesized to optimize activity and pharmacokinetic properties. Herein the efforts to identify and characterize novel, orally available, and inexpensive

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antileishmanial compound(s) by utilizing the phenotype based live/dead approach are reported.

4.2 MATERIALS AND METHODS

4.2.1 PRIMARY SCREEN, IC 50 DETERMINATIONS AND VERO CELL

SELECTIVITY SCREEN

4.2.1.1 Chemicals and Biochemicals

The CNS-Set™ of 10,000 compounds was purchased from ChemBridge

(www.chembridge.com ). The compounds were supplied in 96-well plates at a concentration of 10 µM (200 µL volume) in 100% DMSO; there were 80 compounds per plate, with the two outer columns containing only DMSO. To maintain the integrity of the library, two sets of daughter plates (each containing 50 µL) were made from the parent plates. The parent plates were stored at -80° C in the dark while the daughter plates were stored at -20° C in the dark prior to use. 5 mg of each active compound were purchased for reevaluation from ChemBridge

(www.hit2lead.com ), the compounds were dissolved in DMSO at a concentration of

20 mM and stored in the dark at -20 °C. Where not otherwise noted, all other chemicals and biochemicals were purchased from Sigma (St. Louis, MO).

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4.2.1.2 Primary Screen Against L. donovani Axenic Amastigotes

L. donovani axenic amastigotes (WHO designation: MHOM/SD/62/1S-CL2 D) were maintained by serial passage in a 37° C humidified atmosphere containing 5%

CO 2. The parasites were cultured in the medium described by Werbovetz et al.

[145]. Before addition of fetal bovine serum to a final concentration of 20%, the medium contains 15 mM KCl, 115 mM KH 2PO 4, 10 mM K 2HPO 4, 0.5 mM MgSO 4, 24 mM NaHCO 3, a 1× concentration of RPMI-1640 vitamins and amino acids, 2.0 mM L- glutamine, 22 mM D-, 50 units/mL penicillin, 50 µg/mL streptomycin, 0.1 mM adenosine, 1 µg/mL folate, and 25 mM MES, pH 5.5. The assays were performed in

96-well half area plates (Costar) in the amastigote medium described previously. 1

µL of each compound was added via multi-channel pipette to a 96-well transfer plate; the compounds were then diluted 100-fold by the addition of amastigote medium.

Pentamidine, the control drug used in this assay, was serially diluted in 100% DMSO and added to the first row of the transfer plate. Medium was added to the pentamidine containing wells such that the starting concentration (well A1) was 250

µM. Following compound dilution, assay plates were prepared by adding 27 µL of medium containing L. donovani amastigotes at a concentration of 1.1 × 10 6 cells mL -1

(assay, negative control (A12-E12), and pentamidine control wells (A1-H1)) or 27 µL of medium without parasites (positive control wells (F12-H12)). 3 µL of each diluted compound and pentamidine serial dilutions (starting at 25 µM) were added to the assay plate for a final volume of 30 µL. The general setup for the screen is shown below (Fig. 4.5).

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Figure 4.5 Schematic of primary screen setup

The assay plates were then incubated at 37 °C, in a humidified 5% CO 2 atmosphere for 72 hours. Cell survival after 72 hours was assessed by the addition of 6 µL of

® CellTiter 96 AQ ueous Non-Radioactive Cell Proliferation Assay (Promega). Following

4-8 hours of incubation with the CellTiter reagent at 37 °C the absorbances were read at 490 nm in a SPECTRAmax PLUS spectrophotometer (Molecular Devices).

IC 50 values for pentamidine were determined using SOFTmax Pro software

(Molecular Devices) using the log dose response equation y = a + b / (1 + ( x/c)d where x, drug concentration; y, absorbance at 490 nm; a, lower asymptote; b, the difference between the upper asymptote and the lower asymptote; c, IC 50 ; and d,

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slope. To determine the absorbance value at the 50% inhibition threshold the following formula was used:

(Avg (negative cont.) - Avg (positive cont.) ) + Avg (positive cont.) , wells with absorbance values ≤ the 2

50% inhibition threshold were considered primary hits.

To identify secondary (40-49% inhibition) and tertiary (35-39% inhibition) hit threshold the following equation was used:

0.65 (Avg (negative cont.) − Avg (positive cont.) )+ Avg (positive cont.) . To evaluate the quality of the assay, the Z’-Factor (Fig. 4.6) was determined for each plate [229].

σ = StDev (3σc + + 3σc -) Z' = 1 - µ = Mean µc + −µc -

Figure 4.6 The Z’ -Factor is defined using the values obtained from the positive controls (c+) and negative controls (c-) of each plate.

4.2.1.3 Determination of IC 50 Values for Hit Compounds

Prior to use, L. donovani axenic amastigotes were maintained as outlined in

“materials and methods” section 2.2. IC 50 values (the concentration of compound which inhibits 50% of parasite growth as compared to untreated parasites) for all of the primary hits were determined following the procedure described by Werbovetz et

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al. [152], with some modifications. Briefly, a dilution plate was made in which compounds of interest were diluted to 2× the desired starting assay concentration of

50 µM with amastigote medium in a 96-well plate (Costar). The control drug for this assay, pentamidine, was diluted to 2× the starting concentration of 25 µM and placed in well A1; the diluted compounds were located horizontally adjacent to each other in wells A2-A11 with A12 remaining empty. To prepare the assay plate, 15 µL of amastigote medium was added to all of the wells of a 96-well half-area plate with the exception of the first row wells A1-A11. 15 µL of each compound including pentamidine (well A1) from the dilution plate were transferred concomitantly via multichannel pipette to the first row (wells A1-A11) of the assay plate. An additional

15 µL of compounds from the dilution plate including pentamidine were added to row

B of the assay plate and serially diluted vertically (rows B1-H1, B2-H2, etc.) to a terminal concentration of 0.39 µM. Parasites were diluted to 2× the required density

(2 × 10 6 cells/mL) in amastigote medium. 15 µL of diluted parasites were added to each well with the exception of F12, G12, and H12 (positive control wells). An additional 15 µL of fresh medium were added to the positive control wells, bringing the total volume of each well to 30 µL. The plates were then incubated at 37 °C for

72 hours, IC 50 values were determined as described for pentamidine in section

4.2.1.2.

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4.2.1.4 Vero Cell Assay

Vero cells (ATCC, Rockville, MD) were maintained in a humidified 37 °C atmosphere with 5% CO 2 in minimum essential medium, alpha modification containing Glutamax-I (Invitrogen™, Carlsbad, CA) and supplemented with 10% heat inactivated fetal bovine serum (FBS) 50 units/mL penicillin, and 50 µg/mL streptomycin. The cells were maintained by serial passage in T-75 flasks (Costar).

Briefly, used medium was removed, cells were washed once with HyQ ® Hanks

Balanced Salt Solution/Modified (HyClone Biologicals, Logan, UT), the solution was then removed and the cells were detached via incubation with 2 mL trypsin/EDTA

(0.05% Trypsin, EDTA • 4 Na) (Invitrogen™, Carlsbad, CA) at 37 °C for 5 min. After the cells were detached the trypsin/EDTA solution was quenched with the addition of

6 mL of fresh medium; this dilution was used for passing cells and performing assays. To begin assay, cells were detached as described above and the cell density was adjusted to 2 × 10 4 cells/mL in the Vero cell medium described above.

50 µL of cells were added to each well of a 96-well plate (Costar) except for positive control wells and were allowed to adhere overnight in the aforementioned environment. The following day, selected compounds were diluted to the desired concentration of either 100 µM, 50 µM, or 25 µM in fresh Vero cell medium.

Podophyllotoxin, the control compound used for this assay, was serially diluted from

1 µM to 0.007 µM. Old medium was removed from the assay plate and 100 µL of compounds (internal wells), podophyllotoxin (column 1), or medium (positive and negative controls, column 12) were added to each well. Following 72h of incubation, cell survival, the control compound IC 50 value, and Z’-factor were determined using

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the colorimetric method described previously in section 2.2. Percent cell survival

Acmpd was determined using the formula: ×100 , where A cmpd = the absorbance of X(A12 - E12) the compound at 490 nm and X is the average absorbance value in the given wells

(negative controls).

4.2.1.5 Infected Macrophage Assay

The colorimetric infected macrophage assay described herein was based on the protocol described by Buckner and Wilson [249]. Starch elicited mouse macrophages were harvested as described previously [250,251]. Briefly, 8-12 week old CD-1 female mice (Harlan) were injected with i.p. with 2 mL of a 2% starch solution to elicit macrophage recruitment. Twenty-four hours post-injection, mice were sacrificed and macrophages were harvested by peritoneal lavage with RPMI

1640 medium supplemented with 10% FBS, 50 units/mL penicillin G and 50 units/mL streptomycin (pH 7.4). The peritoneal fluid from the mice was pooled and centrifuged at 350 × g for 10 min at 4 °C. The supernatant was removed and the cells were resuspended in fresh medium and stored on ice until use. Macrophage viability was assessed during counting by Trypan Blue exclusion dye [252]. The cells were diluted to a concentration of 2 × 10 6 cells/mL and were pipetted in a volume of

100 µL into 96-well plates (Costar) and allowed to adhere overnight in a 34 °C humidified incubator with 5 % CO 2. The following day, the medium and unattached macrophages were removed and the attached macrophages were washed once with

Hanks’ balanced salt solution (HBSS). The number of attached macrophages was

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determined by subtracting the number of unattached macrophages. The macrophages were infected with 100 µL of late stage β-lactamase expressing L. amazonensis promastigotes (the promastigotes were a kind gift from Dr. Frederick

Buckner, University of Washington) at a ration of 7.5:1 (parasites:macrophages).

The promastigotes had previously been maintained in RPMI 1640 supplemented with

0.2 mM glutamine, 0.1 mM adenosine, 1 µg/mL folate, 50 units/mL penicillin, 50 units/mL streptomycin and 10 % FBS in a T-25 flask at 25 °C. The infection was allowed to take for 24 hours, after which the medium was removed and the cells were washed once with HBSS to remove any unphagocytized parasites. The medium was replaced with 200 µL of macrophage medium containing dilutions of compounds to be tested. After another 72 hours of incubation, the medium was removed and the cells were washed once with sterile PBS. Following the wash, 100

µL of a lysis solution containing 0.1 % Triton-X and 100 µM nitrocefin (Calbiochem,

La Jolla, CA) in PBS was added to each well. After a 3-4 hour incubation at 37 °C, parasitic burden was assessed colorimetrically by measuring absorbance at 490 nm.

IC 50 values were determined as described previously in 4.2.1.2 .

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4.2.2 BIOLOGICAL EVALUATION OF COMPOUND 4.45 AGAINST L. donovani

AXENIC AMASTIGOTES

4.2.2.1 Transmission Electron Microscopy

L. donovani axenic amastigotes maintained as previously described were seeded into T-75 flasks (Costar) at a cell density of 5 × 10 6 cells/mL (total volume 25 mL/flask). Two flasks were used for each group; groups included control (no compound), and treatment groups with compound 4.45 at 10 µM, 5 µM, 2.5 µM, and

1 µM. Post-treatment the cells were incubated for 24 hr at 37 °C in a humidified atmosphere containing 5% CO 2. Following treatment the cells were harvested by centrifugation at 1500 × g at 4 °C for 15 min. The resulting supernatant was carefully removed and the pellet was resuspended in 1 mL of fixative containing 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.1 M sucrose. The cells were fixed at room temperature for 3 hr. Cells were washed four times with 0.1 M phosphate buffer containing 0.1 M sucrose then incubated for 1 h in 1% osmium tetroxide in phosphate buffer, rinsed twice, and set with 2% agarose which had been previously chilled in an ice bath for 10 min. The agarose was removed from the centrifuge tube, cut into 1 mm blocks and placed in buffer at 4 °C overnight. The sample was warmed to ambient temperature and rinsed once in buffer and 4 times in diH 2O. The samples were then stained with 2% aqueous uranyl acetate for 1 hr and rinsed three times with diH 2O. The samples

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were then dehydrated for 10 min in each of the following ethanol gradients: 50%,

70%, 80%, 95%, 100%, and 100%. The samples were treated for 10 min in propylene oxide, then propylene oxide/epon resin (1:1 for 1 hr, then 1:2 overnight), and finally embedded in epon resin which was allowed to polymerize overnight at 60

°C. The resin was sectioned at 70 nm on a Leica EM UC6 Ultramicrotome.

Samples were observed on a FEI Technai Spirit TEM at 80 kV.

4.2.2.2 Cell Cycle Analysis by Flow Cytometry

L. donovani axenic amastigotes were maintained in amastigote medium as described previously. 5 mL of 5 × 10 6 cells/mL were incubated for 24 hr with compound 4.45 at

10 µM, 5 µM, 2.5 µM, or 0 µM (control). Cells were centrifuged at 3200 × g for 15 min at 4 °C. The resulting supernatant was removed and the cells were fixed with

70% methanol (500 µL) in PBS at -20 °C for 2 hr. Following fixation, cells were centrifuged at 3200 × g for 15 min at 4 °C, the fixative was removed, the cells were resuspended in 500 µL solution containing 10 µg/mL propidium iodide, 0.1% Triton –

X 100, and 5 µg/mL RNase A and incubated at 37 °C for 30 min. Cells were then collected by centrifugation and resuspended in PBS to a concentration of 1 × 10 7 cells/mL. Fluorescence intensity in individual cells was analyzed by flow cytometry using a Becton Dickinson FACSCalibur instrument (Rutherford, NJ) [145].

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4.3 RESULTS AND DISCUSSION

4.3.1 PRIMARY SCREEN, IC 50 DETERMINATIONS AND VERO CELL

SELECTIVITY SCREEN

4.3.1.1 Assay Validation and Optimization

Assays for high throughput screening require small sample volume, high throughput, robustness, adequate sensitivity, reproducibility, and accuracy in order to discriminate among a very large number of compounds that span the entire range of activity[229]. In our screen, each compound is tested only once, as such, it was essential that our assay be optimized and reproducible as not to miss any potential hits. Traditional methods of assessing the quality of an assay include the signal-to-

mean signal - mean background noise ratio [253], defined as: S/N = , and the signal standard deviation of background

mean signal to background ratio [254], defined as: S/B = . The inherent mean background problem with using S/N or S/B ratios is that neither takes into full account both the variability in the sample and background measurements and the signal dynamic range [229]. The Z-factor was developed as a screening window coefficient which is reflective of both the assay signal dynamic range and the data variation associated

3SD of sample + 3SD of control with signal measurements; it is defined as: Z = -1 mean of sample - mean of control

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[229]. The Z’-factor as defined in the Materials and Methods section (section 2.2 ) is similar to the Z-factor except that it is used to define the quality of the assay itself, independent of test compounds. We chose this parameter because we had previously defined a hit limit of 50% inhibition, as such, we were only interested in ensuring that the window of separation in our assay was great enough to clearly distinguish 50% inhibition from (+) and (-) controls. A Z’-factor which is <0.5 indicates that the band of separation is too small and the assay should be performed in duplicate. A Z’-factor ≥ 0.5 and <1 indicates an excellent assay.

Although our lab has performed numerous assays evaluating compounds against Leishmania in vitro [145,152,165], the assay was developed to determine specific activity (IC 50 values) for a relatively small number of compounds. Both previous assays and the high throughput screen were performed in half-area 96-well plates (Costar) to reduce surface area, thereby minimizing the unavoidable impact of medium evaporation. It was originally envisioned that the assay was to be performed in 384-well plates, however, after careful consideration it was decided that the transition from the 96-well plate format which the library was supplied in to the

384-well plate format for assaying would add unnecessary confusion and little benefit. Traditionally, in vitro antileishmanial assays were performed in a total volume of 60 µL, however, to reduce the amount of reagents required it was proposed that the assay be performed with a final volume of 30 µL. To insure that the reduced final volume would not significantly impact the assay results or decrease the band of separation below acceptable levels, a head-to-head comparison was

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performed with the standard drug pentamidine using both 60 µL and 30 µL volumes

(Table 4.2).

Volume (µL) Pentamidine IC 50 (µM) StDev Z'-factor StDev 30 1.4 0.3 0.56 0.12 60 1.1 0.06 0.82 0.09

Table 4.2 Compariso n of 30 and 60 µL assay volumes (Each value represents the average of three independent experiments )

As there was little variability between the assay when conducted with the two different assay volumes in terms of IC 50 values and since the Z’-factor indicated a good band of separation with both assays, the 30 µL assay was chosen to minimize reagent use.

4.3.1.2 Primary Screen

While performing a high throughput screen, large amounts of data are collected in a relatively short timespan. One potential bottleneck is the interpretation of these data sets. Our data sets were in the form of absorbance values obtained from the half-area 96-well microtiter plates. There were a total of 125 plates in the library we purchased, each plate containing 80 compounds. To rapidly identify hits from our assay, absorbance values were pasted into Excel. The formulas described

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previously in the Materials and Methods section were applied to determine what absorbance corresponded to 50% or greater inhibition and which absorbance values corresponded to 35-49% inhibition; once the values were determined, conditional formatting was set such that primary hits were highlighted by red text with a yellow background, while secondary and tertiary hits were highlighted by black text with a yellow background. Figures 4.7 shows the transition from the assay plate following incubation with MTS/PMS values to the absorbance values in Excel with primary, secondary, and tertiary hits highlighted.

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A490

1 2 3 4 5 6 7 8 9 10 1112

A 0.1 1.3 1.2 1.1 1.2 1.2 1.3 1.3 1.3 1.1 1.1 1.0 (-)Controls B 0.1 1.3 1.1 1.0 1.0 1.2 1.1 1.1 1.0 0.6 1.0 1.0 C 0.1 1.2 1.2 1.1 1.1 1.2 1.1 1.1 1.1 1.1 1.0 1.0 D 0.4 1.3 1.3 0.1 1.2 1.2 0.1 1.2 1.2 1.1 1.0 1.1

E 1.0 0.1 1.2 1.2 1.2 0.1 1.1 1.1 1.1 1.1 1.0 1.0 (+)Controls

Pentamidine Pentamidine F 1.0 1.1 1.1 1.1 1.2 1.2 1.1 1.1 1.1 1.1 1.0 0.1 G 1.1 1.1 1.1 1.2 1.1 1.1 1.1 1.1 1.0 1.0 1.0 0.1 H 1.0 0.9 1.2 1.1 1.1 1.1 1.1 1.0 1.0 1.0 0.9 0.1

Compounds

Figure 4.7 Assay plate following 6 hr incubation with colorimetric dye. After reading the absorbance at 490 nm, values were pasted into an Excel spreadsheet with conditional formatting set such that primary, secondary, and tertiary hits were unambiguously identified. Wells D4, D7, E2, and E6 are primary hits ( ≥ 50% inhibition); well B10 is a secondary/tertiary hit.

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Of the 10,000 compounds screened, there were a total of 75 primary hits (see appendix for structures), 35 secondary hits, and 21 tertiary hits. The rationale for identifying secondary and tertiary hits was to provide future options should the primary hits be found to be undesirable in the future stages of the development process. As such, their structures are not shown at this time. The assessment parameters from the primary screen (Table 4.3) shows the excellent performance of the assay, providing confidence that compounds possessing antileishmanial activity were accurately identified.

Assessment Parameter Average* StDev* Z'-Factor 0.72 0.18

Pentamidine IC 50 2.8 1.1

Table 4.3 The average Z’ -Factor and internal standard IC 50 values. A Z’-Factor > 0.5 indicates that the assay had a band of separation great enough to identify hits. Pentamidine’s IC 50 is close to the value of 1.9 µM previously reported by our laboratory[175].

*The values represented are the result of 125 individual assays (plates)

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4.3.1.3 Determination of the Activity of Primary Hits

To provide a more precise measure of the antileishmanial activity of the hits from the primary screen, the concentrations of the compounds which inhibited 50% of parasite growth (IC 50 ) were determined. The results from the primary screen and

IC 50 value determinations are summarized in Table 4.4. Of the 75 hits, 47 possessed IC 50 values ≤ 5 µM while 11 compounds appeared to be relatively inactive with IC 50 values > 25 µM. Pentamidine was again used as the control drug, with the average IC 50 value from the assays (26 plates total) being 2.2 µM ± 0.6.

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Primary Primary Primary Compound Inhibition IC 50 * Compound Inhibition IC 50 * Compound Inhibition IC 50 * 4.6 99 6.9 4.31 100 3.2 4.56 100 1.4 4.7 63 >25 4.32 87 1.5 4.57 87 3.5 4.8 75 4 4.33 100 1.3 4.58 58 5.3 4.9 77 4.8 4.34 98 2.4 4.59 59 4.7 4.10 76 >25 4.35 99 0.64 4.60 95 4.2 4.11 100 0.88 4.36 100 1.7 4.61 93 4.7 4.12 63 20 4.37 96 1.5 4.62 65 >25 4.13 100 3.9 4.38 62 12 4.63 88 3.3 4.14 66 >25 4.39 94 5.9 4.64 91 4.6 4.15 99 0.74 4.40 100 1.2 4.65 95 1.3 4.16 61 >25 4.41 99 3 4.66 98 1.2 4.17 66 8.3 4.42 99 1.6 4.67 93 3.3 4.18 86 8.8 4.43 99 1.5 4.68 98 1.1 4.19 98 >25 4.44 100 1 4.69 55 >25 4.20 99 8.3 4.45 100 1.7 4.70 52 >25 4.21 100 1.3 4.46 87 6.8 4.71 57 3.7 4.22 99 4.2 4.47 100 3.2 4.72 99 2.2 4.23 52 17 4.48 99 4.6 4.73 54 >25 4.24 92 5 4.49 100 0.62 4.74 78 >25 4.25 99 1.2 4.50 100 0.77 4.75 51 >25 4.26 81 8.4 4.51 100 0.96 4.76 90 2.6 4.27 94 6.6 4.52 91 3 4.77 60 5.4 97 7.8 100 2.6 90 6.5 4.28 4.53 4.78 4.29 62 6.7 4.54 99 2.9 4.79 55 8.2 4.30 91 3.8 4.55 60 20 4.80 98 2.6

Table 4.4 The percent inhibition values from the primary scre en (Primary Inhibition) and the IC 50 values of the 75 primary hits identified from the screen.

*Values are the average of at least 3 individual experiments

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4.3.1.4 Vero Cell Selectivity Screen

The 47 compounds with IC 50 values ≤ 5 µM were screened against Vero cells

(a mammalian cell line from monkey kidney) to determine if they were specific for

Leishmania or were instead broad spectrum cytotoxic compounds. The goal of this screen was to identify compounds possessing 25-fold or greater selectivity for

Leishmania parasites. In the first experiment, all compounds were assayed at a concentration of 100 µM. In subsequent experiments, if needed, the assay concentrations were lowered such that they were 25 × the compound’s IC 50 against

L. donovani (i.e. a compound with an IC 50 of 1 µM would be screened against Vero cells at 25 µM). The results from the selectivity screen are shown in Table 4.5. Of the 47 compounds screened in this assay, 17 were found to possess 25-fold or greater selectivity for Leishmania ; the compound structures and their clogPs are shown in Table 4.6. Of the 17 selective compounds identified there were 12 distinct structural classes. Compounds 4.22 , 4.31 , 4.45 , and 4.53 all contain a pyrrolidinedione moiety, whereas compounds 4.40 , 4.43 , and 4.44 all contain a quinoxalinone 4-oxide moiety.

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Vero Cell % Adjusted Amastigote Inhibition Concentration

Com pound IC 50 (µM) (100 µM) (µM) % Inhibition S.E. S.I.* 4.8 4.01 98 100 98 0.33 4.9 4.82 27 100 32 13 >21 4.11 0.884 87 25 63 6.8 4.13 3.87 100 100 100 0 4.15 0.738 100 25 52 5.8 4.21 1.25 84 25 73 2.4 4.22 4.16 35 100 34 13.7 >24 4.24 4.99 68 100 66 3.2 4.25 1.22 100 25 93 3.9 4.30 3.83 80 100 79 1.5 4.31 3.15 0 100 5 3.2 >31 4.32 1.54 64 50 43 12 >33 4.33 1.27 100 25 73 15 4.34 2.38 99 50 94 1.9 4.35 0.639 100 25 100 0.25 4.36 1.73 100 50 99 0.25 4.37 1.46 100 25 78 5.5 4.40 1.17 60 50 28 3.7 >42 4.41 3.03 96 100 98 0.63 4.42 1.6 100 50 100 0.2 4.43 1.52 74 50 31 8.7 >33 4.44 1.02 37 100 49 4.6 >100 4.45 1.7 0 100 10 3.8 >59 4.47 3.24 99 100 98 0.26 4.48 4.64 73 100 65 5.1 4.49 0.619 100 25 100 0.25 4.50 0.771 100 25 99 0.18

Table 4.5 Results from selectivity screen against Vero cells. Compounds possessing selectivity are indicated by bold text and grey background. Percent inhibition and standard error (S.E.) values reported are the average of no less than 5 experiments. The selectivity index (S.I.) is reported as the number of times more cytotoxic the compounds are for Leishmania parasites compared to Vero cells. (CONTINUED)

* These data were generated by dividing the compound concentration used in the Vero cell screen (all of which produced <50 % inhibition) by the IC 50 of the compound against L. donovani . IC 50 values were not determined for Vero cells, as such, actual selectivity indices are higher than indicated

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Table 4.5 CONTINUED

Vero Cell % Adjusted Amastigote Inhibition Concentration Compound IC50 (µM) (100 µM) (µM) % Inhibition S.E. S.I.* 4.51 0.957 100 25 99 0 4.52 2.96 46 100 47 0.91 >33 4.53 2.64 0 100 5 3.5 >38 4.54 2.85 60 50 50 15 >17 4.56 1.38 97 25 47 13 >17 4.57 3.51 100 100 51 15 4.59 4.69 99 100 100 0.17 4.60 4.22 99 100 99 0.3 4.61 4.68 51 100 44 9.7 >21 4.63 3.34 46 100 44 5.9 >30 4.64 4.64 97 100 55 14 4.65 1.33 100 25 87 3.1 4.66 1.21 99 25 93 2.4 4.67 3.27 89 100 41 15 >30 4.68 1.1 97 25 84 5.5 4.71 3.66 9 100 13 5.1 >27 4.72 2.17 85 50 93 2.5 4.76 2.55 4 100 4 2.6 >39 4.80 2.52 100 50 95 1

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Compound Name Structure cLogP

O

2-[(4-tert- HN 4.9 butylbenzyl)thio]- 3.28 S 4(3H)-pyrimidinone N

O 3-(diethylamino)-1- O 4.22 (4-ethoxyphenyl)-2,5- 3.28 N N pyrrolidinedione O

O

3-[2-(2- N N hydroxyethyl)-1- 4.31 1.94 piperidinyl]-1-phenyl- O 2,5-pyrrolidinedione

HO O 7-allyl-8-(1,3- N benzoxazol-2-ylthio)- N N NH 4.32 3-methyl-3,7- 2.75 dihydro-1H-purine- O S N O 2,6-dione

HO O 3-(3-bromophenyl)- N 1-hydroxy-2(1H)- 4.40 1.4 quinoxalinone 4- + oxide N O- Br

Table 4.6 Structures, nomenclature and calculate d partition coefficient (cLogP) of compounds displaying selectivity for L. donovani axenic amastigotes over mammalian Vero cells. (CONTINUED)

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Table 4.6 CONTINUED

Compound Name Structure cLogP

HO O 1-hydroxy-3-(4- N propoxyphenyl)- 4.43 1.5 2(1H)-quinoxalinone O + 4-oxide N O-

F 1-hydroxy-3-[3- F -O (trifluoromethyl)phe F N+ 4.44 nyl]-2(1H)- 2.91 quinoxalinone 4- oxide N O OH

O 1-(2-chlorophenyl)-3- Cl N [2-(2-hydroxyethyl)- N 4.45 2.53 1-piperidinyl]-2,5- O pyrrolidinedione HO

1'-(2- O methoxybenzyl)-5'- O O 4.52 methylspiro[1,3- 2.98 dioxane-2,3'-indol]- N 2'(1'H)-one O

3-[2-(2- O hydroxyethyl)-1- N 4.53 piperidinyl]-1-(4- N 2.5 methylphenyl)-2,5- O pyrrolidinedione HO

(CONTINUED)

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Table 4.6 CONTINUED

Compound Name Structure cLogP

3-methylphenyl 5- Cl chloro-2- O 4.54 (methylsulfonyl)-4- N 1.38 O pyrimidinecarboxylat O N S e O

F 4-[2-(4- F F bromophenyl)vinyl]- 4.56 Br 2.71 6-(trifluoromethyl)- HN 2(1H)-pyrimidinone N O

N-(3-{[(5-methyl- H 1,3,4-thiadiazol-2- S N 4.61 NH 1.5 yl)amino]carbonyl}p N O henyl)-2-furamide N O O

N-(2-furylmethyl)-N- O (8- 4.63 3.26 quinolinylsulfonyl)bu N N S O tanamide O O

N N-(4-fluorophenyl)-2- N O S {[5-(2-pyridinyl)-4H- 4.67 N 1.75 1,2,4-triazol-3- F NH H yl]thio}acetamide N

(CONTINUED)

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Table 4.6 CONTINUED

Compound Name Structure cLogP

2,4-diphenyl-1,2- O dihydro-3H- 4.71 N 2.77 pyrazolo[3,4- b]pyridin-3-one HN N

2,5-dimethyl-6-(2- N methylbenzyl)[1,2,4] O N 4.76 triazolo[1,5- N 1.14 a]pyrimidin-7(4H)- HN one

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4.3.1.5 Confirmation of Activity and Selectivity

To further evaluate the 17 compounds which showed selectivity for L. donovani amastigotes, 5 mg of each compound was purchased from ChemBridge

(www.hit2lead.com ). The compounds were dissolved in DMSO at a concentration of

20 mM and were reevaluated against both L. donovani axenic amastigotes and Vero cells. The assays were performed as described in the “Materials and Methods” section. Pentamidine and podophyllotoxin were again used as standards for the

Leishmania and Vero cell assays respectively; the IC 50 values obtained with these compounds fell within the expected range indicating the assay was working well

(data not shown). Table 4.7 shows the IC 50 values against L. donovani axenic amastigotes. The results of the selectivity screen against Vero cells are summarized in Table 4.8. As can be seen in Table 4.7, the IC 50 values obtained with the compounds supplied in the library correspond closely to those obtained with the compounds which were purchased individually. With the exception of compound

4.67 , which appeared to be more active against Vero cells in the retest assay, all of the compounds appear to retain their respective selectivities for L. donovani over vero cells. Being as compound 4.67 was not exceptionally active against

Leishmania and possessed marginal selectivity in the primary assay, it was not investigated further.

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Compound Primary IC 50 * StDev* Retest IC 50 * StDev* 4.9 4.8 0.57 3.1 0.68 4.22 4.2 0.96 2.4 0.76 4.31 3.2 0.48 2.4 0.36 4.32 1.5 0.41 2.9 1.5 4.40 1.2 0.49 2.6 0.26 4.43 1.5 0.2 2.1 0.3 4.44 1 0.47 0.84 0.16 4.45 1.7 0.01 1.4 0.52 4.52 3 0.27 3.2 0.56 4.53 2.6 0.48 2.9 1.3 4.54 2.9 0.76 1.8 0.35 4.56 1.4 0.13 1.1 0.13 4.61 4.7 0.76 5.4 0.87 4.63 3.3 0.54 3 0.62 4.67 3.3 0.2 3.3 0.19 3.7 1.8 9.6 7 4.71 4.76 2.6 0.54 2.7 1.1

Table 4.7 IC 50 values of selective compounds against L. donovani. IC 50 values for original compounds supplied in library (Primary IC 50 ) and for 5 mg quantity of separately purchased compounds (Retest IC 50 ) are given in µM units.

*Results based on the average of no less than 3 independent experiments

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Primary % Retest % Compound Conc. (µM) Inhibition* S.E. Inhibition** Range (±) 4.9 100 32 13 20 1.5 4.22 100 34 14 21 19 4.31 100 5 3.2 0 0 4.32 50 43 12 63 14 4.40 50 28 3.7 16 10 4.43 50 31 8.7 22 7 4.44 100 49 4.6 53 10 4.45 100 10 3.8 5 5 4.52 100 47 0.91 37 2 4.53 100 5 3.5 0 0 4.54 50 50 15 62 6 4.56 25 47 13 66 2 100 44 9.7 48 3 4.61 4.63 100 44 5.9 59 10 4.67 100 41 15 83 8

4.71 100 13 5.1 17 5 4.76 100 4 2.6 0 0

Table 4.8 % Inhibition of selective compounds against Vero cells. %

Inhibition for original compounds supplied in library (Primary % Inhibition) and 5 mg quantity of separately purchased compounds (Retest %

Inhibition); the standard error (S.E.) and range are reported for the compounds respectively.

* Results are the average for no fewer than 4 individual experiments

** Results are the average for two individual experiments

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4.3.1.6 Infected Macrophage Assay

All 17 of the compounds which showed selectivity for parasitic cells were analyzed in the infected macrophage assay. Drugs targeting intracellular face an additional set of challenges in that they must not only contend with the environment present within the host but also must gain entry to- and contend with the environment within the host cells. Thus, axenic in vitro drug susceptibility assays are twice-removed from the relevant biological context presented in vivo . The infected macrophage assay is a means to shorten the gap between axenic in vitro and in vivo conditions. Although the compounds do not have to clear all of the obstacles they would in an in vivo Leishmania model, the infected macrophage assay insures that they are capable of permeating the macrophage membrane, gain access to the phagolysosome and also can confirm that the compound demonstrates selective cytotoxicity towards the parasite. Traditionally, parasite clearance in this assay was assessed by optical enumeration of Giemsa stained amastigotes and macrophages.

Optical enumeration has drawbacks, most notable of which is the time required to count cells. When assaying multiple compounds over a range of concentrations, data acquisition becomes a major bottleneck. In an effort to remove this bottleneck, we have recently begun utilizing L. amazonensis transfected with a β-lactamase reporter gene [249]. Nitrocefin, which changes from yellow to a red color in the presence of β-lactamase, can be used to quantify the parasite burdens spectrophotometrically by measuring the absorbance at 490 nm. Coupled to a 96- well plate reader, this colorimetric method allows for the rapid and accurate

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quantification of parasite burden in the infected macrophage assay. The 17 selective compounds were all evaluated in the infected macrophage assay. Unfortunately, only compound 4.44 showed marginal activity in clearing parasites (IC 50 ~25 µM). It should be noted, however, that these results are only from one experiment. To be sure of these results, the assay will have to be repeated multiple times.

4.3.2 BIOLOGICAL EVALUATION OF COMPOUND 4.45

4.3.2.1 Transmission Electron Microscopy

Transmission electron microscopy (TEM) was used in an effort to gain some insight into the mechanism by which compound 4.45 exerts its leishmaniacidal activity. Although promastigotes have been more extensively characterized morphologically, amastigotes were chosen due to the fact that none of the compounds identified in the HTS have been evaluated against promastigotes as of yet. Compound 4.45 was incubated with L. donovani amastigotes at concentrations of 2.5, 5 and 10 µM for twenty-four hours. Examination of the cell samples by TEM confirmed what we have previously observed by light microscopy, that indeed, cultures of axenic amastigotes in fact contain both amastigotes and promastigotes.

This observation indicatess the requirement of a host cell to ensure that promastigotes undergo a uniform and complete metamorphosis to amastigotes; this further underscores the importance of the infected macrophage assay to close the gap between in vitro and in vivo activities. There appear to be three notable morphological changes in treated cells when compared to controls: aberrant Golgi

146

morphology, increased cytoplasmic vacuolization and nuclear disintegration (Figure

4.8).

The induction of an aberrant Golgi morphology by compound 4.45 may have implications in the parasite’s protein secretory pathway. Although protein secretory pathways in kinetoplastid parasites are not completely understood [see 255 for rev.], it is known that much like in mammalian systems, proteins are synthesized in the rough endoplasmic reticulum (ER) and transported to the for processing and translocation. Unlike mammalian systems, a complex of tubular clusters/translucent vesicles, the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, is the only site of endo- and exocytosis [256]. It is difficult to discern from the electron micrographs if treatment of cells with compound 4.5 is causing the Golgi apparatus to become distended and disintegrated or if it is causing an increase in secretory activity. If the former event is occurring this would indicate that compound 4.5 is interfering with cellular protein trafficking via disruption of the Golgi apparatus. If treatment with compound 4.5 results in exacerbated protein production this may be a last effort of the cells in attempt to survive as seen in L. amazonensis treated with the natural product parthenolide. This intense exocytic activity may help to explain the enlarged flagellar pockets observed in some of the treated parasites. The implications of this aberrant morphology are somewhat unclear in that they may be directly result in cytotoxicity by interfering with protein translocation yet it is also possible that they are the result of an indirect response by the parasite in an attempt to survive.

147

Much like the appearance of an aberrant Golgi apparatus, the implications of increased vacuolization may be somewhat ambiguous. An increase in the presence of acidocalcisomes may represent an attempt by the organism to respond to and cope with unfavorable osmotic conditions. The acidocalcisome, which is present in kinetoplastids [257] has been shown to be an important organelle for osmoregulation in T. cruzi [258]. It is more likely however, that the increased vacuolization is a result of, not a response to a cytotoxic environment. Numerous leishmaniacidal compounds have been shown to increase the number of vacuoles prior to cell death

[259,260,261]. It has further been suggested that vacuolization may be indicative of autophagic cell death [261].

Nuclear disintegration, unlike the former two morphological observations, is fairly unambiguous in that it is not a survival response but is indicative of impending cell death. To better investigate the effects of compound 4.45 on the nuclear membrane and DNA, it would be useful to perform a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, which can detect DNA fragmentation in end stage apoptosis [262]. Taken together, these observations are useful in explaining the cytotoxic effects of 4.45 on L. donovani parasites, however, they provide little insight into the actual mechanism of action.

148

A B

500 nm 500 nm

C

500 nm

Figure 4.8 Electron micrograph images of Leishmania donovani axenic amastigotes treated with compound 4.45. A control cell is shown in A; B and C show cells treated with 2.5 µM and 10 µM of compound 4.45 respectively for 24 h.

149

4.3.2.2 Flow Cytometry

In an attempt to gain further insight into the mechanism by which compound

4.45 exerts its cytotoxic effects on Leishmania , cell cycle analysis was performed by flow cytometry. Cells were treated with 2.5 µM and 10 µM compound 4.45 . As can be seen in figure 4.9, compound 4.45 does not appear to interfere with the progression of the cell cycle. The cells treated with 2.5 µM appear similar to the control. The variation in the 10 µM histogram is indicative of large populations of dead/dying cells; visual inspection confirmed that indeed, treatment of cells with 10

µM of compound 4.45 for 24 hr results in a very small percentage of surviving cells.

A B C

G0/G 1

Counts G2/M

Fluorescence

Figure 4.9 Histogram of cell cycle analysis by flow cytometry. Cells were treated with compound 4.45 for 24 hr at concentrations of 2.5 µM (B) and 10 µM (C). Control cells were incubated with 1 % DMSO (A)

150

4.4 CONCLUSIONS AND FUTURE DIRECTIONS

Leishmaniasis continues to place a large burden on developing nations and the unmet need for efficacious, inexpensive and orally available treatments persists.

The ChemBridge CNS set was chosen because it contains compounds that have attributes found in many drugs which are orally available. The compounds identified by screen and found to be selective for parasite cells will continue to be evaluated in the infected macrophage assay. The infected macrophage assay will be used as a tool to select which compounds will be evaluated in vivo . It should be noted, however, that although the infected macrophage assay will work well in establishing the hierarchy in which the compounds are evaluated in vivo it should not necessarily be used to exclude any of the compounds should they fail to show activity. The lack of activity in the infected macrophage assay may be a result of the compounds failing to localize in the phagolysosome. Fortunately, the properties of the compounds are such that structural modifications to alter properties and add functional groups can be performed without violating Lipinski’s rule of 5. As such, it will be possible to synthesize structural analogues of the primary hits in attempt to find a structural derivative which demonstrates better activity in the infected macrophage assay. An alternative option to analogue synthesis is to evaluate the compounds under in vivo conditions to look for activity. Prior to evaluating the compounds in the infected mouse assay, they will be administered to uninfected mice to look for signs of toxicity. If no overt signs of toxicity are observed by day 5 following the cessation of treatment, the compounds will be advanced to the infected model. Although our end goal is an orally active drug, during the first round the compounds will be

151

administered i.p. Intraperitoneal injection circumvents 1 st pass metabolism, thus a compound which may need some structural modifications to retain activity when administered orally will not be overlooked. Compounds demonstrating activity when administered i.p. will be reevaluated for oral activity. If oral activity is observed, the pharmacokinetic properties of the compound will be further examined, more specifically in terms of ADMET (Absorption, Distribution, Metabolism, Excretion and

Toxicity). It may also be useful to analyze the compound in an alternative animal model such as the hamster. The mouse model of infection does not exactly mimic the infection in humans, however, it is still useful for preliminary in vivo evaluation. In the likely event that compound optimization is required, this will be performed and the compounds will be reevaluated in the cyclical progression shown in figure 4.10.

In vitro In vivo Evaluation Evaluation

Analogue Biological Synthesis Evaluation

Figure 4.10 Cyclical progression of compound development

152

Aside from evaluation against parasites, it will be necessary to perform biological evaluation of the compounds in an effort to determine their mechanism of action. Being that no single compound showed exceptional activity in the infected macrophage assay it was decided to move forward and attempt to determine the mechanism of action of the selective compounds. Target identification will make it possible to intuitively synthesize analogues capable of penetrating into the phagolysosome without sacrificing activity against the parasites. Compound 4.45 was chosen first for evaluation because it demonstrates extremely good activity and selectivity. Furthermore, three other pyridinediones of similar structures were identified as hit compounds; this indicates that structural modification of the scaffold is fairly well tolerated, this is optimal for analogue synthesis. Preliminary biological evaluation of compound 4.45 indicatess that it may elicit its cytotoxic effects by disrupting protein trafficking, however, more work needs to be done to confirm this and also to identify the target(s) of 4.45 . Biological evaluation of the compounds will continue in hopes of identifying molecular targets; this information will be useful in designing future generation analogues and getting us closer to our ultimate goal of identifying and developing an orally available antileishmanial compound.

153

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